Recombinant yeast cells and their use in the production of thaumatin

Abstract

The present disclosure relates to the technical field of biological fermentation, and in particular, to a recombinant yeast cell and its use in producing thaumatin. The recombinant yeast cell comprises nucleic acids corresponding to the genes of thaumatin, PDI and MSN4, and can functionally express the same. The present disclosure provides a recombinant yeast cell co-expressing chaperone PDI and transcription factor MSN4 to improve the secretion expression of thaumatin protein. Surprisingly, under the synergistic effect of chaperone PDI and transcription factor MSN4, the secretion expression of exogenous thaumatin protein is significantly increased by 3.25 times.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to the field of biological fermentation, and in particular, to a recombinant yeast cell and its use in producing thaumatin. BACKGROUND

[0002] Thaumatin is the sweetest substance in the world, which is a protein extracted from the fruit of Thaurnato-coccus daniellii Benth originally from West Africa. The sweetness of thaumatin is about 2500-3000 times that of sucrose. Compared with traditional carbohydrate-saccharide and chemically synthesized sweeteners, thaumatin has the advantages of high sweetness, good taste, low heat, safety and non-toxicity, and can improve food flavor, increase milk flavor, mask bitter and astringent taste. Thaumatin can be degraded into amino acids needed by the human body and has certain nutrition. It is considered to be a promising food additive due to its unique advantages of not causing blood sugar to rise and the like. However, the method of extracting from the fruit of Thaurnato-coccus daniellii Benth has limited yield and high cost, which is difficult to meet the needs of people. Genetic engineering expression is a feasible method.

[0003] Pichia pastoris is a kind of yeast widely used in industrial production of exogenous proteins. Compared with bacteria, Pichia pastoris cells have some specific advantages, such as not producing endotoxin, having post-translational modification, being able to express and secrete, being easy to genetically manipulate, and the like, so that the production cost can be greatly reduced. At present, more than 5000 kinds of exogenous proteins have been successfully expressed in Pichia pastoris, and the expression of thaumatin by Pichia pastoris is a selectable method. Since thaumatin molecules contain 8 disulfide bonds, it is difficult to express correctly folded thaumatin protein by recombinant secretion. 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, which 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) as an important transcription factor can respond to heat shock, oxidative stress, nutrient deficiency and the like, and help cells to cope with various environmental stresses by regulating the expression of a series of target genes. At present, the amount of recombinant secretion of thaumatin by Pichia pastoris reported in the literature is low, which is difficult to meet the needs of industrial production. SUMMARY

[0004] According to an aspect of the present disclosure, there is provided a recombinant yeast cell comprising nucleic acids corresponding to the SOMATOSTATIN, PDI and MSN4 genes and capable of functionally expressing the same.

[0005] According to still another aspect of the present disclosure, there is provided a method for producing SOMATOSTATIN, comprising: i) culturing the recombinant yeast cell as described above in a suitable medium, ii) isolating the SOMATOSTATIN from the medium.

[0006] The present disclosure provides a recombinant yeast cell co-expressing chaperone PDI and transcription factor MSN4 to improve the secretion expression of SOMATOSTATIN protein, and unexpectedly, the secretion expression of exogenous SOMATOSTATIN protein is significantly increased by 3.25 times under the synergistic effect of chaperone PDI and transcription factor MSN4. BRIEF DESCRIPTION OF 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 needed in the specific embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present disclosure, and those skilled in the art can also obtain other drawings according to these drawings without creative effort.

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

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

[0010] Reference will now be made in detail to the present disclosure, one or more examples of which are illustrated in the drawings. Each example is provided as an explanation and not a limitation of the present disclosure. Indeed, it will be apparent to one skilled in the art that numerous modifications and variations can be made in the present disclosure without departing from its scope or spirit. For example, features illustrated or described as part of one embodiment can be used with another embodiment to yield a still further embodiment.

[0011] Unless otherwise indicated, all terms, including technical and scientific terms, used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. By further guidance, the following definitions are set forth to better define the present teachings. The terminology used in the specification of the present disclosure herein is used only in a descriptive sense and not intended to limit the present disclosure.

[0012] In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Also, the terms and experimental operation steps related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology used herein are the terms and conventional steps widely used in the corresponding field. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided as follows.

[0013] The selection range of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, including any two related listed items, any more related listed items, or all related listed items.

[0014] The terms "containing", "including" and "comprising" used in the present disclosure are synonymous terms, which are inclusive or open, and do not exclude additional, unmentioned members, elements or method steps.

[0015] The numerical range expressed by endpoints in the present disclosure includes all numerical values and fractions contained in the range, as well as the mentioned endpoints.

[0016] In the present disclosure, the concentration values involved include fluctuations within a certain range. For example, it can fluctuate within a corresponding accuracy range. For example, 2%, it can be allowed to fluctuate within ±0.1%. For numerical values that are larger or do not need to be controlled too finely, it is also allowed that the meaning includes larger fluctuations. For example, 100mM, it can be allowed to fluctuate within ±1%, ±2%, ±5%, etc. For molecular weight, it is allowed that the meaning includes ±10% fluctuation.

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

[0018] In the present disclosure, the description of "a plurality of", "a plurality of", etc. refers to greater than or equal to 2 in number, unless otherwise specified.

[0019] In the present disclosure, in the technical features described in an open way, both the closed technical solutions consisting of the enumerated features and the open technical solutions containing the enumerated features are included.

[0020] In the context of the present disclosure, the term "increased expression" or "overexpression" means that the amount of brazzein protein produced by a cell is higher as compared to 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 a functional transcription of the relevant nucleic acid sequence, allowing the nucleic acid sequence to actually be transcribed, for example leading to the synthesis of a protein.

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

[0023] As used herein, the term "nucleic acid" or "nucleotide" refers to the monomeric units of deoxyribonucleotide or ribonucleotide polymers (i.e., polynucleotides) in either single- or double-stranded form, and encompasses known analogs having properties substantially similar to the naturally-occurring nucleotides (e.g., peptide nucleic acids) unless otherwise limited. For example, an enzyme of some sort defined by a nucleotide sequence encoding the enzyme includes (unless otherwise limited) nucleotide sequences that hybridize with the reference nucleotide sequence encoding the enzyme. A polynucleotide can be a full-length or subsequence of a natural or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes a reference to the complementary sequence to a given sequence, as well. Thus, DNA or RNA with a backbone that is modified is a polynucleotide as the term is contemplated herein for reasons of stability or otherwise. Furthermore, DNA or RNA comprising rare bases such as inosine or modified bases such as tritylated bases, to name two examples, are polynucleotides as the term is used herein. It will be understood that a wide variety of modifications to DNA and RNA have been made 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 that are specific to viruses and cells, including simple and complex cells.

[0024] The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein. One 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 particular sequence (e.g., "SEQ ID NO: X") refers to a polynucleotide and / or nucleic acid sequence comprising the particular sequence, provided that one or more nucleic acids are mutated, substituted, deleted, added, and / or inserted, and the polynucleotide encodes a sequence having the same (qualitatively) function (e.g., the function of expressing a chaperone protein, as a transcription factor, or as 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 that 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, e.g., as displayed by an amino acid sequence. These terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. A natural property of such analogues of naturally occurring amino acids is that the protein, when incorporated into a protein, is specifically reactive with antibodies raised against the same protein consisting entirely of naturally occurring amino acids. The terms "polypeptide," "peptide," and "protein" also encompass modifications, including but not limited to glycosylation, lipid attachment, sulfation, Y-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0027] All the documents mentioned in the present disclosure are cited as references in the present disclosure as if each document is individually cited as a reference. Unless and to the extent that the cited documents conflict with the purpose and / or technical solutions of the present disclosure, the cited documents are cited in the present disclosure in their entirety, in all purposes. When the present disclosure refers to the cited documents, the definitions of the relevant technical features, terms, names, phrases, etc. in the cited documents are also cited in the present disclosure. When the present disclosure refers to the cited documents, the examples, preferred modes of the relevant technical features cited are also cited in 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 is correct or is modified according to the description in the present disclosure adaptively.

[0028] In the present disclosure, PDI refers to endoplasmic reticulum protein disulfide isomerase. It is a multifunctional protein from the thioredoxin superfamily that catalyzes disulfide bond formation and helps 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 having a typical zinc finger domain, and mainly regulates STREs (stress response element)-like genes in response to stress processes.

[0030] The first aspect of the present disclosure relates to a recombinant yeast cell comprising nucleic acids corresponding to the genes of thaumatin, PDI and MSN4, and capable of functionally expressing the same.

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

[0032] The nucleic acids corresponding to the genes of thaumatin, PDI and MSN4 referred to in the present disclosure refer to any nucleic acid sequence capable of expressing a protein having similar or identical activity of thaumatin, PDI and MSN4 (exemplary thaumatin, PDI and MSN4 active protein sequences are as shown in SEQ ID NOs: 4-6). That is, it comprises a functionally equivalent sequence of the sequences shown in SEQ ID NOs: 1-3.

[0033] Exemplary functional equivalent sequences also include those having 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: 1-3, and which can be isolated from any cell; but preferably a eukaryotic cell, particularly a yeast cell. The percentage of sequence identity can be obtained by known bioinformatics algorithms, including the Myers and Miller algorithm (Bioinformatics, 4(l):ll-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 Karlin and Altschul algorithm (Altschul et al., J. Mol. Biol., 215(3):403-410, 1990; PNAS, 90:5873-5877, 1993). These are familiar to those skilled in the art.

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

[0035] Exemplary functional equivalent sequences also include those obtained by codon optimization of the above sequences.

[0036] Functional equivalent sequences also clearly include sequences of the above sequences that have been subjected to general nucleic acid modifications. General nucleic acid modifications can be made using well known methods, such as adding tails, shortening sequences, lengthening sequences, moving sequences up or down stream by a few 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), inverted dT, inverted 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 permissible as long as they do not affect the function of the expressed protein.

[0037] The yeast cells described herein can be derived from a host cell by any technique known to those of skill in the art to be suitable. Such techniques can include any one or more of mutagenesis, recombinant DNA techniques (including but not limited to CRISPR-CAS techniques), selective and / or adaptive evolution, vector transformation, conjugation, cell fusion, and / or cytoduction between yeast strains. Suitably, one or more desired genes are incorporated into the yeast cell by a combination of one or more of the above techniques. In some embodiments, the nucleic acid is integrated into the genome and / or expressed extrachromosomally, with extrachromosomal expression preferably using a replicative expression vector. The term "expression vector" refers to a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest under the control of (i.e., operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments can include a promoter and terminator sequences, and can optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or can contain elements of both. In particular, an expression vector comprises nucleic acid sequences in the 5' to 3' direction which comprise and are operably linked to: (a) yeast-recognized transcription and translation initiation regions, (b) a coding sequence of a polypeptide of interest, and (c) yeast-recognized transcription and translation termination regions. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and are available from commercial suppliers. Individual expression cassettes for fungal expression are known and consist essentially 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 pPICZA, pAO815, pGAPZA, and the like.

[0038] In some embodiments, the recombinant yeast cell further comprises at least one other additional recombinant secretion-promoting gene, for example a gene for a chaperone, a foldase and / or a glycosylation-promoting protein.

[0039] In some other embodiments, the recombinant yeast cell does not comprise an exogenously introduced chaperone other than PDI.

[0040] In some other embodiments, the recombinant yeast cell does not comprise an exogenously introduced transcription factor other than MSN4.

[0041] In some embodiments, the expression is constitutive expression and / or inducible expression. "Constitutive expression" is understood herein as the continuous transcription of a nucleic acid sequence. That is, the nucleic acid sequence is transcribed in a constant manner. Genes expressed constitutively are always "on". "Inducible expression" is, in turn, the transcription of a nucleic acid sequence only in the presence of an inducer, the transcription usually being induced by an inducible promoter.

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

[0043] Other example yeasts include Torulaspora, such as Torulaspora delbrueckii; Kluyveromyces, such as K. 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 anomala; Metschnikowia; Issatchenkia, such as Issatchenkia orientalis; Kloeckera, such as Kloeckera apiculata; and Aureobasidium, such as Aureobasidium pullulans.

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

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

[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, ii) isolating thaumatin from the culture medium.

[0047] The recombinant protein can be isolated and purified by various separation methods according to the physical, chemical and other characteristics thereof, 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 lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various 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 with reference to the examples. It should be understood that these examples are only used to illustrate the present disclosure and not to limit the scope of the present disclosure. The experimental methods not specified in the following examples are preferably referred to the guidance given in the present disclosure, and can also be carried out according to the experimental manuals or conventional conditions in the art, or can be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturers.

[0050] In the following specific examples, the measurement parameters of the raw material components may, without specific instructions, have slight deviations within the weighing accuracy range. With respect to the temperature and time parameters, acceptable deviations caused by the instrument testing accuracy or operation accuracy are allowed.

[0051] Materials and reagents

[0052] Restriction enzymes, high-fidelity DNA polymerase were purchased from NEB, homologous recombination kit -Basic Seamless Cloning and Assembly Kit was purchased from Beijing Zoman Biotechnology Co., Ltd., primers MSN4-f and MSN4-r, primers pGAP-MSN4-f and pGAP-MSN4-r, primers FPDM and RPDM, primers pGAP-M-H-f and pGAP-M-H-r (all synthesized by Suzhou Jingezhi Biotechnology Co., Ltd.), DNA Marker, 2x PCR Master Mix, Agarose Gel DNA Recovery Kit, Plasmid Miniprep Kit were purchased from Tiangen Biotech (Beijing) Co., Ltd., Endotoxin-free Plasmid Midiprep Kit was purchased from Promega, Protein Marker was purchased from Thermo Fisher Scientific, Zeocin Selective Antibiotic was purchased from InvivoGen, and the rest of the reagents were domestic or imported analytical pure. 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 rest of the reagents were domestic or imported analytical pure.

[0053] Yeast 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) 100 mmol / 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 solid:

[0061] 2% glucose (w / v), 1.34% YNB (w / v), 4 x 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 genetically engineered bacteria ZAPHST expressing thaumatin protein

[0064] Search for Thaumatin (thaumatin) gene sequence of Thaumatococcus daniellii in literature and GenBank, select mature protein segment of GenBank: AAA93095.1, optimize codons after (shown in SEQ ID NO: 1), synthesized by General Biological Systems (Anhui) Co., Ltd., insert thaumatin gene into pPICZA vector, and the recombinant plasmid is named as pPICZA-PHST.

[0065] 2. Transformation and screening

[0066] 5-10 μg plasmid pPICZA-PHST was digested with restriction endonuclease Pme I, and after recovery, the volume was adjusted to 5-10 μL by concentration. The method for preparing Pichia pastoris competent cells and electroporation was referred to the operation manual Catalog no. V190-20 (Preparation of Pichia for Electroporation, Transformation by Electroporation) of Invitrogen Corporation. 5-10 μg of linearized plasmid was mixed with 80 μL of Pichia pastoris competent (GS115), transferred to a pre-cooled 0.2 cm electroporation cup, and electroporated at a voltage of 1500 V, a capacitance of 25 μF, and a resistance of 200 Q. After electroporation, 1 mL of ice-precooled 1 mol / L sorbitol solution was immediately added, mixed, incubated at 30°C for 2 h, and an appropriate amount of the transformation liquid was spread on a YPD + 100 μg / mL Zeocin plate and cultured at 30°C for 2-4 days to obtain positive transformants.

[0067] 3. Shake flask fermentation verification

[0068] The single transformants were inoculated into BMGY medium, and after 24 h of 30°C, 200 rpm shaking culture, they were transferred into BMMY medium, and after 30°C, 200 rpm shaking culture, 1% methanol was added every 24 h. After 3 d of induction and expression, centrifugation was performed, and the supernatant was subjected to sweet protein electrophoresis determination. A Pichia pastoris genetically engineered bacterium with secretory expression of sweet protein was screened, and was named ZAPHST. Figure 2 lane 1).

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

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

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

[0072] 2. Transformation and screening

[0073] 5-10 μg plasmid pAO815-PDI was digested with restriction enzyme Sail, and after recovery, the volume was adjusted to 5-10 μL by concentration. The method for preparing Pichia competent cells and electroporation was referred to the operation manual Catalog no. V190-20 (Preparation of Pichia for Electroporation, Transformation by Electroporation) of Invitrogen Company. 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 at a voltage of 1500 V, a capacitance of 25 μF, and a resistance of 200 Q. After electroporation, 1 mL of ice-precooled 1 mol / L sorbitol solution was immediately added, mixed, incubated at 30°C for 2 h, and an appropriate amount of the transformed liquid was spread on an MD solid plate and cultured at 30°C for 2-3 days.

[0074] 3. Shake flask fermentation verification

[0075] A single transformant was picked and inoculated into BMGY medium, and after 24 h of 30°C, 200 rpm shaking culture, it was transferred into BMMY medium, and after 30°C, 200 rpm shaking culture, 1% methanol was added every 24 h. After 3 d of induction and expression, centrifugation was performed, and the supernatant was subjected to sweet protein electrophoresis determination. A Pichia genetic engineering bacteria with 1.13-fold increase in sweet protein secretion and expression compared with the original strain ZAPHST was screened, and was named ZAPHST-P. Figure 2

[0076] Example 3 Construction of genetic engineering bacteria ZAPHST-M expressing transcription factor MSN4

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

[0078] The MSN4 fragment was amplified from the Pichia 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 subjected to Gibson assembly with the pGAPZ A yeast expression vector linearized by BstBI and AgeI. The assembled plasmid was named pGAPZ A-MSN4 plasmid,

[0079] ​Then, the pGAP-MSN4-H fragment was amplified from the pGAPZ A-MSN4 plasmid using primers pGAP-M-H-f and pGAP-M-H-r (pGAP-M-H-f: 5'-CCGAAAAGTGCCACCTGGAGATCTTTTTTGTAGAAATGTCTTG-3', pGAP-M-H-r: 5'-GAAGCTATGGTGTGTGGGGGATCCGCACAAACGAAGGTCTCAC-3') (the primers were synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.), and Gibson assembly was performed using the large fragment of the pPICA-Hyg plasmid (Note: the pPICA-Hyg plasmid was synthesized by Anhui General Biotechnology Co., Ltd.) digested with BglII and BamHI to obtain the pGAP-MSN4-HygR.

[0080] 2. Transformation and screening

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

[0082] 3. Shake flask fermentation verification

[0083] The single transformants were inoculated into BMGY medium, and after 24 hours of 30°C, 200 rpm shaking culture, they were transferred into BMMY medium, and after 30°C, 200 rpm shaking culture, 1% methanol was added every 24 hours. After 3 days of induction and expression, centrifugation was performed, and the supernatant was subjected to sweet protein electrophoresis determination, and a Pichia pastoris genetically engineered bacterium with 0.85-fold increase in sweet protein secretion and expression compared to the original strain ZAPHST was screened, and was named ZAPHST-M. Figure 2

[0084] ​Construction of somatin protein genetically engineered bacteria co-expressing chaperone protein PDI and transcription factor MSN4

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

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

[0087] The MSN4 fragment was amplified from the Pichia pastoris GS115 genome (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.

[0088] Then, the pGAP-MSN4 fragment was amplified from the pGAPZ A-MSN4 plasmid (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 the pAO815-PDI linearized with BamHI. The assembled plasmid was named pAO815-PDI-MSN4.

[0089] 2. Transformation and screening

[0090] 5-10 μg plasmid pAO815-PDI-MSN4 was digested with restriction enzyme Sal I, and after recovery, the volume was adjusted to 5-10 μL. The method for preparing Pichia competent cells and electroporation was according to the operation manual Catalog no. V190-20 (Preparation of Pichia for Electroporation, Transformation by Electroporation) of Invitrogen Corporation. 5-10 μg of linearized plasmid was mixed with 80 μL of Pichia competent cells (Pichia genetic engineering bacteria ZAPHST), and transferred to a pre-cooled 0.2 cm electroporation cup. The electroporation was carried out at a voltage of 1500 V, a capacitance of 25 μF, and a resistance of 200 Q. After the electroporation, 1 mL of ice-precooled 1 mol / L sorbitol solution was immediately added, mixed, and incubated at 30 °C for 2 h. An appropriate amount of the transformed liquid was spread on an MD solid plate, and cultured at 30 °C for 2-3 days. Four transformed clones A, B, C, and D were picked and subjected to PCR identification (using primers FPDM and RPDM) (primer sequences: FPDM: 5'-CTTTAAGAATTCGCCTTAGACATGACTGTTC-3', RPDM: 5'-TCAACATCAAAATCCGAAGATACAAAGCTA-3'). The results were all positive clones (lane 3 to lane 6). Figure 1

[0091] 3. Shake flask fermentation verification

[0092] Clone D was picked and inoculated into BMGY medium, and cultured at 30 °C with 200 rpm shaking for 24 h. Then, it was transferred into BMMY medium, and cultured at 30 °C with 200 rpm shaking. 1% methanol was added every 24 h. After 3 d of induction, the bacteria were removed by centrifugation, and the supernatant was subjected to a thaumatin electrophoresis assay. The results showed that compared with the original strain ZAPHST, the genetic engineering bacteria ZAPHST-P expressing chaperone protein PDI, and the genetic engineering bacteria ZAPHST-M expressing transcription factor MSN4, the clone had a higher ability to secretively express thaumatin (Table 1, Figure 2 The genetic engineering bacteria of Pichia co-expressing chaperone protein PDI and transcription factor MSN4, which was screened and had a high ability to secretively express thaumatin, was named ZAPHST-PM.

[0093] The expression level was as follows:

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

[0095] Table 1 Electrophoresis scanning results of Pichia pastoris strains secreting and expressing thaumatin

[0096] SEQ ID NO Strain Electrophoretic scan band area number 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 examples, we explored the expression of chaperone protein PDI gene or transcription factor MSN4 gene and the co-expression of chaperone protein PDI and transcription factor MSN4 to improve the secretion expression of thaumatin protein, and screened the Pichia pastoris strain ZAPHST secreting and expressing thaumatin protein as the starting strain. Molecular biology techniques were used to insert the chaperone protein PDI gene or the transcription factor MSN4 gene and simultaneously insert the chaperone protein PDI gene and the transcription factor MSN4 gene into the genome of the Pichia pastoris strain ZAPHST, thereby constructing the genetically engineered strain ZAPHST-P expressing chaperone protein PDI, the genetically engineered strain ZAPHST-M expressing transcription factor MSN4, and the genetically engineered strain ZAPHST-PM co-expressing chaperone protein PDI and transcription factor MSN4. Compared with the original strain ZAPHST, the secretion expression of the exogenous thaumatin protein of ZAPHST-P increased by 2.13 times, the secretion expression of the exogenous thaumatin protein of ZAPHST-M increased by 1.85 times, and the secretion expression of the exogenous thaumatin protein of ZAPHST-PM significantly increased by 3.25 times under the synergistic effect of chaperone protein PDI and transcription factor MSN4.

[0102] The above-described embodiments are merely illustrative of several embodiments of the present disclosure, which are described in a relatively specific and detailed manner, but should not be construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present disclosure, which are all within the scope of the present disclosure. Therefore, the scope of protection of the patent of the present disclosure should be subject to the appended claims, and the description and drawings can be used to interpret the content of the claims.

Claims

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

1.

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

2.

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

3.

4. The recombinant yeast cell according to any one of claims 1 to 3, the nucleic acids are integrated into the genome and / or expressed extrachromosomally as expression cassettes, wherein the extrachromosomal expression uses a replicative expression vector.

5. The recombinant yeast cell according to any one of claims 1 to 3, further comprising at least one other additional recombinant secretion promoting gene.

6. The recombinant yeast cell according to claim 5, the other additional recombinant secretion promoting gene is selected from the group of genes for chaperones, foldases and / or glycosylation promoting proteins.

7. The recombinant yeast cell according to any one of claims 1 to 3, the expression is constitutive and / or inducible.

8. A method for producing thaumatin, comprising: i) cultivating the recombinant yeast cell according to any one of claims 1 to 7 in a suitable culture medium, ii) isolating thaumatin from the culture medium.

9. The method according to claim 8, the culture medium comprises BMGY medium and / or BMMY medium.

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