Recombinant yeast cells and their use in production of thaumatin

Abstract

The invention relates to the technical field of biological fermentation, in particular to a recombinant yeast cell and application thereof in production of thaumatin. The recombinant yeast cell contains nucleic acids corresponding to thaumatin, PDI and MSN4 genes, and can functionally express the thaumatin, PDI and MSN4 genes. The invention provides the recombinant yeast cell for co-expressing the chaperonin PDI and the transcription factor MSN4 to improve the secretory expression of the thaumatin, and accidentally, under the synergistic effect of the chaperonin PDI and the transcription factor MSN4, the secretory expression quantity of the exogenous thaumatin is remarkably increased by 3.25 times.

Description

Technical Field

[0001] The present disclosure generally relates to the field of biofermentation technology, and in particular, to a recombinant yeast cell and use thereof in producing thaumatin. Background Art

[0002] Thaumatin is the sweetest substance in the world. It was first extracted from the fruit of African arrowroot (Thaurnato-coccus daniellii Benth) native to western Africa. Its sweetness is about 2500 to 3000 times that of sucrose of the same mass. Compared with traditional carbohydrates-sugars and chemically synthesized sweeteners, thaumatin has the advantages of high sweetness, good taste, low calories, safety and non-toxicity, and can improve food flavor, increase milk flavor, and mask bitterness and astringency. Thaumatin can be degraded into amino acids required by the human body and has certain nutritional value. Because of its unique advantages such as not causing an increase in blood sugar, it is considered to be a food additive with development prospects. However, the method of extracting from African arrowroot fruit has limited yield and high cost, which is difficult to meet people's needs. Genetic engineering expression is a feasible method.

[0003] Pichia pastoris is a yeast widely used in industrial production of foreign proteins. Compared with bacteria, Pichia pastoris cells have some specific advantages, such as no endotoxin production, post-translational modification, secretory expression, and easy genetic manipulation, which can greatly reduce production costs. At present, more than 5,000 foreign proteins have been successfully expressed in Pichia pastoris. Using Pichia pastoris to express thaumatin is an optional method. Since the thaumatin molecule contains 8 disulfide bonds, it is difficult to recombinantly secrete and express the correct conformation of thaumatin protein. Molecular chaperones can help the correct folding of proteins or correct the misfolding of proteins and reduce the degradation rate of misfolded proteins. In yeast, protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily. It can catalyze the formation of disulfide bonds and promote the correct folding of proteins, thereby improving the expression level of target proteins in yeast. In yeast, multicopy suppressor of SNF1 mutation protein 4 (MSN4) is an important transcription factor that can respond to heat shock, oxidative stress, nutrient deficiency and other conditions, and regulate the expression of a series of target genes to help cells cope with various environmental stresses. Currently, the literature reports that the amount of thaumatin secreted by Pichia pastoris is low, which is difficult to meet the needs of industrial production. Summary of the invention

[0004] According to one aspect of the present disclosure, it relates to a recombinant yeast cell, which comprises nucleic acids corresponding to thaumatin, PDI and MSN4 genes and is capable of functionally expressing them.

[0005] According to yet another aspect of the present disclosure, it relates to a method for producing thaumatin, comprising: i) culturing the recombinant yeast cell as described above in a suitable culture medium, and ii) separating thaumatin from the culture medium.

[0006] The present disclosure provides a recombinant yeast cell that co-expresses chaperone protein PDI and transcription factor MSN4 to increase the secretory expression of thaumatin. Unexpectedly, under the synergistic effect of chaperone protein PDI and transcription factor MSN4, the secretory expression amount of exogenous thaumatin protein is significantly increased to 3.25 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0008] Figure 1 PCR identification of transformant clones co-expressing chaperone protein and transcription factor genes; M: Marker; Lane 1: Negative control; Lane 2: ZAPHST; Lane 3: transformant clone A co-expressing chaperone protein and transcription factor genes; Lane 4: transformant clone B co-expressing chaperone protein and transcription factor genes; Lane 5: transformant clone C co-expressing chaperone protein and transcription factor genes; Lane 6: transformant clone D co-expressing chaperone protein and transcription factor genes.

[0009] Figure 2 The results of SDS-PAGE detection of expressed proteins; Lane 1: ZAPHST; Lane 2: ZAPHST-P; Lane 3: ZAPHST-M; Lane 4: ZAPHST-PM. DETAILED DESCRIPTION

[0010] References to embodiments of the present disclosure will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0011] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present disclosure is the same as that commonly understood by those of ordinary skill in the art to which the present disclosure belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0012] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0013] The terms "and / or", "or / and", and "and / or" used in this article have a selection scope that includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, said any and all combinations including any two related listed items, any more related listed items, or a combination of all related listed items.

[0014] As used in this disclosure, the terms "comprising," "including," and "including" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0015]

[0013] Numerical ranges recited in this disclosure as endpoints include all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0016] The present disclosure relates to concentration values, and the meaning includes fluctuations within a certain range. For example, fluctuations can be allowed within a corresponding precision range. For example, 2%, a fluctuation within the range of ±0.1% can be allowed. For values ​​that are large or do not require too fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, fluctuations within the range of ±1%, ±2%, ±5%, etc. can be allowed. Involving molecular weight, the meaning is allowed to include fluctuations of ±10%.

[0017] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0018] In the present disclosure, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0019] In the present disclosure, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0020] In the context of the present disclosure, the term "increased expression" or "overexpression" refers to the amount of thaumatin produced by a cell that is higher than when expressed in a control cell showing normal, unaltered or baseline expression.

[0021] In the context of the present disclosure, the term "functionally expressed" means that there is functional transcription of the relevant nucleic acid sequence, allowing the nucleic acid sequence to actually be transcribed, for example resulting in the synthesis of a protein.

[0022] As used herein, the term "gene" refers to a nucleic acid sequence that can be transcribed into mRNA that is then translated into a protein. A gene encoding a certain protein refers to one or more nucleic acid sequences encoding the protein.

[0023] As used herein, the term "nucleic acid" or "nucleotide" refers to a monomer unit in a deoxyribonucleotide or ribonucleotide polymer (i.e., polynucleotide) in a single-stranded or double-stranded form, and unless otherwise limited, known analogs with the essential properties of natural nucleotides are encompassed because they hybridize with single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). For example, a certain enzyme defined by a nucleotide sequence encoding an enzyme includes (unless otherwise limited) a nucleotide sequence hybridized with a reference nucleotide sequence encoding an enzyme. A polynucleotide can be the full length or subsequence of a natural or heterologous structure or regulatory gene. Unless otherwise indicated, the term includes reference to a specified sequence and its complementary sequence. Therefore, a DNA or RNA with a modified backbone for stability or other reasons is a term "polynucleotide" as intended herein. In addition, a DNA or RNA containing a rare base (such as inosine) or a modified base (such as a tritylated base) (just to name two examples) is a term polynucleotide as used herein. It will be understood that a variety of modifications have been made to DNA and RNA for many useful purposes known to those skilled in the art. The term polynucleotide as used herein includes such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells (especially including simple and complex cells).

[0024] The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein. An example of a nucleic acid sequence is a DNA sequence.

[0025] As used herein, the term "functional homolog" (or simply "homolog") of a polynucleotide and / or nucleic acid sequence having a specific sequence (e.g., "SEQ ID NO: X") refers to a polynucleotide and / or nucleic acid sequence comprising the specific sequence, provided that one or more nucleic acids are mutated, substituted, deleted, added and / or inserted, and the polynucleotide encodes a sequence having (qualitatively) the same function (e.g., expression of a chaperone protein, function as a transcription factor or a sweetener). With respect to nucleic acid sequences, the term functional homolog is intended to include nucleic acid sequences that differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

[0026] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, such as those exhibited by an amino acid sequence. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. An essential property of such analogues of naturally occurring amino acids is that, when incorporated into a protein, the protein has specific reactivity with antibodies elicited by proteins consisting of the same but entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0027] All documents mentioned in the present disclosure are cited as references in the present disclosure, just as each document is cited as a reference individually. Unless they conflict with the invention objectives and / or technical solutions of the present disclosure, the cited documents involved in the present disclosure are cited with all their contents and all their purposes. When the present disclosure involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present disclosure involves cited documents, the examples and preferred methods of the cited relevant technical features may also be incorporated into the present disclosure as references, but are limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present disclosure, the present disclosure shall prevail or be modified adaptively according to the description of the present disclosure.

[0028] In the present disclosure, PDI refers to endoplasmic reticulum protein disulfide isomerase, which is a multifunctional protein from the thioredoxin superfamily that catalyzes disulfide bond formation and helps in the correct folding of proteins.

[0029] In the present disclosure, MSN4 refers to a multicopy suppressor of SNF1 mutation protein 4, which is a transcription factor with a typical zinc finger domain and mainly regulates STREs (stress response element) genes in the stress response process.

[0030] A first aspect of the present disclosure relates to a recombinant yeast cell, which comprises nucleic acids corresponding to thaumatin, PDI and MSN4 genes and is capable of functionally expressing them.

[0031] The nucleic acids corresponding to the thaumatin, PDI and MSN4 genes are well known in the art, for example: in some embodiments, the nucleic acid sequence of thaumatin is shown in SEQ ID NO: 1. In some embodiments, the nucleic acid sequence of PDI is shown in SEQ ID NO: 2. In some embodiments, the nucleic acid sequence of MSN4 is shown in SEQ ID NO: 3.

[0032] The nucleic acids corresponding to the thaumatin, PDI and MSN4 genes referred to in the present disclosure refer to any nucleic acid sequences capable of expressing proteins having similar or identical thaumatin, PDI and MSN4 activities (exemplary thaumatin, PDI and MSN4 activity protein sequences are shown in SEQ ID NOs: 4 to 6). That is, they contain functional equivalent sequences of the sequences shown in the above SEQ ID NOs: 1 to 3.

[0033] Functional equivalent sequences also include sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to any of the sequences shown in SEQ ID NOs: 1 to 3, and can be isolated from any cell; but preferably eukaryotic cells, especially yeast cells. The percentage of sequence identity can be obtained by well-known bioinformatics algorithms, including the Myers and Miller algorithm (Bioinformatics, 4 (1): 11-17, 1988), the Needleman-Wunsch global alignment method (J. Mol. Biol., 48 (3): 443-53, 1970), the Smith-Waterman local alignment method (J. Mol. Biol., 147: 195-197, 1981), the Pearson and Lipman similarity search method (PNAS, 85 (8): 2444-2448, 1988), and the algorithm of Karlin and Altschul (Altschul et al., J. Mol. Biol., 215 (3): 403-410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to those skilled in the art.

[0034] Functional equivalent sequences obviously also include DNA sequences that can hybridize with the DNA of the above sequences under stringent conditions. "Stringent conditions" used in the present disclosure are well known, including hybridization at 60°C for 12 to 16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH 6.4) and 1mM EDTA, followed by washing at 65°C with a washing solution containing 0.1% SDS and 0.1% SSC for 15 to 60 minutes.

[0035] Functionally equivalent sequences obviously also include sequences obtained after codon optimization of the above sequences.

[0036] Functional equivalent sequences obviously also include sequences in which the above sequences have been modified by general nucleic acids. General nucleic acid modifications can be carried out by known methods, such as adding tails, shortening sequences, extending sequences, moving the sequence up and downstream by several bases, or any combination thereof. Base modifications such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2'-deoxyadenosine, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2'-deoxyadenosine-5'-triphosphate, 2,6-diaminopurine (2-amino-dA), reverse dT, reverse dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's), and including at least one mismatched base at one of the bases, or replacing at least one of the bases with an RNA base, are all allowed as long as they do not affect the function of the expressed protein.

[0037] Yeast cells described herein can be derived from host cells by any suitable technology known to those skilled in the art. Such technology can include any one or more of mutagenesis, recombinant DNA technology (including but not limited to CRISPR-CAS technology), selective and / or adaptive evolution, vector transformation, conjugation, cell fusion and / or cell guidance between yeast strains. Suitably, one or more desired genes are incorporated into yeast cells by a combination of one or more of the above technologies. In some embodiments, the nucleic acid is integrated into the genome and / or extrachromosomal expression as an expression cassette, wherein the extrachromosomal expression preferably uses a replicative expression vector. The term "expression vector" refers to a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest, which is under the control of another nucleic acid segment that provides its transcription (i.e., operably connected thereto). Such another segment can include a promoter and a terminator sequence, and can optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmids or viral DNA, or can contain elements of both. In particular, the expression vector comprises a nucleic acid sequence that comprises and is operably linked in the 5' to 3' direction to: (a) a transcription and translation initiation region recognized by yeast, (b) a coding sequence for a target polypeptide, and (c) a transcription and translation termination region recognized by yeast. Vectors, strains and protocols for expression in Saccharomyces and Pichia are known in the art and can be obtained from commercial suppliers. Each expression cassette for fungal expression is known and is essentially composed of a promoter, a fungal gene and a terminator. Alternatively or additionally, the gene can be expressed extrachromosomally, preferably using a replicative expression vector, such as a shuttle vector. Preferred expression vectors of the present disclosure include pPICZ A, pAO815, pGAPZA, etc.

[0038] In some embodiments, the recombinant yeast cell further comprises at least one other additional recombinant secretion-promoting gene, such as genes for molecular chaperones, folding enzymes and / or glycosylation-promoting proteins.

[0039] In other embodiments, the recombinant yeast cell does not contain exogenously introduced molecular chaperones other than PDI.

[0040] In other embodiments, the recombinant yeast cell does not contain exogenously introduced transcription factors other than MSN4.

[0041] In some embodiments, the expression is constitutive expression and / or inducible expression. "Constitutive expression" is understood herein as continuous transcription of the nucleic acid sequence. In other words, the nucleic acid sequence is transcribed in a continuous manner. A gene that is constitutively expressed is always "on". "Inducible expression" means that the nucleic acid sequence is transcribed only in the presence of an inducer, and transcription is usually initiated by induction of an inducible promoter.

[0042] The recombinant yeast cell is preferably a yeast cell, or is derived from a yeast cell, that is, the yeast cell is from the genus of Saccharomycetaceae or the genus of Schizosaccharomycetaceae. That is, preferably, the host cell from which the recombinant yeast cell is derived is a yeast cell from the genus of Saccharomycetaceae or the genus of Schizosaccharomycetaceae. Examples of suitable yeast cells include Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces jurei, Saccharomyces pastorianus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum, and Saccharomyces bayanus. Examples of suitable yeast cells further include the genus Schizosaccharomyces, such as Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, and Schizosaccharomyces cryophilus.

[0043] Other exemplary yeasts include Torulaspora, such as Torulaspora delbrueckii; Kluyveromyces, such as Kluyveromyces marxianus; Zygosaccharomyces, such as Zygosaccharomyces bailii; Brettanomyces, such as Brettanomyces intermedius; Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis, and Dekkera heteromorpha. anomala; Metschmkowia, Issatchenkia, such as Issatchenkia orientalis, Kloeckera, such as Kloeckera apiculata; and Aureobasidium, such as Aureobasidium pullulans.

[0044] Preferably, the yeast cells are industrial yeast cells. The living environment of yeast cells in industrial processes is significantly different from the living environment in the laboratory. Industrial yeast cells must be able to perform well under a variety of environmental conditions, which may change during the period. Such changes include changes in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have a potential impact on the cell growth and ethanol production of yeast cells.

[0045] Preferably, the yeast cell is of the genus Pichia, such as Pichia stipitis, Pichia pastoris or Pichia angus.

[0046] A second aspect of the present disclosure relates to a method for producing thaumatin, comprising: i) culturing the recombinant yeast cell as described above in a suitable culture medium, and ii) isolating thaumatin from the culture medium.

[0047] The recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties as needed. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0048] In some embodiments, the culture medium comprises BMGY medium and / or BMMY medium.

[0049] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present disclosure, and can also be based on the experimental manuals or conventional conditions in the art, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0050] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0051] Materials and reagents

[0052] Restriction endonucleases and high-fidelity DNA polymerase were purchased from NEB, and homologous recombination kit -BasicSeamless Cloning and Assembly Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd., primers MSN4-f and MSN4-r, primers pGAP-MSN4-f and pGAP-MSN4-r, primers FPDM and RPDM, primers pGAP-MHf and pGAP-MHr (all synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), DNAMarker, 2-fold concentration PCR premix reagent, agarose gel DNA recovery kit, plasmid mini-extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and endotoxin-free plasmid mini-extraction kit PureYield TM Plasmid Midiprep System was purchased from Promega, Protein Marker was purchased from Thermo Fisher Scientific, Zeocin selective antibiotic was purchased from InvivoGen, and the remaining reagents were domestically produced or imported analytical grade.

[0053] Yeast culture medium:

[0054] YPD:

[0055] Peptone 2% (W / v), yeast extract 1% (W / v), glucose 2% (W / v).

[0056] BMGY:

[0057] Yeast extract 1% (W / v), peptone 2% (W / v), potassium phosphate buffer (pH 6.0) 100mmol / L, YNB 1.34% (w / v), biotin 0.00004% (w / v), glycerol 1% (v / v). Note: YNB is yeast nitrogen base.

[0058] BMMY:

[0059] Yeast extract 1% (W / v), peptone 2% (W / v), potassium phosphate buffer (pH 6.0) 100 mmol / L, YNB 1.34% (W / v), biotin 0.00004% (W / v), methanol 0.5% (v / v). Note: YNB is yeast nitrogen base.

[0060] MD Solids:

[0061] 2% glucose (w / v), 1.34% YNB (w / v), 4×10 -5 % (w / v) biotin (filter sterilized), 2.0% (w / v) agar. Note: YNB is yeast nitrogen base.

[0062] Example 1 Construction of engineered bacteria ZAPHST

[0063] 1. Construction of the genetically engineered strain ZAPHST expressing thaumatin

[0064] The Thaumatin gene sequence of Thaumatin from Thaumatococcus daniellii was retrieved from the literature and GenBank, and the mature protein segment of GenBank: AAA93095.1 was selected. After codon optimization (shown in SEQ ID NO: 1), the whole gene was synthesized by General Biosystems (Anhui) Co., Ltd. The thaumatin gene was inserted into the pPICZ A vector, and the recombinant plasmid was named pPICZA-PHST.

[0065] 2. Transformation and screening

[0066] 5-10 μg of plasmid pPICZA-PHST was digested with restriction endonuclease PmeI, and the concentrated volume was adjusted to 5-10 μL after recovery. The preparation of Pichia competent cells and electroporation methods refer to the Invitrogen operation manual Catalog no. V190-20 (Preparation of Pichia for Electroporation, Transformation by Electroporation), 5-10 μg of linearized plasmid was mixed with 80 μL of Pichia competent cells (GS115), transferred to a pre-cooled 0.2 cm electroporation cup, and electroporated: voltage 1500V, capacitance 25 μF, resistance 200Q, after the electroporation, immediately add 1 mL of ice-precooled 1 mol / L sorbitol solution, mix, incubate at 30°C for 2h, take an appropriate amount of transformation solution to coat YPD+100 μg / mL Zeocin plates, and culture at 30°C for 2 to 4 days to obtain positive transformants.

[0067] 3. Shake flask fermentation verification

[0068] Single transformants were selected and inoculated into BMGY medium, and cultured at 30°C and 200 rpm for 24 hours. Then, they were transferred to BMMY medium and cultured at 30°C and 200 rpm. 1% methanol was added every 24 hours. After 3 days of induction, the cells were centrifuged and the supernatant was subjected to electrophoresis for sweet protein. A Pichia yeast genetic engineering strain with sweet protein secretion expression was screened and named: ZAPHST ( Figure 2 .lane 1).

[0069] Example 2 Construction of genetically engineered bacteria ZAPHST-P expressing chaperone protein PDI

[0070] 1. Construction of expression vector for chaperone protein PDI

[0071] Referring to the Pichia genome sequence on the NCBI website, the PDI gene (SEQ ID NO: 2) was synthesized by General Biotechnology (Anhui) Co., Ltd. The PDI gene was constructed into the EcoRI site of the plasmid vector pAO815, and the vector was named pAO815-PDI.

[0072] 2. Transformation and screening

[0073] 5-10 μg of plasmid pAO815-PDI was digested with restriction endonuclease SalI, and the concentrated volume was adjusted to 5-10 μL after recovery. The preparation of Pichia competent cells and electroporation methods refer to the Invitrogen company's operation manual Catalog no. V190-20 (Preparation of Pichia for Electroporation, Transformation by Electroporation), 5-10 μg of linearized plasmid was mixed with 80 μL of Pichia competent cells (Pichia genetic engineering bacteria ZAPHST), transferred to a pre-cooled 0.2 cm electroporation cup, and electroporated: voltage 1500V, capacitance 25 μF, resistance 200Q, after the electroporation, immediately add 1 mL of ice-precooled 1 mol / L sorbitol solution, mix, incubate at 30°C for 2h, take an appropriate amount of transformation solution to coat MD solid plates, and culture at 30°C for 2 to 3 days.

[0074] 3. Shake flask fermentation verification

[0075] Single transformants were selected and inoculated into BMGY medium, shaken and cultured at 30°C and 200 rpm for 24 hours, and then transferred to BMMY medium, shaken and cultured at 30°C and 200 rpm, with 1% methanol added every 24 hours. After induced expression for 3 days, centrifugation was performed, and the supernatant was taken for sweet protein electrophoresis determination. A Pichia pastoris genetically engineered strain with a 1.13-fold increase in sweet protein secretion expression compared with the original strain ZAPHST was screened and named: ZAPHST-P ( Figure 2 , lane 2).

[0076] Example 3 Construction of genetically engineered bacteria ZAPHST-M expressing transcription factor MSN4

[0077] 1. Construction of expression vector for expressing transcription factor MSN4

[0078] The MSN4 fragment was amplified using the Pichia pastoris GS115 genome as a template (primer combination: MSN4-f, MSN4-r) (MSN4-f: 5'-AATCAATTGAACAACTATTTCGAAACGATGTCTACAACAAAACCAA-3'; MSN4-r: 5'-TTAGAATCTAGCAAGACCGGTTCACTGCTTACGGTGAGTA-3') (MSN4 gene sequence: SEQ ID NO: 3), and the fragment was Gibson assembled with the pGAPZ A yeast expression vector linearized with BstBI and AgeI. The assembled plasmid was named pGAPZ A-MSN4 plasmid.

[0079] Then, the pGAPZ A-MSN4 plasmid was used as a template and pGAP-MHf and pGAP-MHr were used as primers (pGAP-MHf: 5'-CCGAAAAGTGCCACCTGGAGATCTTTTTTGTAGAAATGTCTTG-3', pGAP-MHr: 5'-GAAGCTATGGTGTGTGGGGGATCCGCACAAACGAAGGTCTCAC-3') (primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.) to combine and amplify the fragment pGAP-MSN4-H, and the fragment was Gibson assembled with the large fragment after BglII and BamHI double restriction of plasmid pPICA-Hyg (Note: pPICA-Hyg was synthesized by Anhui General Biotechnology Co., Ltd.) to form pGAP-MSN4-HygR.

[0080] 2. Transformation and screening

[0081] 5-10 μg of plasmid pGAP-MSN4-HygR was digested with restriction endonuclease AvrII, recovered and concentrated, and the volume was adjusted to 5-10 μL. The preparation of Pichia competent cells and electroporation methods refer to the In vitrogen operating manual Catalog no. V 190-20 (Preparation of Pichia for Electroporation, Transformation by Electroporation). 5-10 μg of linearized plasmid was mixed with 80 μL of Pichia competent cells (Pichia genetically engineered bacteria ZAPHST), transferred to a pre-cooled 0.2 cm electric shock cup, and electroporated: voltage 1500 V, capacitance 25 μF, resistance 200 Ω. After the electroporation, 1 mL of ice-cold 1 mol / L sorbitol solution was immediately added, mixed, incubated at 30°C for 2 h, and an appropriate amount of transformation solution was spread on a YPD solid plate containing 300 μg / ml Hygromycin B, and cultured at 30°C for 2 to 3 days.

[0082] 3. Shake flask fermentation verification

[0083] Single transformants were selected and inoculated into BMGY medium, shaken and cultured at 30°C and 200 rpm for 24 hours, and then transferred to BMMY medium, shaken and cultured at 30°C and 200 rpm, with 1% methanol added every 24 hours. After induced expression for 3 days, centrifuged, and the supernatant was taken for sweet protein electrophoresis determination. A Pichia pastoris genetically engineered strain with a 0.85-fold increase in sweet protein secretion expression compared with the original strain ZAPHST was screened and named: ZAPHST-M ( Figure 2 , lane 3).

[0084] Example 4 Construction of thaumatin gene engineering bacteria co-expressing chaperone protein PDI and transcription factor MSN4

[0085] 1. Construction of expression vector for co-expression of chaperone protein PDI and transcription factor MSN4

[0086] With reference to the Pichia genome sequence on the NCBI website, the PDI gene (SEQ ID NO: 2) was synthesized by General Biotechnology (Anhui) Co., Ltd. The PDI gene was constructed into the EcoRI site of the plasmid vector pAO815.

[0087] The MSN4 fragment was amplified using the Pichia pastoris GS115 genome as a template (primer combination: MSN 4-f, MSN4-r) (MSN4-f: 5'-AATCAATTGAACAACTATTTCGAAACGATGTCTACAACAAAACCAA-3'; MSN4-r: 5'-TTAGAATCTAGCAAGACCGGTTCACTGCTTACGGTGAGTA-3') (MSN4 gene sequence: SEQ ID NO: 3), and the fragment was Gibson assembled with the pGAPZ A yeast expression vector linearized with BstBI and AgeI. The assembled plasmid was named pGAPZ A-MSN4 plasmid.

[0088] Then, the pGAPZ A-MSN4 fragment was amplified using the pGAPZ A-MSN4 plasmid as a template (primer combination: pGAP-MSN4-f, pGAP-MSN4-r) (pGAP-MSN4-f: 5'CGTTCGTTTGTGCGGATCTTTTTTGTAGAAATGTCTTGGTGTCC-3'; pGAP-MSN4-r: 5'-TGATAAACTACCGCATTAGGATCCGCACAAACGAAGGTCTCA-3'), and the fragment was Gibson assembled with pAO815-PDI linearized with BamHI. The assembled plasmid was named pAO815-PDI-MSN4.

[0089] 2. Transformation and screening

[0090] 5-10 μg of plasmid pAO815-PDI-MSN4 was digested with restriction endonuclease SalI, recovered and concentrated to adjust the volume to 5-10 μL. The preparation of Pichia competent cells and electroporation methods refer to the Invitrogen operation manual Catalog no. V190-20 (Preparation of Pichia for Electroporation, Transformation byElectroporation), 5-10μg of linearized plasmid was mixed with 80μL of Pichia pastoris competent state (Pichia pastoris genetic engineering bacteria ZAPHST), and transferred to a pre-cooled 0.2cm electroporation cup, and the electroporation was performed with a voltage of 1500V, a capacitance of 25μF, and a resistance of 200Ω. After the electroporation, 1mL of ice-cold 1mol / L sorbitol solution was immediately added, mixed, and incubated at 30℃ for 2h. An appropriate amount of transformation liquid was coated on the MD solid plate, and cultured at 30℃ for 2-3 days. Four transformant clones A, B, C, and D were selected for PCR identification (identification primers FPDM and RPDM) (primer sequences: FPDM: 5'-CTTTAAGAATTCGCCTTAGACATGACTGTTC-3', RPDM: 5'-TCAACATCAAAATCCGAAGATACAAAGCTA-3'), and all the results were positive clones ( Figure 1 , lane 3 to lane 6).

[0091] 3. Shake flask fermentation verification

[0092] Transformant clone D was selected and inoculated into BMGY medium, and cultured at 30°C and 200 rpm for 24 hours, and then transferred to BMMY medium, cultured at 30°C and 200 rpm, and 1% methanol was added every 24 hours. After induced expression for 3 days, the bacteria were removed by centrifugation, and the supernatant was subjected to thaumatin electrophoresis. The results showed that the clone had a higher ability to secrete and express thaumatin protein than the original strain ZAPHST, the genetically engineered strain ZAPHST-P expressing the chaperone protein PDI, and the genetically engineered strain ZAPHST-M expressing the transcription factor MSN4 (Table 1, Figure 2 ), the genetically engineered strain of Pichia pastoris that efficiently secretes and expresses thaumatin and co-expresses the chaperone protein PDI and the transcription factor MSN4 was named ZAPHST-PM.

[0093] The expression levels are as follows:

[0094] Compared with the original strain ZAPHST, the genetically engineered bacteria ZAPHST-P expressing the chaperone protein PDI significantly increased the secretion expression of thaumatin (increased the secretion expression of exogenous protein sweet protein by 1.13 times), the genetically engineered bacteria ZAPHST-M expressing the transcription factor MSN4 significantly increased the secretion expression of thaumatin (increased the secretion expression of exogenous protein sweet protein by 0.85 times), and the genetically engineered bacteria ZAPHST-PM co-expressing the chaperone protein PDI and the transcription factor MSN4 significantly increased the secretion expression of thaumatin and increased the secretion expression of exogenous protein sweet protein by 2.25 times (using Image J software for quantitative analysis of the band area, Table 1) ( Figure 2 ).

[0095] Table 1 Electrophoresis scanning results of thaumatin secreted by Pichia pastoris strains

[0096] Serial number strains Electrophoresis scanning band area 1 ZAPHST 17801.50 2 ZAPHST-P 37907.92 3 ZAPHST-M 32927.97 4 ZAPHST-PM 57918.77

[0097] Note:

[0098] (37907.92-17801.50) / 17801.50=1.13

[0099] (32927.97-17801.50) / 17801.50=0.85

[0100] (57918.77-17801.50) / 17801.50=2.25

[0101] According to the above embodiments, we explored the expression of chaperone protein PDI gene or transcription factor MSN4 gene and co-expression of chaperone protein PDI and transcription factor MSN4 to improve the secretion expression of thaumatin protein, and used the screened Pichia pastoris strain ZAPHST that secretes and expresses thaumatin protein as the starting strain. We used molecular biological techniques to insert the chaperone protein PDI gene or the transcription factor MSN4 gene and the chaperone protein PDI gene and the transcription factor MSN4 gene into the genome of the Pichia pastoris strain ZAPHST, respectively, and simultaneously insert the chaperone protein PDI gene and the transcription factor MSN4 gene, thereby constructing the genetically engineered bacteria ZAPHST-P expressing the chaperone protein PDI, the genetically engineered bacteria ZAPHST-M expressing the transcription factor MSN4, and the genetically engineered bacteria ZAPHST-PM co-expressing the chaperone protein PDI and the transcription factor MSN4. Compared with the original strain ZAPHST, the secretion expression level of exogenous thaumatin protein in ZAPHST-P increased by 2.13 times, and the secretion expression level of exogenous thaumatin protein in ZAPHST-M increased by 1.85 times. Under the synergistic action of the chaperone protein PDI and the transcription factor MSN4, the secretion expression level of exogenous thaumatin protein in ZAPHST-PM increased significantly to 3.25 times.

[0102] The above-described embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be based on the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A recombinant yeast cell comprising nucleic acids corresponding to thaumatin, PDI and MSN4 genes and capable of functionally expressing them. 2 . The recombinant yeast cell according to claim 1 , wherein the nucleic acid sequence of thaumatin is as shown in SEQ ID NO:

1. 3 . 3 . The recombinant yeast cell according to claim 1 , wherein the nucleic acid sequence of PDI is as shown in SEQ ID NO:

2. 4 . The recombinant yeast cell according to claim 1 , wherein the nucleic acid sequence of MSN4 is shown in SEQ ID NO:

3.

5. The recombinant yeast cell according to any one of claims 1 to 4, wherein the nucleic acid is integrated into the genome and / or expressed extrachromosomally as an expression cassette, wherein the extrachromosomal expression preferably uses a replicable expression vector.

6. The recombinant yeast cell according to any one of claims 1 to 4, further comprising at least one other additional recombinant secretion-promoting gene, such as genes for molecular chaperones, folding enzymes and / or glycosylation-promoting proteins.

7. The recombinant yeast cell according to any one of claims 1 to 4, wherein the expression is constitutive expression and / or inducible expression.

8. The recombinant yeast cell according to any one of claims 1 to 4, which is Pichia pastoris.

9. A method for producing thaumatin, comprising: i) culturing the recombinant yeast cell according to any one of claims 1 to 8 in a suitable culture medium, and ii) separating thaumatin from the culture medium.

10. The method according to claim 9, wherein the culture medium comprises BMGY medium and / or BMMY medium.

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