Protein combination influencing yeast growth rate and foreign protein expression quantity

Abstract

The invention relates to a protein combination for influencing the growth rate of yeast and the expression quantity of foreign proteins. After genes in a peptidase A1 family, a peptidase M1 family, a peptidase M18 family, a peptidase M28 family, a peptidase S54 family, a peptidase S10 family, a peptidase S16 family, a peptidase S14 family and a peptidase S8 family and protein defect related genes are knocked out as transcription activating factors, the growth rate can be increased, and the expression quantity of foreign proteins can be increased.

Description

Technical Field

[0001] The present invention relates to the field of proteins, in particular to a protein combination that affects yeast growth rate and exogenous protein expression amount. Background Art

[0002] The yeast expression system has advantages such as high fermentation density, strong secretion ability, and low glycosylation degree. However, it is easy to be attacked by yeast proteolytic enzymes and degraded during the expression of foreign proteins. This is one of the main factors affecting the efficient expression of foreign proteins in Pichia pastoris. Studies have shown that the amount of various proteolytic enzymes in yeast cells will change with changes in nutritional conditions during the culture process, such as starvation stress during the culture process, changes in carbon and nitrogen sources, etc. Especially in the high-density fermentation process, higher levels of proteases can often be detected in the culture medium.

[0003] During the fermentation process, when the cells are under stress, the proteases located in the yeast vacuole are overexpressed or the cells are autolyzed and ruptured and released to the extracellular space, thereby degrading the recombinant proteins secreted to the extracellular space.

[0004] Sweeteners are food additives that give food or feed a sweet taste. According to their sources, they can be divided into natural sweeteners and artificial sweeteners. With the development of organic chemistry, artificial sweeteners have gradually become dominant, but because some chemically synthesized sweeteners are suspected of having safety issues, many countries in the world have discouraged or even banned the use of certain chemical sweeteners. In view of this, it is extremely important to find safe, non-toxic, and pure natural sweeteners.

[0005] Taking brazzein as an example of foreign protein expressed in yeast, brazzein (sometimes translated as Brazzein in Chinese) is the main sweet protein of Oubli fruit (Pentadiplandra brazzeana Baillon) in West Africa. It is present in the extracellular pulp tissue around the seeds. For thousands of years, locals have used it as a sweetener. Since this juice is also used to help infants wean, Brazzein has been proven to have a long history of safe use for both infants and adults. Brazzein is a single-chain polypeptide composed of 54 amino acid residues, containing 8 cysteines, forming 4 pairs of intramolecular disulfide bonds. The relative molecular mass of brazzein is 6500, and its sweetness is 2000 times that of the same mass of sucrose. Compared with other sweet proteins, brazzein has the smallest molecular weight and the best water solubility. The aqueous solution still retains sweetness after 4h heat treatment at 80 degrees Celsius, and has good thermal stability and pH stability.

[0006] Salt is the most common condiment, and NaCl is one of its main ingredients. However, excessive salt intake can lead to an increase in sodium ion concentration. This may lead to increased osmotic pressure, increased vascular tone and cardiac output, and ultimately increase the risk of cardiovascular disease. Studies have shown that a low-salt diet can effectively lower blood pressure in both hypertensive and normotensive patients.

[0007] On October 24, 2024, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) issued the "Definition and Requirements of a Healthy Diet", which recommends that adults use no more than 5 grams of salt per day, but most countries in the world exceed this amount. Generally speaking, the industry uses food additives to reduce the sodium ion concentration in salt, among which potassium chloride is the most widely used food additive. Although potassium ions, which also have a salty taste, can reduce the risk of hypertension while ensuring the saltiness, the increase in potassium is not good for patients with kidney disease, and it is likely to interact with drugs for the treatment of cardiovascular diseases and aggravate the patient's condition. There are certain health risks, and when the concentration exceeds 15%, potassium ions have a bitter taste in the mouth. In addition to salt substitutes such as metal salts, taste enhancers can also be added to activate taste receptors located in the human mouth and tongue to compensate for the taste differences caused by the reduction in sodium ion concentration, among which umami and salty taste perception are most closely related. Currently, the most widely produced and used food flavor enhancer in my country is sodium L-glutamate (MSG). Although it is low-cost, has a strong flavor, and can economically and effectively reduce the amount of salt added, MSG still contains sodium ions and cannot meet the true meaning of "reducing sodium salt".

[0008] The concept of salty peptides was proposed by Tada et al. in 1984. The team discovered Orn-Tau-HCl and Lys-Tau-HCl in the process of synthesizing N-terminal analogs of casein hydrolysate. These two peptides do not contain Na + , but has a similar or even higher salinity than NaC1. Summary of the invention

[0009] One of the present invention provides PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_0913, PAS_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_chr3_0934, PAS_chr3_0633, PRC1 Use of a combination of at least two proteins among PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 or a single PAS_chr3_0689 protein in affecting the growth rate of yeast and / or the expression amount of exogenous protein; wherein the amino acid sequence of the PAS_chr3_1087 protein is shown in SEQ ID No.2, the amino acid sequence of the PAS_chr4_0584 protein is shown in SEQ ID No.4, the amino acid sequence of the PAS_chr2-1_0652 protein is shown in SEQ ID No.6, the amino acid sequence of the PAS_chr4_0113 protein is shown in SEQ ID No.8, the amino acid sequence of the PAS_chr2-2_0380 protein is shown in SEQ ID No.10, the amino acid sequence of the PAS_chr4_0913 protein is shown in SEQ ID No. The amino acid sequence of the PAS_chr1-4_0013 protein is shown in SEQ ID No.26, the amino acid sequence of the PAS_chr1-1_0174 protein is shown in SEQ ID No.28, the amino acid sequence of the PAS_chr3_0979 protein is shown in SEQ ID No.30, the amino acid sequence of the PAS_chr1-4_0048 protein is shown in SEQ ID No.31, the amino acid sequence of the PAS_chr3_0934 protein is shown in SEQ ID No.32, the amino acid sequence of the PAS_chr3_0633 protein is shown in SEQ ID No.33, the amino acid sequence of the PRC1 PAS_chr1-4_0013 protein is shown in SEQ ID No.34, the amino acid sequence of the PAS_chr1-1_0174 protein is shown in SEQ ID No.35, the amino acid sequence of the PAS_chr3_0979 protein is shown in SEQ ID No.36. The amino acid sequence of the PAS_chr1-1_0226 protein is shown in SEQ ID No.30, the amino acid sequence of the PAS_chr1-1_0226 protein is shown in SEQ ID No.32, and the amino acid sequence of the PAS_chr3_0689 protein is shown in SEQ ID No.34.The exogenous protein can be obtained by transferring a gene encoding the exogenous protein into the yeast, and expressing the transferred gene by the yeast.

[0010] In a specific embodiment, PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_0913, PAS_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_chr3_0934, PAS_chr3_0633, PRC1 Compared with before the expression of at least two genes among PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 or the PAS_chr3_0689 gene alone in yeast is silenced, the expression of PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_0913, PAS_chr1-4_0 The expression of at least two proteins among PAS_chr3_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_chr3_0934, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 or the expression of PAS_chr3_0689 protein alone is silenced to increase the growth rate of the yeast and / or the expression amount of the exogenous protein in the yeast.

[0011] In a specific embodiment, the nucleotide sequence of the PAS_chr3_1087 gene is shown in SEQ ID No.1, the nucleotide sequence of the PAS_chr4_0584 gene is shown in SEQ ID No.3, the nucleotide sequence of the PAS_chr2-1_0652 gene is shown in SEQ ID No.5, the nucleotide sequence of the PAS_chr4_0113 gene is shown in SEQ ID No.7, the nucleotide sequence of the PAS_chr2-2_0380 gene is shown in SEQ ID No.9, the nucleotide sequence of the PAS_chr4_0913 gene is shown in SEQ ID No.11, the nucleotide sequence of the PAS_chr1-4_0611 gene is shown in SEQ ID No.13, the nucleotide sequence of the PAS_chr3_0953 gene is shown in SEQ ID No.15, the nucleotide sequence of the PAS_chr1-1_0194 gene is shown in SEQ ID The nucleotide sequence of the PAS_chr1-4_0048 gene is shown in SEQ ID No.17, the nucleotide sequence of the PAS_chr1-4_0048 gene is shown in SEQ ID No.19, the nucleotide sequence of the PAS_chr3_0934 gene is shown in SEQ ID No.21, the nucleotide sequence of the PAS_chr3_0633 gene is shown in SEQ ID No.23, the nucleotide sequence of the PRC1 PAS_chr1-4_0013 gene is shown in SEQ ID No.25, the nucleotide sequence of the PAS_chr1-1_0174 gene is shown in SEQ ID No.27, the nucleotide sequence of the PAS_chr3_0979 gene is shown in SEQ ID No.29, the nucleotide sequence of the PAS_chr1-1_0226 gene is shown in SEQ ID No.31, and the nucleotide sequence of the PAS_chr3_0689 gene is shown in SEQ IDNo.33.

[0012] In one embodiment, by knocking out PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_0913, PAS_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_chr3_0934, PAS_chr3_0633, PRC1 At least two genes among PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 or the PAS_chr3_0689 gene alone are used to silence the expression of the corresponding protein.

[0013] In a specific embodiment, the PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 genes in the yeast are knocked out simultaneously.

[0014] In a specific embodiment, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633 and PRC1 PAS_chr1-4_0013 genes in the yeast are knocked out simultaneously.

[0015] In a specific embodiment, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953 and PAS_chr2-2_0380 genes in the yeast are knocked out simultaneously.

[0016] In a specific embodiment, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380 and PAS_chr1-4_0611 genes in the yeast are knocked out simultaneously.

[0017] In a specific embodiment, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, PAS_chr1-1_0226 and PAS_chr3_1087 genes in the yeast are knocked out simultaneously.

[0018] In a specific embodiment, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611 and PAS_chr1-1_0194 genes in the yeast are knocked out simultaneously.

[0019] In a specific embodiment, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584 and PAS_chr3_1087 genes in the yeast are knocked out simultaneously.

[0020] In a specific embodiment, the exogenous protein is at least one of brazilian tamarind, reduced salt protein, semaglutide, casein and whey protein.

[0021] In a specific embodiment, the amino acid sequence of the brazilian tamarind protein is shown as SEQ ID No.36.

[0022] In a specific embodiment, the gene encoding the brazilian tamanin is introduced into the yeast so that the yeast expresses the brazilian tamanin.

[0023] In a specific embodiment, the nucleotide sequence of the gene encoding the brazilian tamarind protein is shown as SEQ ID No. 35.

[0024] In a specific embodiment, the amino acid sequence of the salt-reducing protein is shown as SEQ ID No.38.

[0025] In a specific embodiment, the gene encoding the salt-reducing protein is transferred into the yeast so that the yeast expresses the salt-reducing protein.

[0026] In a specific embodiment, the nucleotide sequence of the gene encoding the salt-reducing protein is shown as SEQ ID No.37.

[0027] In a specific embodiment, the amino acid sequence of semaglutide is shown as SEQ ID No.40.

[0028] In a specific embodiment, the gene encoding the semaglutide is transferred into the yeast so that the yeast expresses the semaglutide.

[0029] In a specific embodiment, the nucleotide sequence of the gene encoding semaglutide is shown as SEQ ID No.39.

[0030] In one embodiment, the yeast is Pichia pastoris.

[0031] In a specific embodiment, the starting strain of the yeast is Pichia pastoris GS115 strain or SMD1163 strain.

[0032] The second aspect of the present invention provides an engineered yeast, which is obtained by knocking out the gene in the yeast in any one of the applications in the first aspect of the present invention. These engineered bacteria can have different commercial applications, including but not limited to providing a testing and comparison platform for interested persons or companies, or using this platform to express different exogenous and endogenous protein and polypeptide products.

[0033] In a specific embodiment, the yeast engineered bacteria is a yeast engineered bacteria obtained by using the Pichia pastoris GS115 strain as a starting strain and knocking out the PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 genes therein.

[0034] In a specific embodiment, the yeast engineered bacteria is a yeast engineered bacteria obtained by taking the Pichia pastoris GS115 strain as the starting strain and knocking out the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633 and PRC1 PAS_chr1-4_0013 genes therein.

[0035] In a specific embodiment, the yeast engineering bacteria is a yeast engineering bacteria obtained by taking Pichia pastoris GS115 strain as the starting strain, and knocking out the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953 and PAS_chr2-2_0380 genes therein.

[0036] In a specific embodiment, the yeast engineering bacteria is a yeast engineering bacteria obtained by taking Pichia pastoris GS115 strain as the starting strain, and knocking out the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380 and PAS_chr1-4_0611 genes therein.

[0037] In one embodiment, the yeast engineering bacteria is based on the Pichia pastoris GS115 strain as the starting strain, and PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013 are knocked out at the same time. , PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, PAS_chr1-1_0226 and PAS_chr3_1087 genes.

[0038] In a specific embodiment, the yeast engineering bacteria is a yeast engineering bacteria obtained by taking Pichia pastoris GS115 strain as the starting strain, and knocking out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611 and PAS_chr1-1_0194 genes therein.

[0039] In a specific embodiment, the yeast engineering bacteria is a yeast engineering bacteria obtained by taking Pichia pastoris GS115 strain as the starting strain, and knocking out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584 and PAS_chr3_1087 genes therein.

[0040] In a specific embodiment, the yeast engineered bacteria is a yeast engineered bacteria obtained by taking the Pichia pastoris GS115 strain as a starting strain and knocking out the PAS_chr3_0689 gene therein.

[0041] In a specific embodiment, a gene for expressing a foreign protein is transferred into the yeast engineered bacteria to express the foreign protein.

[0042] In a specific embodiment, the exogenous protein is at least one of brazilian tamarind, reduced salt protein, semaglutide, casein and whey protein.

[0043] In a specific embodiment, the amino acid sequence of the brazilian tamarind protein is shown as SEQ ID No.36.

[0044] In a specific embodiment, the gene encoding the brazilian tamanin is introduced into the yeast so that the yeast expresses the brazilian tamanin.

[0045] In a specific embodiment, the nucleotide sequence of the gene encoding the brazilian tamarind protein is shown as SEQ ID No. 35.

[0046] In a specific embodiment, the amino acid sequence of the salt-reducing protein is shown as SEQ ID No.38.

[0047] In a specific embodiment, the gene encoding the salt-reducing protein is transferred into the yeast so that the yeast expresses the salt-reducing protein.

[0048] In a specific embodiment, the nucleotide sequence of the gene encoding the salt-reducing protein is shown as SEQ ID No.37.

[0049] In a specific embodiment, the amino acid sequence of semaglutide is shown as SEQ ID No.40.

[0050] In a specific embodiment, the gene encoding the semaglutide is transferred into the yeast so that the yeast expresses the semaglutide.

[0051] In a specific embodiment, the nucleotide sequence of the gene encoding semaglutide is shown as SEQ ID No.39.

[0052] Sweetener: The term "sweetener" is used herein to refer to a product or composition in a sweetening form that can be directly applied to food, beverages and / or pharmaceutical products for human consumption. A sweetener may contain a single active ingredient, i.e., a single substance with a sweet taste, or it may contain several such active ingredients, i.e., a blend of substances that contribute to sweet taste. The sweetener may be an active ingredient in its substantially pure form, such as a sweet protein that is currently in a form that can be applied to a product intended for human consumption, separated from its production cells. Alternatively, the sweetener may also contain other substances, such as, for example, fillers (e.g., lactose), in addition to the active ingredient. The sweetener may be further blended with other substances before being applied to food or beverage products, or before being sold to final consumers for home purposes such as sweetening of tea or coffee.

[0053] Brazilian sweet: also known as Brazilian sweet protein, bunazhen sweet protein or braziin, refers to the extractable form of the fruit of the West African climbing plant Pentadiplandra brazzeana (Baillon), or its recombinantly produced form. In nature, Brazilian sweet appears in three different forms, with or without glutamine residues or pyroglutamic acid attached to its N-terminus. In the context of the present invention, the Brazilian sweet wild-type sequence preferably comprises the amino acid sequence of SEQ ID NO: 36, without glutamine residues or pyroglutamic acid attached to its N-terminus.

[0054] Coding sequence: When used herein, the term "coding sequence" means a polynucleotide sequence that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are usually determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG, and ends with a stop codon such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA, RNA, synthetic or recombinant nucleotide sequence.

[0055] Expression: in the context of the present invention includes any step involved in the production of the sweet protein of the present invention, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0056] Expression vector: refers to a linear or circular nucleic acid molecule construct, which comprises a polynucleotide encoding a protein such as the Brazilian sweet provided herein, and the polynucleotide is operably connected to the additional nucleotides provided for its expression. This additional nucleotide sequence includes, for example, a promoter, a suitable transcription initiation and termination sequence. "Promoter" is a DNA sequence dna that RNA polymerase recognizes, combines and begins to transcribe, and it contains the conserved sequence required for RNA polymerase specific binding and transcription initiation, and most of them are located upstream of the transcription start point, and the promoter itself is not transcribed. In addition, there is the host internal replication ability usually given by the replication origin in the expression vector, and / or with a selection gene that helps to identify the transformant. Usually, the expression vector utilized in the recombinant DNA technique is often a "plasmid" form, i.e., refers to a circular double-stranded DNA ring. Obviously, the vector derived from viruses such as retroviruses, adenoviruses, etc. can also be used.

[0057] Amino acid sequence: Synonymous with the terms "polypeptide", "protein" and "peptide", and used interchangeably. The conventional one-letter code or the three-letter code for amino acid residues is used, with the amino acid sequence presented in the standard amino to carboxyl terminal orientation (ie, N→C).

[0058] Signal peptide: A short (5 to 30 amino acids in length) peptide chain that guides newly synthesized proteins to the secretory pathway. It usually refers to the amino acid sequence at the N-terminus of a newly synthesized polypeptide chain that is used to guide the transmembrane transfer (localization) of proteins.

[0059] Primer design: Use Snapgene software to design the corresponding primers for the constructed plasmid. When designing primers, attention should be paid to the GC content, Tm value, hairpin structure, primer length, primer dimer, primer mismatch, and the introduction of restriction sites.

[0060] The terms "comprising," "including," "having" and their conjugations mean "including but not limited to," but also consist of only the listed elements.

[0061] The definitions provided herein are intended to facilitate understanding of certain terms frequently used herein and are not meant to limit the scope of the present disclosure.

[0062] Since exogenous proteins are easily attacked by yeast proteolytic enzymes during the expression process and degraded, which in turn affects the efficient expression of exogenous proteins in Pichia pastoris, the amount of various proteolytic enzymes in yeast cells will change with the change of nutritional conditions during the culture process, especially during high-density fermentation. A higher content of protease can often be detected in the culture medium. By optimizing the culture conditions, the degradation of recombinant proteins can be reduced to a certain extent. The main measures include: 1) changing the pH, temperature or time of the culture to reduce the activity of the protease; 2) adding some specific amino acids or casein hydrolysates as competitive substrates for the protease or adding specific protease inhibitors. However, these methods are usually limited in effect and are not applicable to all recombinant proteins. Using genetic engineering to knock out the gene encoding a specific proteolytic enzyme in the host cell is an effective way to fundamentally solve the problem of recombinant protein degradation. This method has been widely used in a variety of expression systems including Escherichia coli and yeast.

[0063] Due to the structural complexity of Brazilian sweet, especially the presence of numerous cysteine ​​residues, Brazilian sweet expressed in traditional systems such as Escherichia coli and Pichia pastoris cannot fold into the correct conformation. Coupled with the degradation of its own proteases, the prepared Brazilian sweet has low sweetness. Ultimately, the method of preparing Brazilian sweet by genetic engineering cannot meet the demand for low cost of industrial production. In view of the above problems, the present invention proposes a method for preparing Brazilian sweet with high sweetness. The present invention knocks out the gene encoding a specific proteolytic enzyme in the host cell, fuses the signal peptide sequence and the Brazilian sweet protein sequence, and uses a protease-deficient Pichia pastoris strain host to express the fusion sequence. The Brazilian sweet protein finally prepared can be fermented at high density, which is convenient for downstream extraction and purification, saving production costs. At the same time, the present invention also applies the prepared Brazilian sweet to scenes such as yogurt drinks and sparkling water, proving that it can well replace sucrose as a sugar substitute sweetener.

[0064] Beneficial effects of the present invention: The present invention found that in the knockout of Pichia pastoris PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_0913, PAS_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_chr3_0934, PAS_chr3_0633, PRC1 After at least two genes among PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 or knocking out the PAS_chr3_0689 gene alone, the growth rate of Pichia pastoris can be increased and the expression yield of foreign proteins can be improved.

[0065] In order to meet the growing demand for low-sugar and reduced-sugar consumption, the inventors used the gene-deficient strain of the present invention to express high-sweetness Brazilian sweet, which has a higher yield and significantly higher sweetness than Brazilian sweet prepared by the prior art. In addition, the expressed protein has no obvious sweetness delay, making the protein closer to sucrose in taste. At the same time, the preparation method meets the needs of slowing down the fermentation degradation of Brazilian sweet protein and meets the needs of industrial production. Since its sweetness is significantly enhanced, its production cost is also significantly reduced.

[0066] The present invention artificially synthesizes a DNA sequence, clones it into an expression plasmid PHKA, and uses a protease-deficient Pichia pastoris strain for recombinant expression. Then, the protein with a purity higher than 90% is obtained by separation and purification by ion exchange chromatography and affinity chromatography. The growth curve experiment of the protease-deficient Pichia pastoris strain proves that the protease-deficient Pichia pastoris strain of the present invention can effectively promote the growth rate of cells. It is used for shake flask experiment test, and compared with the control group, the protease-deficient Pichia pastoris strain can significantly enhance the growth rate of cells. Therefore, the protease-deficient Pichia pastoris strain obtained by the present invention can slow down the protein degradation problem during high-density fermentation to a certain extent during high-density fermentation, and can also reduce the fermentation time, save fermentation cost, thereby reducing production cost, facilitating downstream extraction, and providing high-purity protein.

[0067] The expressed brasil sweet protein of the present invention is very suitable for use as a natural high-intensity non-caloric sugar substitute sweetener. In addition, studies have shown that brasil sweet protein can block the bitterness of other natural and artificial sweeteners. When used in combination with other sweeteners, the taste, intensity and duration of other sweeteners can be significantly improved. Therefore, in many food and beverage applications, brasil sweet protein can not only be used as an independent sweetener, but also as a taste modifier together with other sweeteners and flavorings, and its potential application range is wide and of great significance.

[0068] The salt-reducing protein expressed by the present invention is a salty peptide, which is derived from the food itself. It is a type of oligopeptide with salty taste among flavor polypeptides. Adding an appropriate amount of salty peptide can reduce the salt content from 0.9% to 0.3%, and basically maintain palatability. Salty peptide is a healthy and safe salty taste enhancement product with umami taste; they are composed of amino acids and are harmless to the human body. The salt-reducing protein of the present invention can be prepared in large quantities by microbial fermentation. Salty peptide is a natural salting agent with certain salt-enhancing and fresh-enhancing effects. While responding to the United Nations WHO's low-sodium and salt-reducing recommendations, it also meets consumers' demand for natural products. Therefore, the salt-reducing protein developed by the present invention is of great significance in responding to the global demand for low-sodium and salt-reducing. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Growth curves of each knockout strain are shown.

[0070] Figure 2 The protein expressions of MF001-29-Bra2-KRx3, MF001-94-Bra2-KRx3, MF001-136-Bra2-KRx3, MF001-143-Bra2-KRx3, MF001-256-Bra2-KRx3, MF001-249-Bra2-KRx3, MF001-251-Bra2-KRx3, MF001-169-Bra2-KRx3, GS115-Bra2-KRx4, and SMD1163-Bra2-KRx3 at different times are shown. Among them, M protein molecular weight Marker (3.3kD-31.0kD), lane 1 represents the sample before induction; lane 2 represents the sample for 6 hours of induction; lane 3 represents the sample for 24 hours of induction; lane 4 represents the sample for 48 hours of induction; lane 5 represents the sample for 72 hours of induction; lane 6 represents the sample for 96 hours of induction; lane 2 represents the sample for 6 hours of induction; lane 7 represents the Brazilian sweet protein standard; lane 8 represents the sample before induction; lane 9 represents the sample for 6 hours of induction; lane 10 represents the sample for 24 hours of induction; lane 11 represents the sample for 48 hours of induction; lane 12 represents the sample for 72 hours of induction; lane 13 represents the sample for 96 hours of induction.

[0071] Figure 3The protein expressions of MF001-29-Bra4-KRx3, MF001-94-Bra4-KRx3, MF001-136-Bra4-KRx3, MF001-143-Bra4-KRx3, MF001-256-Bra4-KRx3, MF001-249-Bra4-KRx3, MF001-251-Bra4-KRx3, MF001-169-Bra4-KRx3, GS115-Bra4-KRx3, and SMD1163-Bra4-KRx3 at different times are shown. Among them, M protein molecular weight Marker (3.3kD-31.0kD), lane 1 represents the sample before induction; lane 2 represents the sample after 48 hours of induction; lane 3 represents the sample after 72 hours of induction; lane 4 represents the sample after 96 hours of induction; lane 5 represents the Brazilian sweet protein standard; lane 6 represents the sample before induction; lane 7 represents the sample after 48 hours of induction; lane 8 represents the sample after 72 hours of induction; lane 9 represents the sample after 96 hours of induction; lane 10 represents the sample before induction; lane 11 represents the sample after 48 hours of induction; lane 12 represents the sample after 72 hours of induction; lane 13 represents the sample after 96 hours of induction.

[0072] Figure 4 The HPLC peak times and areas of the brasilin standard and MF001-29-Bra4-KRx3 fermentation 72H are shown.

[0073] Figure 5 Growth curves of MF001-29-Bra2-KRx3, MF001-94-Bra2-KRx3, MF001-136-Bra2-KRx3, MF001-143-Bra2-KRx3, MF001-249-Bra2-KRx3, MF001-251-Bra2-KRx3, MF001-256-Bra2-KRx3, MF001-169-Bra2-KRx3, GS115-Bra2-KRx4, and SMD1163-Bra2-KRx3 are shown.

[0074] Figure 6 Growth curves of MF001-29-Bra4-KRx3, MF001-94-Bra4-KRx3, MF001-136-Bra4-KRx3, MF001-143-Bra4-KRx3, MF001-249-Bra4-KRx3, MF001-251-Bra4-KRx3, MF001-256-Bra4-KRx3, MF001-169-Bra4-KRx3, GS115-Bra4-KRx3, and SMD1163-Bra4-KRx3 are shown.

[0075] Figure 7 The growth curve and biomass curve of MF001-94-PF are shown.

[0076] Figure 8 The results of SDS-PAGE electrophoresis of the supernatant after MF001-94-PF induced fermentation are shown. Among them, M represents the protein molecular weight marker (3.3kD-31.0kD), lane 1 represents the 1g / L BSA sample; lane 2 represents the 22H sample after MF001-94-PF fermentation induction; lane 3 represents the 46H sample after MF001-94-PF fermentation induction; lane 4 represents the 70H sample after MF001-94-PF fermentation induction; lane 5 represents the 94H sample after MF001-94-PF fermentation induction; lane 6 represents the 22H sample after GS115 fermentation induction; lane 7 represents the 46H sample after GS115 fermentation induction; lane 8 represents the 70H sample after GS115 fermentation induction; lane 9 represents the 94H sample after GS115 fermentation induction.

[0077] Fig. 9 The growth curve of MF001-94-P29-SUMO is shown.

[0078] Fig.10 The SDS-PAGE electrophoresis results of the supernatant protein of MF001-94-P29-SUMO are shown. Among them, M1 represents the protein molecular weight marker (3.3kD-31.0kD); lane 1 represents the sample 22 hours after GS115 fermentation induction; lane 2 represents the sample 46 hours after GS115 fermentation induction; lane 3 represents the sample 46 hours after GS115 fermentation induction; lane 4 represents the sample before MF001-94-PF induction; lane 5 represents the sample 22 hours after MF001-94-PF fermentation induction; lane 6 represents the sample 46 hours after MF001-94-PF fermentation induction; lane 7 represents the sample 70 hours after MF001-94-PF fermentation induction. DETAILED DESCRIPTION

[0079] The disclosure below provides many different embodiments or examples to realize different implementations of the present invention. In order to simplify the disclosure of the present invention, specific embodiments or examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the examples of various specific processes and materials provided by the present invention, those of ordinary skill in the art can be aware of the applicability of other processes and / or the use of other materials. Unless otherwise stated, the implementation of the present invention will adopt the traditional techniques in the fields of chemistry, molecular biology, etc. within the capabilities of those skilled in the art. In addition, unless otherwise stated, in this article, nucleic acids are written from left to right in the direction of 5' to 3', and amino acid sequences are written from left to right in the direction of amino terminal to carboxyl terminal.

[0080] Unless otherwise specified, the strains, plasmids and reagents in the embodiments of the present invention can be purchased through commercial channels.

[0081] The yeast strains used in the following examples of the present invention are Pichia pastoris GS115 and SMD1163 strains purchased from Invitrogen / ThermoFisher Scientific, GS115 Catalog # C18100, SMD1163 Catalog # C17500.

[0082] The Pichia protease-related genes used in the following embodiments of the present invention include:

[0083] The PAS_chr3_1087 gene in the peptidase A1 family has a nucleic acid sequence as shown in SEQ ID No.1, and an amino acid sequence of the protein encoded by it is shown in SEQ ID No.2; the PAS_chr4_0584 gene has a nucleic acid sequence as shown in SEQ ID No.3, and an amino acid sequence of the protein encoded by it is shown in SEQ ID No.4.

[0084] The PAS_chr2-1_0652 gene in the peptidase M1 family has a nucleic acid sequence as shown in SEQ ID No.5, and an amino acid sequence of its encoded protein as shown in SEQ ID No.6; the PAS_chr4_0113 gene has a nucleic acid sequence as shown in SEQ ID No.7, and an amino acid sequence of its encoded protein as shown in SEQ ID No.8; the PAS_chr2-2_0380 gene has a nucleic acid sequence as shown in SEQ ID No.9, and an amino acid sequence of its encoded protein as shown in SEQ ID No.10.

[0085] The PAS_chr4_0913 gene in the peptidase M18 family has a nucleic acid sequence as shown in SEQ ID No.11 and an amino acid sequence of the encoded protein as shown in SEQ ID No.12.

[0086] The PAS_chr1-4_0611 gene in the peptidase M28 family, whose nucleic acid sequence is shown in SEQ ID No.13, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No.14; the PAS_chr3_0953 gene, whose nucleic acid sequence is shown in SEQ ID No.15, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No.16; the PAS_chr1-1_0194 gene, whose nucleic acid sequence is shown in SEQ ID No.17, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No.18.

[0087] The PAS_chr1-4_0048 gene in the peptidase S54 family has a nucleic acid sequence as shown in SEQ ID No.19, and an amino acid sequence of the protein encoded by it is shown in SEQ ID No.20; the PAS_chr3_0934 gene has a nucleic acid sequence as shown in SEQ ID No.21, and an amino acid sequence of the protein encoded by it is shown in SEQ ID No.22.

[0088] The PAS_chr3_0633 gene in the Peptidase S10 family, whose nucleic acid sequence is shown in SEQ ID No.23, and whose encoded protein amino acid sequence is shown in SEQ ID No.24;

[0089] The PRC1 PAS_chr1-4_0013 gene, the nucleic acid sequence of which is shown in SEQ ID No.25, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No.26.

[0090] The PAS_chr1-1_0174 gene in the peptidase S16 family has a nucleic acid sequence as shown in SEQ ID No.27, and an amino acid sequence of the encoded protein as shown in SEQ ID No.28.

[0091] The PAS_chr3_0979 gene in the peptidase S14 family has a nucleic acid sequence as shown in SEQ ID No.29 and an amino acid sequence of the encoded protein as shown in SEQ ID No.30.

[0092] The PAS_chr1-1_0226 gene in the peptidase S8 family has a nucleic acid sequence as shown in SEQ ID No.31 and an amino acid sequence of the encoded protein as shown in SEQ ID No.32.

[0093] The protein defect-related gene used in the following examples of the present invention is the transcription activator PAS_chr3_0689 gene, whose nucleic acid sequence is shown in SEQ ID No.33, and whose encoded protein amino acid sequence is shown in SEQ ID No.34.

[0094] The nucleic acid sequence of the brasilien gene used in the following examples of the present invention is shown in SEQ ID No.35, and the amino acid sequence of the protein is shown in SEQ ID No.36.

[0095] The nucleic acid sequence of the salt-reducing protein gene used in the following examples of the present invention is shown in SEQ ID No.37, and the amino acid sequence of the protein is shown in SEQ ID No.38.

[0096] The nucleic acid sequence of the semaglutide gene mentioned in the present invention is shown as SEQ ID No.39, and the amino acid sequence of the protein is shown as SEQ ID No.40.

[0097] Culture media, reagents, and experimental methods:

[0098] LB liquid culture medium: 0.5 g (0.5% w / v) yeast extract, 1 g (1% w / v) tryptone, 1 g (1% w / v) NaCl, add distilled water and make up to a total volume of 100 mL, sterilize under high pressure at 115°C for 20 min before use.

[0099] LBK resistance screening plate: 0.5g yeast extract (0.5% w / v), 1g tryptone (1% w / v), 1g NaCl (1% w / v), 2g agar powder (2% w / v) were added to distilled water and the total volume was adjusted to 100mL, and then sterilized by high pressure at 115℃ for 20min. When the temperature of the culture medium dropped to about 50 to 55℃, kanamycin (25mg / mL) was added to the final concentration of the antibiotic in the clean bench to 100μg / mL, and the plate was poured after shaking evenly.

[0100] LBLZ resistance screening plate: 0.5g yeast extract (0.5% w / v), 1g NaCl (1% w / v), 1g tryptone (1% w / v), 2g agar powder (2% w / v) were added to distilled water and the total volume was adjusted to 100mL, and then autoclaved at 115°C for 20min and poured into the plate. After the culture medium was cooled to 50 to 55°C, bleomycin (25mg / mL) was added in a clean bench to a final concentration of 25μg / mL of the antibiotic, and the plate was poured after shaking evenly.

[0101] MD solid plate medium: 2 g (2% w / v) of glucose, 1.34 g (1.34% w / v) of YNB (without amino acids), and 2 g (2% w / v) of agar powder were added with distilled water and the total volume was adjusted to 100 mL. After high-pressure sterilization at 115°C for 20 min, the plate was poured.

[0102] YPD liquid medium: 1 g (1% w / v) yeast extract, 2 g (2% w / v) glucose, 2 g (2% w / v) tryptone, add distilled water and make up to a total volume of 100 mL, and sterilize by high pressure at 115°C for 20 min.

[0103] YPDZ solid plate medium: 1g (1% w / v) yeast extract, 2g (2% w / v) glucose, 2g (2% w / v) tryptone, 2g (2% w / v) agar powder, add distilled water and dilute to a total volume of 100mL, 115℃, autoclave for 20min and pour into the plate. When the temperature of the culture medium drops to about 50 to 55℃, add bleomycin (25mg / mL) to a final concentration of 100μg / mL of the antibiotic in a clean bench, shake evenly and pour into the plate.

[0104] BMGY liquid medium: 1% (w / v) yeast extract, 2% (w / v) tryptone, 1.34% (w / v) YNB, 1% (v / v) glycerol, 10% (v / v) 1M pH 6.0 phosphate buffer, dissolved with a certain amount of distilled water and made up to the final volume, sterilized at 115°C for 20 min.

[0105] BMMY liquid culture medium: 1% (w / v) yeast extract, 2% (w / v) tryptone, 1.34% (w / v) YNB, 10% (v / v) 1M pH 6.0 phosphate buffer, dissolved with a certain amount of distilled water and adjusted to the final volume, autoclaved at 115°C for 20 min, and then added methanol at a ratio of 1% (v / v).

[0106] 1M phosphate buffer (pH 6.0): Mix 868mL of 1M KH2PO4 and 132mL of 1M K2HPO4 evenly, adjust the pH to 6.0 with phosphoric acid, sterilize at 121℃ for 20min, and place at room temperature for use.

[0107] The Pichia pastoris genome was extracted according to the instructions of Yeast DNAiso Kit (Takara Code: D9082).

[0108] The DNA polymerase used in the PCR amplification reaction was Phanta Max ultra-high-fidelity DNA polymerase.

[0109] Linearization of recombinant plasmid and transformation of yeast by electroporation

[0110] 1. Linearization of recombinant vector

[0111] The plasmid to be transformed is digested by the enzyme linearization system, and then the linearization product is recovered. The concentration of the recovered linearized DNA product is measured and stored at -20°C for future use.

[0112] 2. Preparation of GS115 electroporated competent cells

[0113] 1) Pick a single colony of GS115 from the streaked activated YPD solid plate and inoculate it into 5 mL YPD liquid medium, and culture it overnight at 30°C and 250 rpm;

[0114] 2) Take 1 mL of bacterial solution and inoculate it into 100 mL of YPD liquid medium, and culture it at 30°C and 250 rpm for 6 to 8 hours until the OD 600 0.6 to 0.8;

[0115] 4) Transfer the bacterial solution to a 50 mL sterile centrifuge tube, centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, and collect the bacteria;

[0116] 5) Add sterile deionized water to 50 mL, wash the cells twice, centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, and collect the cells;

[0117] 6) Add 25 mL of LDST solution, let stand at room temperature for 30 min, centrifuge at 4°C and 5000 rpm for 10 min, discard the supernatant, and collect the bacteria;

[0118] 7) Add 50 mL of 1 M pre-cooled sorbitol to wash, gently mix and resuspend the cells, centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, and collect the cells;

[0119] 8) Add 25 mL of 1 M pre-cooled sorbitol to wash, gently mix and resuspend the cells, centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, and collect the cells;

[0120] 9) Then resuspend the cells in 0.5 mL 1 M pre-cooled sorbitol and store at 4°C;

[0121] 10) It is best to prepare competent cells for electroporation immediately before use. If you want to freeze competent cells, dispense 80 μL per tube into a sterile 1.5 mL centrifuge tube and store in a -80°C refrigerator.

[0122] 3. Linearized plasmid electroporation into Pichia pastoris competent cells

[0123] 1) Add about 0.5 to 1 μg of linearized DNA to the prepared yeast competent cells, gently mix the linearized DNA and yeast competent cells, transfer them to an ice-bathed electroporation cup, and then place them on ice for 5 minutes;

[0124] 2) Set the voltage of the electric shock instrument to 1.5Kv, the resistance to 200Ω, the capacitance to 25mF, and the electric shock mode to the fungus mode, and then perform electric shock on the mixed solution;

[0125] 3) Add 1M ice-bath precooled sorbitol to the electroporation cup, and transfer the mixed bacterial solution to a sterilized 1.5mL EP tube with a pipette, incubate it in a 30℃ incubator for 1.5h, then centrifuge it at 6000rpm for 1 to 2min, and discard a portion. The supernatant and about 200μL of liquid are left, and the bacteria are suspended and coated on the corresponding screening plates. MD solid plates are used to screen histidine auxotrophic strains, and YPDZ solid plates are used for bleomycin resistance screening. The plates are inverted and cultured in a 30℃ incubator for 2 to 4 days.

[0126] Shake flask fermentation of recombinant yeast strains

[0127] Pick a single yeast colony with good growth on the solid plate and inoculate it into a 50mL Erlenmeyer flask containing 5mL of BMGY medium. Place it in a shaker at 30°C and 250rpm for about 24h, and measure the OD of the seed solution. 600 value, calculate and extract the actual OD 600 Transfer to 25 mL BMMY, centrifuge at 6000 rpm for 2 min at room temperature, discard the supernatant and collect the bacteria, then wash the bacteria 2 to 3 times with 800 μL BMMY liquid culture medium, and then transfer all the washed bacteria to a 250 mL Erlenmeyer flask with 25 mL BMMY, place it in a shaker at 30°C and 250 rpm for culture, and add 1% methanol every 24 hours.

[0128] SDS-PAGE

[0129] Culture supernatants or purified samples were analyzed by SDS-PAGE, using FuturePAGE gel. TMProtein precast gel 4% to 20% (ACE). On the gel, load 20 μl of sample (10 μl of each sample mixed with 10 μl of loading buffer), apply 10 μl of Marker: (pre-stained SDS-PAGE standard, GenScript Bio, #M00624-250). In the matching special electrophoresis buffer: MOPS-SDS Running Buffer (Cat. No.: BR0001-02), run the gel at a constant voltage of 160V for 80 min. Stain the protein band with Bio-Safe Coomassie dye (Bio-Rad).

[0130] Analysis of Brazilian melamine by HPLC

[0131] HPLC was performed as described: using a Thermo Scientific Vanquish HPLC system equipped with 5um C18(2) 250×4.6mm chromatographic column. Use stationary phase buffer A (water + 0.1% TFA) and mobile phase buffer B (ACN + 0.1% TFA). UV detector monitors absorbance at 220nm. Column temperature is set to 37℃, injection volume is 10μL. 15-minute gradient program settings are shown in Table 1.

[0132] Table 1

[0133] time Flow rate (mL / min) ％A ％B 0.000 0.5 70 30 0.100 0.5 70 30 10.000 0.5 10 90 10.100 0.5 70 30 15.000 0.5 70 30

[0134] The content analysis of brazilian tamarind was carried out by calculating the content of brazilian tamarind in shake flask fermentation based on the standard product.

[0135] Knockout of protease genes in Pichia pastoris

[0136] Example 1

[0137] PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 were knocked out simultaneously.

[0138] Construction of plasmid containing crRNA fragment

[0139] Two PAM sites were selected from the PAS_chr3_0934 gene, and two crRNA fragments corresponding to the selected PAM sites were determined. One of the crRNA fragments was connected to the pCrRNA plasmid (Xinying Zhang, Songjie Gu, Xueyun Zheng et al, A Novel and Efficient Genome Editing Tool Assisted by CRISPR-Cas12a / Cpf1 for Pichia pastoris; ACS Synthetic Biology. 2021, 10, 2927-2937) to obtain pCrRNA-0934-1, wherein the base sequence of the crRNA fragment is shown in SEQ ID No. 41; the other crRNA fragment was connected to the pCrRNA plasmid to obtain pCrRNA-0934-2, wherein the base sequence of the crRNA fragment is shown in SEQ ID No. 42.

[0140] Similarly, two PAM sites were selected from the PAS_chr4_0913 gene, and two CrRNA fragments corresponding to the selected PAM sites were determined. The base sequence of one CrRNA fragment was shown in SEQ ID No.43, and it was connected to the pCrRNA plasmid to obtain pCrRNA-0913-1; the base sequence of the other CrRNA fragment was shown in SEQ ID No.44, and it was connected to the pCrRNA plasmid to obtain pCrRNA-0913-2.

[0141] Similarly, two PAM sites were selected from the PAS_chr1-4_0048 gene, and two CrRNA fragments corresponding to the selected PAM sites were determined. The base sequence of one CrRNA fragment was shown in SEQ ID No.45, and it was connected to the pCrRNA plasmid to obtain pCrRNA-0048-1; the base sequence of the other CrRNA fragment was shown in SEQ ID No.46, and it was connected to the pCrRNA plasmid to obtain pCrRNA-0048-2.

[0142] Preparation of PAS_chr3_0934 gene donor DNA

[0143] Using the Pichia pastoris GS115 genome as a template, 0934-LF (as shown in SEQ ID No.47) and 0934-D-RF (as shown in SEQ ID No.48) as a primer pair, PCR amplification is performed to obtain a 0934 left-wing PCR product; using the Pichia pastoris GS115 genome as a template, 0934-D-LF (as shown in SEQ ID No.49) and 0934-RF (as shown in SEQ ID No.50) as a primer pair, PCR amplification is performed to obtain a 0934 right-wing PCR product; using a mixture of the 0934 left-wing PCR product and the 0934 right-wing PCR product as a template, 0934-LF and 0934-RF as a primer pair, PCR amplification is performed to obtain a 0934 donor DNA fragment.

[0144] Preparation of PAS_chr4_0913 gene donor DNA

[0145] Using the Pichia pastoris GS115 genome as a template, 0913-LF (as shown in SEQ ID No.51) and 0913-D-RF (as shown in SEQ ID No.52) as a primer pair, PCR amplification is performed to obtain a 0913 left-wing PCR product; using the Pichia pastoris GS115 genome as a template, 0913-D-LF (as shown in SEQ ID No.53) and 0913-RF (as shown in SEQ ID No.54) as a primer pair, PCR amplification is performed to obtain a 0913 right-wing PCR product; using a mixture of the 0913 left-wing PCR product and the 0913 right-wing PCR product as a template, 0913-LF and 0913-RF as a primer pair, PCR amplification is performed to obtain a 0913 donor DNA fragment.

[0146] Preparation of PAS_chr1-4_0048 gene donor DNA

[0147] Using the Pichia pastoris GS115 genome as a template, 0048-LF (as shown in SEQ ID No.55) and 0048-D-RF (as shown in SEQ ID No.56) as a primer pair, PCR amplification is performed to obtain the 0048 left-wing PCR product; using the Pichia pastoris GS115 genome as a template, 0048-D-LF (as shown in SEQ ID No.57) and 0048-RF (as shown in SEQ ID No.58) as a primer pair, PCR amplification is performed to obtain the 0048 right-wing PCR product; using a mixture of the 0048 left-wing PCR product and the 0048 right-wing PCR product as a template, 0048-LF and 0048-RF as a primer pair, PCR amplification is performed to obtain the 0048 donor DNA fragment.

[0148] Gene knockout

[0149] First, the CRISPR CAS12 gene editing auxiliary plasmid pGAP-Cpf1 (Xinying Zhang, Songjie Gu, Xueyun Zheng et al, A Novel and Efficient Genome Editing Tool Assisted by CRISPR-Cas12a / Cpf1 for Pichia pastoris; ACS Synthetic Biology. 2021, 10, 2927-2937) was linearized by single enzyme digestion.

[0150] The linearized plasmid pGAP-Cpf1 was transformed into Pichia pastoris GS115 competent cells by electroporation, and the positive strain GS115-Cpf1 was obtained after culture and identification.

[0151] pCrRNA-0934-1, pCrRNA-0934-2, pCrRNA-0913-1, pCrRNA-0913-2, pCrRNA-0048-1, pCrRNA-0048-2, 0934 donor DNA fragment, 0913 donor DNA fragment and 0048 donor DNA fragment were co-transformed into Pichia pastoris GS115-Cpf1 competent cells, cultured on YPDS plates containing 100 μg / mL bleomycin, and then identified by PCR and sequencing to obtain simultaneous knockout of three genes The positive transformants were further cultured on YPD solid plates without antibiotics until single colonies grew out. Single colonies were picked from the YPD solid plates without antibiotics and cultured again on YPD solid plates with bleomycin. The colonies that could not grow on the bleomycin plates were the strains with the plasmid eliminated. The strains were again verified by colony PCR and sequencing, and finally a defective strain with PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 genes knocked out was obtained, which was numbered MF001-29.

[0152] Example 2

[0153] PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, and PRC1 PAS_chr1-4_0013 were knocked out simultaneously.

[0154] The base sequence of a CrRNA fragment of the knocked-out PAS_chr2-1_0652 gene is shown in SEQ ID No.59, which is connected to the pCrRNA plasmid to obtain pCrRNA-0652-1; the base sequence of another CrRNA fragment is shown in SEQ ID No.60, which is connected to the pCrRNA plasmid to obtain pCrRNA-0652-2.

[0155] The base sequence of a CrRNA fragment of the knocked-out PAS_chr3_0633 gene is shown in SEQ ID No.61, which is connected to the pCrRNA plasmid to obtain pCrRNA-0633-1; the base sequence of another CrRNA fragment is shown in SEQ ID No.62, which is connected to the pCrRNA plasmid to obtain pCrRNA-0633-2.

[0156] The base sequence of a CrRNA fragment of the knocked-out PRC1 PAS_chr1-4_0013 gene is shown in SEQ ID No.63, which is connected to the pCrRNA plasmid to obtain pCrRNA-0013-1; the base sequence of another CrRNA fragment is shown in SEQ ID No.64, which is connected to the pCrRNA plasmid to obtain pCrRNA-0013-2.

[0157] Preparation of PAS_chr2-1_0652 gene donor DNA

[0158] Using the Pichia pastoris GS115 genome as a template, 0652-LF (as shown in SEQ ID No.65) and 0652-D-RF (as shown in SEQ ID No.66) as a primer pair, PCR amplification is performed to obtain the 0652 left-wing PCR product; using the Pichia pastoris GS115 genome as a template, 0652-D-LF (as shown in SEQ ID No.67) and 0652-RF (as shown in SEQ ID No.68) as a primer pair, PCR amplification is performed to obtain the 0652 right-wing PCR product; using a mixture of the 0652 left-wing PCR product and the 0652 right-wing PCR product as a template, 0652-LF and 0652-RF as a primer pair, PCR amplification is performed to obtain the 0652 donor DNA fragment.

[0159] Preparation of PAS_chr3_0633 gene donor DNA

[0160] Using the genome of Pichia pastoris GS115 as a template, 0633-LF (as shown in SEQ ID No.69) and 0633-D-RF (as shown in SEQ ID No.70) as a primer pair, PCR amplification was performed to obtain the 0633 left wing PCR product; using the genome of Pichia pastoris GS115 as a template, 0633-D-LF (as shown in SEQ ID No.71) and 0633-RF (as shown in SEQ ID No.72) as a primer pair, PCR amplification was performed to obtain the 0633 right wing PCR product; using a mixture of the 0633 left wing PCR product and the 0633 right wing PCR product as a template, 0633-LF and 0633-RF as a primer pair, PCR amplification was performed to obtain the 0633 donor DNA fragment

[0161] Preparation of PRC1 PAS_chr1-4_0013 gene donor DNA

[0162] Using the genome of Pichia pastoris GS115 as a template, 0013-LF (as shown in SEQ ID No.73) and 0013-D-RF (as shown in SEQ ID No.74) as a primer pair, PCR amplification was performed to obtain the 0013 left-wing PCR product; using the genome of Pichia pastoris GS115 as a template, 0013-D-LF (as shown in SEQ ID No.75) and 0013-RF (as shown in SEQ ID No.76) as a primer pair, PCR amplification was performed to obtain the 0013 right-wing PCR product; using a mixture of the 0013 left-wing PCR product and the 0013 right-wing PCR product as a template, 0013-LF and 0013-RF as a primer pair, PCR amplification was performed to obtain the 0013 donor DNA fragment.

[0163] The linearized plasmid pGAP-Cpf1 was transformed into Pichia pastoris MF001-29 competent cells by electroporation, and the positive strain MF001-29-Cpf1 was obtained after culture and identification; pCrRNA-0652-1, pCrRNA-0652-2, pCrRNA-0633-1, pCrRNA-0633-2, pCrRNA-0013-1, pCrRNA-0013-2, 0652 donor DNA fragment, 0633 donor DNA fragment and 0013 donor DNA fragment were further transformed into Pichia pastoris MF001-29-Cpf1 competent cells, cultured on a bleomycin-containing resistance plate to obtain positive transformants, and then cultured on an antibiotic-free plate to eliminate the plasmid, and finally a gene-deficient strain in which 6 genes were simultaneously knocked out was obtained, and the strain number was MF001-94.

[0164] Example 3

[0165] Simultaneously knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380.

[0166] The base sequence of a CrRNA fragment of the knocked-out PAS_chr3_0979 gene is shown in SEQ ID No.77, and the base sequence of another CrRNA fragment is shown in SEQ ID No.78; the primers for preparing the donor DNA are 0979-LF (shown in SEQ ID No.79), 0979-D-RF (shown in SEQ ID No.80), 0979-D-LF (shown in SEQ ID No.81) and 0979-RF (shown in SEQ ID No.82).

[0167] The base sequence of a CrRNA fragment of the knocked-out PAS_chr4_0113 gene is shown in SEQ ID No.83, and the base sequence of another CrRNA fragment is shown in SEQ ID No.84; the primers for preparing the donor DNA are 0113-LF (shown in SEQ ID No.85), 0113-D-RF (shown in SEQ ID No.86), 0113-D-LF (shown in SEQ ID No.87) and 0113-RF (shown in SEQ ID No.88).

[0168] The base sequence of a CrRNA fragment of the knocked-out PAS_chr1-1_0174 gene is shown in SEQ ID No.89, and the base sequence of another CrRNA fragment is shown in SEQ ID No.90; the primers for preparing the donor DNA are 0174-LF (shown in SEQ ID No.91), 0174-D-RF (shown in SEQ ID No.92), 0174-D-LF (shown in SEQ ID No.93) and 0174-RF (shown in SEQ ID No.94).

[0169] The base sequence of a CrRNA fragment of the knocked-out PAS_chr3_0953 gene is shown in SEQ ID No.95, and the base sequence of another CrRNA fragment is shown in SEQ ID No.96; the primers for preparing the donor DNA are 0953-LF (shown in SEQ ID No.97), 0953-D-RF (shown in SEQ ID No.98), 0953-D-LF (shown in SEQ ID No.99) and 0953-RF (shown in SEQ ID No.100).

[0170] The base sequence of a CrRNA fragment of the knocked-out PAS_chr2-2_0380 gene is shown in SEQ ID No.101, and the base sequence of another CrRNA fragment is shown in SEQ ID No.102; the primers for preparing the donor DNA are 0380-LF (shown in SEQ ID No.103), 0380-D-RF (shown in SEQ ID No.104), 0380-D-LF (shown in SEQ IDNo.105) and 0380-RF (shown in SEQ ID No.106).

[0171] The above-mentioned PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, and PAS_chr2-2_0380 were divided into two groups. First, one of the groups was knocked out using MF001-94 as the starting strain, and then the other group was further knocked out using the obtained gene knockout strain as the starting strain, and finally a gene-deficient strain with 11 genes knocked out at the same time was obtained, and its strain number was MF001-136.

[0172] The rest is the same as in Example 1.

[0173] Example 4

[0174] Simultaneously knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611.

[0175] The construction of plasmids containing CrRNA fragments of PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, and PAS_chr2-2_0380 genes and the preparation of donor DNA were the same as in Example 3.

[0176] The base sequence of a CrRNA fragment of the knocked-out PAS_chr1-4_0611 gene is shown in SEQ ID No.107, and the base sequence of another CrRNA fragment is shown in SEQ ID No.108; the primers for preparing the donor DNA are 0611-LF (shown in SEQ ID No.109), 0611-D-RF (shown in SEQ ID No.110), 0611-D-LF (shown in SEQ IDNo.111) and 0611-RF (shown in SEQ ID No.112).

[0177] The PAS_chr1-4_0611 gene was knocked out using MF001-136 as the starting strain, and eventually a gene-deficient strain with 12 genes knocked out at the same time was obtained, and its strain number was MF001-143.

[0178] The rest is the same as in Example 1.

[0179] Example 5

[0180] Simultaneously knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194.

[0181] The construction of the CrRNA fragment plasmid containing the PAS_chr1-4_0611 gene and the preparation of the donor DNA are the same as in Example 4.

[0182] The base sequence of a CrRNA fragment of the knocked-out PAS_chr1-1_0194 gene is shown in SEQ ID No.113, and the base sequence of another CrRNA fragment is shown in SEQ ID No.114; the primers for preparing the donor DNA are 0194-LF (shown in SEQ ID No.115), 0194-D-RF (shown in SEQ ID No.116), 0194-D-LF (shown in SEQ IDNo.117) and 0194-RF (shown in SEQ ID No.118).

[0183] Using MF001-136 as the starting strain, the PAS_chr1-4_0611 and PAS_chr1-1_0194 genes were knocked out simultaneously, and finally a gene-deficient strain with 13 genes knocked out simultaneously was obtained, and its strain number was MF001-249.

[0184] The rest is the same as in Example 1.

[0185] Example 6

[0186] Simultaneously knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, P AS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, PAS_chr3_1087.

[0187] The base sequence of a CrRNA fragment of the knocked-out PAS_chr4_0584 gene is shown in SEQ ID No.119, and the base sequence of another CrRNA fragment is shown in SEQ ID No.120; the primers for preparing the donor DNA are 0584-LF (shown in SEQ ID No.121), 0584-D-RF (shown in SEQ ID No.122), 0584-D-LF (shown in SEQ ID No.123) and 0584-RF (shown in SEQ ID No.124).

[0188] The base sequence of a CrRNA fragment of the knocked-out PAS_chr3_1087 gene is shown in SEQ ID No.125, and the base sequence of another CrRNA fragment is shown in SEQ ID No.126; the primers for preparing the donor DNA are 1087-LF (shown in SEQ ID No.127), 1087-D-RF (shown in SEQ ID No.128), 1087-D-LF (shown in SEQ ID No.129) and 1087-RF (shown in SEQ ID No.130).

[0189] Using MF001-249 as the starting strain, the PAS_chr4_0584 and PAS_chr3_1087 genes were knocked out simultaneously, and finally a gene-deficient strain with 15 genes knocked out simultaneously was obtained, and its strain number was MF001-251.

[0190] The rest is the same as in Example 1.

[0191] Example 7

[0192] Simultaneously knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2- 2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, PAS_chr3_1087, PAS_chr1-1_0226.

[0193] The construction of plasmids containing CrRNA fragments of PAS_chr4_0584 and PAS_chr3_1087 genes and the preparation of donor DNA are the same as in Example 6.

[0194] The base sequence of one crRNA of the knocked-out PAS_chr1-1_0226 gene is shown in SEQ ID No.131, and the base sequence of the other crRNA is shown in SEQ ID No.132; the base sequence of the donor DNA is shown in 136).

[0195] Using MF001-249 as the starting strain, the PAS_chr4_0584, PAS_chr3_1087 and PAS_chr1-1_0226 genes were knocked out simultaneously, and finally a gene-deficient strain with 16 genes knocked out simultaneously was obtained, and its strain number was MF001-256.

[0196] The rest is the same as in Example 1.

[0197] Example 8

[0198] Knockout PAS_chr3_0689.

[0199] The base sequence of a CrRNA fragment of the knocked-out PAS_chr3_0689 gene is shown in SEQ ID No.137, and the base sequence of another CrRNA fragment is shown in SEQ ID No.138; the primers for preparing the donor DNA are 0689-LF (shown in SEQ ID No.139), 0689-D-RF (shown in SEQ ID No.140), 0689-D-LF (shown in SEQ ID No.141) and 0689-RF (shown in SEQ ID No.142).

[0200] The rest is the same as in Example 1.

[0201] The gene-deficient strain finally obtained was numbered MF001-169.

[0202] Growth rate determination of knockout strains

[0203] The growth curve is the group growth law of single-cell microorganisms when cultured in liquid under certain environmental conditions. When measuring, a certain number of pure microbial strains are generally inoculated into a certain volume of sterilized, suitable fresh culture medium, cultured under suitable temperature conditions, and the number of bacteria in the culture medium is measured by taking samples at regular intervals. 600 (OD is the optical density of the bacterial suspension measured by a photoelectric colorimeter based on the relationship that the number of bacterial cells in the culture solution is proportional to the turbidity and inversely proportional to the transmittance) as the ordinate and the growth time as the abscissa to draw a growth curve. The growth curve is generally divided into four periods: the delay period, the logarithmic period, the stable period, and the decay period. Determining the growth curve of microorganisms under certain culture conditions has certain guiding significance for actual production.

[0204] MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169 were activated and inoculated into 50 mL YPD liquid culture medium at a volume ratio of 1%, respectively. The starting strain GS115 was used as control-1, and the SMD1163 strain was used as control-2. Samples were taken at 0, 6, 9, 24 h, 30 h, 48 h, and 54 h to measure OD 600 OD of each strain 600 The results are shown in Table 2. Then, the sampling time was used as the horizontal axis and the OD of each strain was used as the horizontal axis. 600The value is used as the vertical axis to draw the growth curve, see Figure 1 , for comparison of growth rates.

[0205] Table 2

[0206] strain 0h 6h 9h 24h 30h 48h MF001-29 0.1+ / -0 0.92+ / -0.01 2.6+ / -0.01 40.48+ / -0.29 42.93+ / -0.42 41.2+ / -0.16 MF001-94 0.1+ / -0.01 0.92+ / -0.06 2.72+ / -0.01 39.3+ / -0.16 43.23+ / -0.12 41.5+ / -0.08 MF001-136 0.1+ / -0 1.02+ / -0.02 1.96+ / -0.02 38.7+ / -0.33 43.5+ / -0.33 40.8+ / -0.08 MF001-143 0.1+ / -0.01 0.95+ / -0.01 1.93+ / -0.01 39.17+ / -0.29 44.7+ / -0.16 40.5+ / -0.33 MF001-249 0.1+ / -0 0.97+ / -0.01 2.68+ / -0.04 39.15+ / -0.2 43.7+ / -0.16 40.5+ / -0.16 MF001-251 0.1+ / -0.01 0.91+ / -0.01 2.58+ / -0.03 39.13+ / -0.45 43.83+ / -0.29 40.4+ / -0.16 MF001-256 0.1+ / -0 0.96+ / -0.01 2.62+ / -0.02 38.87+ / -0.29 42.5+ / -0.16 40.77+ / -0.33 MF001-169 0.1+ / -0.01 0.9+ / -0.01 1.95+ / -0.02 39.2+ / -0.24 42.9+ / -0.33 41.3+ / -0.16 GS115 0.1+ / -0 0.75+ / -0.01 1.78+ / -0.02 34.3+ / -0.16 37.3+ / -0.16 37.7+ / -0.33 SMD1163 0.1+ / -0.01 0.96+ / -0.01 1.66+ / -0.02 44.8+ / -0.37 59.6+ / -0.24 56.87+ / -0.49

[0207] From the above data analysis, it can be seen that the growth rates of MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169 strains are higher than or do not affect the growth rate of their starting strain GS115.

[0208] Construction of expression plasmids with different copy numbers of brazilian thaumatin

[0209] Example 9

[0210] In yeast, including Saccharomyces pastoris, the alpha mating factor is expressed as tandem repeats. The alpha mating factor is separated by the amino acid sequence KR(EA)x. For Pichia pastoris, this gene has 10 repeats of the mature alpha mating factor, and the spacer sequence can vary from 1 to 9 EA repeats. By expressing a polypeptide sweetener that mimics the tandem repeats of the yeast alpha mating factor, the production of braisedin produced by P. pastoris can be increased.

[0211] The brazilian tadalafil in the present invention is synthesized according to the codon preference of Pichia pastoris and cloned into a plasmid PHKA vector. The components are connected to the PHKA vector in series in the following order:

[0212] 1) For the PHKA-Bra2-KR plasmid: the α-factor secretion signal, the brazilin gene, the KR spacer sequence and the brazilin gene were concatenated.

[0213] 2) For the PHKA-Bra2-KRx plasmid: the α-factor secretion signal, the brazilin gene, the spacer sequence 1 (as shown in SEQ ID No. 143) and the brazilin gene were connected in series.

[0214] 3) For the PHKA-Bra2-KRx2 plasmid: the α-factor secretion signal, the brazilin gene, the spacer sequence 2 (as shown in SEQ ID No. 144) and the brazilin gene were connected in series.

[0215] 4) For the PHKA-Bra2-KRx3 plasmid: the α-factor secretion signal, the brazilin gene, the spacer sequence 3 (as shown in SEQ ID No. 145) and the brazilin gene were connected in series.

[0216] 5) For the PHKA-Bra2-KRx4 plasmid: the α-factor secretion signal, the brazilin gene, the spacer sequence 4 (as shown in SEQ ID No. 146) and the brazilin gene were connected in series.

[0217] 6) For the PHKA-Bra2-KRx5 plasmid: the α-factor secretion signal, the brazilin gene, the spacer sequence 5 (as shown in SEQ ID No. 147) and the brazilin gene were connected in series.

[0218] 7) For the PHKA-Bra4-KR plasmid: concatenate the α-factor secretion signal, the brazilian tadalafil gene, the KR spacer sequence, the brazilian tadalafil gene, the KR spacer sequence, the brazilian tadalafil gene, the KR spacer sequence, and the brazilian tadalafil gene.

[0219] 8) For the PHKA-Bra4-KRx plasmid: the α-factor secretion signal, the brazilian thauma protein gene, the spacer sequence 1 (as shown in SEQ ID No. 143), the brazilian thauma protein gene, the spacer sequence 1 (as shown in SEQ ID No. 143), the brazilian thauma protein gene, the spacer sequence 1 (as shown in SEQ ID No. 143), and the brazilian thauma protein gene are concatenated.

[0220] 9) For the PHKA-Bra4-KRx2 plasmid: the α-factor secretion signal, the brazilian thauma protein gene, the spacer sequence 2 (as shown in SEQ ID No. 144), the brazilian thauma protein gene, the spacer sequence 2 (as shown in SEQ ID No. 144), the brazilian thauma protein gene, the spacer sequence 2 (as shown in SEQ ID No. 144), and the brazilian thauma protein gene are concatenated.

[0221] 10) For the PHKA-Bra4-KRx3 plasmid: the α-factor secretion signal, the brazilian thauma protein gene, the spacer sequence 3 (as shown in SEQ ID No. 145), the brazilian thauma protein gene, the spacer sequence 3 (as shown in SEQ ID No. 145), the brazilian thauma protein gene, the spacer sequence 3 (as shown in SEQ ID No. 145), and the brazilian thauma protein gene are concatenated.

[0222] 11) For the PHKA-Bra4-KRx4 plasmid: the α-factor secretion signal, the brazilian thauma protein gene, the spacer sequence 4 (as shown in SEQ ID No. 146), the brazilian thauma protein gene, the spacer sequence 4 (as shown in SEQ ID No. 146), the brazilian thauma protein gene, the spacer sequence 4 (as shown in SEQ ID No. 146), and the brazilian thauma protein gene are concatenated.

[0223] 12) For the PHKA-Bra4-KRx5 plasmid: the α-factor secretion signal, the brazilian thauma protein gene, the spacer sequence 5 (as shown in SEQ ID No. 147), the brazilian thauma protein gene, the spacer sequence 5 (as shown in SEQ ID No. 147), the brazilian thauma protein gene, the spacer sequence 5 (as shown in SEQ ID No. 147), and the brazilian thauma protein gene are concatenated.

[0224] Expression of brazilian thaumatin in Pichia pastoris lacking protease gene

[0225] Example 10

[0226] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transformed into the gene-deficient strain MF001-29, and the free plasmids were eliminated to obtain MF001- 29-Bra2-KR, MF001-29-Bra2-KRx, MF001-29Bra2-KRx5, MF001-29-Bra2-KRx2, MF001-29-Bra2-KRx3, MF001-29-Bra2-KRx4, MF0 01-29-Bra4-KR, MF001-29-Bra4-KRx, MF001-29-Bra4-KRx5, MF001-29-Bra4-KRx2, MF001-29-Bra4-KRx3 and MF001-29-Bra4-KRx4.

[0227] Embodiment 11

[0228] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transferred into the gene-deficient strain MF001-94, and the free plasmids were eliminated to obtain MF001- 94-Bra2-KR, MF001-94-Bra2-KRx, MF001-94-Bra2-KRx5, MF001-94-Bra2-KRx2, MF001-94-Bra2-KRx3, MF001-94-Bra2-KRx4, MF0 01-94-Bra4-KR, MF001-94-Bra4-KRx, MF001-94-Bra4-KRx5, MF001-94-Bra4-KRx2, MF001-94-Bra4-KRx3 and MF001-94-Bra4-KRx4.

[0229] Example 12

[0230] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transferred into the gene-deficient strain MF001-136, and the free plasmids were eliminated to obtain MF001-136-Br a2-KR、MF001-136-Bra2-KRx、MF001-136-Bra2-KRx5、MF001-136-Bra2-KRx2、MF001-136-Bra2-KRx3、MF001-136-Bra2-KRx4、MF001- 136-Bra4-KR, MF001-136-Bra4-KRx, MF001-136-Bra4-KRx5, MF001-136-Bra4-KRx2, MF001-136-Bra4-KRx3 and MF001-136-Bra4-KRx4.

[0231] Example 13

[0232] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transferred into the gene-deficient strain MF001-143, and the free plasmids were eliminated to obtain MF001-143-Br a2-KR、MF001-143-Bra2-KRx、MF001-143-Bra2-KRx5、MF001-143-Bra2-KRx2、MF001-143-Bra2-KRx3、MF001-143-Bra2-KRx4、MF001- 143-Bra4-KR, MF001-143-Bra4-KRx, MF001-143-Bra4-KRx5, MF001-143-Bra4-KRx2, MF001-143-Bra4-KRx3 and MF001-143-Bra4-KRx4.

[0233] Embodiment 14

[0234] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transformed into the gene-deficient strain MF001-249, and the free plasmids were eliminated to obtain MF001-249-Br a2-KR、MF001-249-Bra2-KRx、MF001-249-Bra2-KRx5、MF001-249-Bra2-KRx2、MF001-249-Bra2-KRx3、MF001-249-Bra2-KRx4、MF001- 249-Bra4-KR, MF001-249-Bra4-KRx, MF001-249-Bra4-KRx5, MF001-249-Bra4-KRx2, MF001-249-Bra4-KRx3 and MF001-249-Bra4-KRx4.

[0235] Embodiment 15

[0236] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transformed into the gene-deficient strain MF001-251, and the free plasmids were eliminated to obtain MF001-251-Br a2-KR、MF001-251-Bra2-KRx、MF001-251-Bra2-KRx5、MF001-251-Bra2-KRx2、MF001-251-Bra2-KRx3、MF001-251-Bra2-KRx4、MF001- 251-Bra4-KR, MF001-251-Bra4-KRx, MF001-251-Bra4-KRx5, MF001-251-Bra4-KRx2, MF001-251-Bra4-KRx3 and MF001-251-Bra4-KRx4.

[0237] Example 16

[0238] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transferred into the gene-deficient strain MF001-256, and the free plasmids were eliminated to obtain MF001-256-Br a2-KR、MF001-256-Bra2-KRx、MF001-256-Bra2-KRx5、MF001-256-Bra2-KRx2、MF001-256-Bra2-KRx3、MF001-256-Bra2-KRx4、MF001- 256-Bra4-KR, MF001-256-Bra4-KRx, MF001-256-Bra4-KRx5, MF001-256-Bra4-KRx2, MF001-256-Bra4-KRx3 and MF001-256-Bra4-KRx4.

[0239] Embodiment 17

[0240] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transferred into the gene-deficient strain MF001-169, and the free plasmids were eliminated to obtain MF001-169-Br a2-KR、MF001-169-Bra2-KRx、MF001-169-Bra2-KRx5、MF001-169-Bra2-KRx2、MF001-169-Bra2-KRx3、MF001-169-Bra2-KRx4、MF001- 169-Bra4-KR, MF001-169-Bra4-KRx, MF001-169-Bra4-KRx5, MF001-169-Bra4-KRx2, MF001-169-Bra4-KRx3 and MF001-169-Bra4-KRx4.

[0241] Comparative Example 1

[0242] The plasmids PHKA-Bra2-KR, PHKA-Bra2-KRx, PHKA-Bra2-KRx5, PHKA-Bra2-KRx2, PHKA-Bra2-KRx3, PHKA-Bra2-KRx4, PHKA-Bra4-KR, PHKA-Bra4-KRx, PHKA-Bra4-KRx5, PHKA-Bra4-KRx2, PHKA-Bra4-KRx3, and PHKA-Bra4-KRx4 were respectively transformed into the gene-deficient strain GS115 and sterilized. In addition to the free plasmid, GS115-Bra2-KR, GS115-Bra2-KRx, GS115-Bra2-KRx5, GS115-Bra2-KRx2, GS115-Bra2-KRx3, GS115-Bra2-KRx4, GS115-Bra4-KR, GS115-Bra4-KRx, GS115-Bra4-KRx5, GS115-Bra4-KRx2, GS115-Bra4-KRx3 and GS115-Bra4-KRx4 were obtained respectively.

[0243] Comparative Example 2

[0244] The plasmids PHKA-Bra2-KRx3 and PHKA-Bra4-KRx3 were respectively transferred into the gene-deficient strain SMD1163, and the free plasmids were eliminated to obtain the corresponding strains SMD1163-Bra2-KRx3 and SMD1163-Bra4-KRx3, respectively.

[0245] Determination of the expression yield of brazilian thauma protein

[0246] A single clone was selected for shake flask fermentation and inoculated into a 250 mL baffled shake flask containing 100 ml of BMGY (Buffered Glycerol-complex Medium). The culture was grown in a shaker at 30 degrees Celsius and 250 to 300 rpm until the OD 600 = 2 to 6 (about 16 to 18 hours) Centrifuge at 1500 to 3000g for 5 minutes at room temperature to collect cells, remove the supernatant, resuspend the cells with 1 / 5 to 1 / 10 of the original culture medium volume of BMMY (Buffered Minimal Methanol YP Medium) (about 10-20 ml) and place in a 100 ml baffled shake flask, cover the bottle mouth with 2 layers of sterile gauze or cheesecloth, and continue to culture in a shaker. Every 24 hours, add methanol to a final concentration of 1% to continue induction. At 6H, 12H, 24H, 48H, 72H, and 96H, take 1 ml of culture medium to a 1 to 5 ml centrifuge tube for analysis of OD at each time point 600 And SDS-PAGE was used to detect the expression of protein in the supernatant of the fermentation broth. After confirming the expression of brazilian tamanin, the brazilian tamanin was purified by ion exchange chromatography gel coupling method, and the purified protein was freeze-dried by low-temperature vacuum drying to obtain freeze-dried powder. Among them, the purity of the freeze-dried powder was determined to be above 90% by the brazilian tamanin standard (purchased from Nanjing Wobo Biotechnology Co., Ltd., item number: Wb2641-1mg). Among them, the SDS-PAGE results of the proteins expressed by some expression strains at various time points are shown in Figure 2 and Figure 3 From the results, it can be seen that some strains such as the protease-deficient strain MF001-136-Bra4-KRx3 showed a 114% increase in brassica protein production at 72 hours compared to the control strain GS115-Bra4-KRx3.

[0247] HPLC was used to quantitatively analyze the lyophilized powder of brazilian tamanu protein standard and the brazilian tamanu protein expressed by each expression strain. The yield of brazilian tamanu protein expressed by each expression strain was detected by comparing with the brazilian tamanu protein standard. It was found that the peak time of the standard product with a concentration of 1g / L was 6.650min and the peak area was 19.3746. The MF001-29-Bra4-KRx3 shake flask fermentation 72H sample was first centrifuged, treated with a 0.22μm membrane, and the sample was tested after filtration. The sample peak time was 6.643min and the peak area was 18.1872. It was calculated that the yield of MF001-29-Bra4-KRx3 fermentation 72H reached 0.939g / L. Its HPLC peak time and area are shown in Table 1. Figure 4 . Among them, MF001-29-Bra2-KRx3, MF001-29-Bra4-KRx3, MF001-94-Bra2-KRx3, MF001-94-Bra4-KRx3, MF001-136-Bra2-KRx3 , MF001-136-Bra4-KRx3, MF001-143-Bra2-KRx3, MF001-143-Bra4-KRx3, MF001-249-Bra2-KRx3, MF001-249-Bra4-KR The results of MF001-251-Bra2-KRx3, MF001-251-Bra4-KRx3, MF001-256-Bra2-KRx3, MF001-256-Bra4-KRx3, MF001-169-Bra2-KRx3, MF001-169-Bra4-KRx3, GS115-Bra2-KRx4, GS115-Bra4-KRx3, SMD1163-Bra2-KRx3 and SMD1163-Bra4-KRx3 are shown in Table 3.

[0248] The above data show that the gene knockout strain of the present invention can significantly increase the expression level of exogenous proteins.

[0249] Table 3

[0250]

[0251] Growth rate determination of strains used to express brasilin

[0252] The strains of Examples 10 to 17 and Comparative Examples 1 and 2 were activated and inoculated into 50 mL YPD liquid culture medium at a volume ratio of 1%, and samples were taken at 0, 6, 9, 24 h, 30 h, 48 h and 54 h to measure OD 600 Then, the sampling time was used as the horizontal axis and the OD of each strain was used as the horizontal axis. 600The growth curves of MF001-29-Bra2-KRx3, MF001-94-Bra2-KRx3, MF001-136-Bra2-KRx3, MF001-143-Bra2-KRx3, MF001-249-Bra2-KRx3, MF001-251-Bra2-KRx3, MF001-256-Bra2-KRx3, MF001-169-Bra2-KRx3, GS115-Bra2-KRx4 and SMD1163-Bra2-KRx3 are shown in the figure. Figure 5 ; The growth curves of MF001-29-Bra4-KRx3, MF001-94-Bra4-KRx3, MF001-136-Bra4-KRx3, MF001-143-Bra4-KRx3, MF001-249-Bra4-KRx3, MF001-251-Bra4-KRx3, MF001-256-Bra4-KRx3, MF001-169-Bra4-KRx3, GS115-Bra4-KRx3 and SMD1163-Bra4-KRx3 are shown in Figure 6 . Figure 5 and Figure 6 The results showed that the insertion of PHKA-Bra2-KRx3 and PHKA-Bra4-KRx3 linearized plasmids did not affect the growth of the strain.

[0253] Construction of the expression plasmid for salt-reducing protein

[0254] Embodiment 18

[0255] For the PHKA-PF plasmid: the α factor secretion signal and the salt-reducing protein gene were connected in series 16 times, the linearized plasmid PHKA-PF was transferred into each gene-deficient strain, and the free plasmid was eliminated to obtain each gene-deficient strain that can express the salt-reducing protein. Among them, the expression strain after transfer into MF001-94 is MF001-94-PF.

[0256] The OD of each expression strain at each time point was measured by high-density fermentation. 600 , biomass and production of salt-reduced protein.

[0257] The following uses the high-density fermentation of the recombinant yeast engineered salt-reducing protein MF001-94-PF strain as an example to illustrate that the gene-deficient strains of the present invention can produce a higher yield of salt-reducing protein than the starting strain GS115.

[0258] 1) Preparation of fermentation seed solution

[0259] Pick the MF001-94-PF strain and streak the activated culture on the YPD plate with a single colony that has grown well, inoculate it into 50 mL of sterilized YPD liquid culture medium, place it in a 30°C shaker, and culture it overnight at 250 rpm for strain activation. Transfer the YPD-activated yeast seed culture liquid at a ratio of 4% to a 500 mL Erlenmeyer flask containing 100 mL of YPD liquid culture medium, place it in a 30°C shaker, and culture it overnight at 250 rpm as the seed liquid for fermentation in a 5 L fermenter.

[0260] 2) High-density culture of recombinant yeast strains in 5L fermenters

[0261] The cultured yeast seed liquid was inoculated into a 5L fully automatic mechanical stirring and ventilated fermentation tank (sterilized) at an inoculation rate of 8%, and glycerol was added to start bacterial growth (the volume of the initial culture medium was 2L, sterilized at 121°C for 30min). Bacterial growth stage: 25% concentrated ammonia water was used to control its pH at 5.5, the dissolved oxygen was maintained at 30% to 60%, the temperature was controlled at 30°C, the upper limit of the stirring speed was 200r / min, the lower limit was 120r / min, and the ventilation volume was set to 2vvm. Initial cell density OD 600 After reaching 400, stop adding glycerol. When the dissolved oxygen DO rebounds to more than 80% again, start methanol feeding induced fermentation. Methanol feeding stage: the temperature is controlled at 30°C, the pH is controlled at 5.5 using 25% concentrated ammonia water, the speed is controlled at 800r / min, the ventilation is controlled at 4L / min, the pressure is controlled at 0.05Mpa, the ventilation is adjusted to 2vvm, and the methanol is added by variable speed feeding, and the dissolved oxygen is maintained at 15% to 25%. The fermentation process is automatically controlled and the fermentation data is collected using the Bailun BLBIO B control system software.

[0262] The relationship between the collection time of MF001-94-PF salt-reduced protein and OD and WCW (biomass) is as follows Figure 7 shown.

[0263] Samples were taken from the fermenter at 22 hours, 46 hours, 70 hours and 94 hours after methanol fermentation induction, centrifuged and the supernatant was subjected to SDS-PAGE electrophoresis. Figure 8 .from Figure 8 From the electrophoresis of the fermentation strain MF001-94-PF salt-reducing protein, the salt-reducing protein gradually accumulated with the fermentation time. Among them, the SDS gel is FuturePAGE TM Protein precast gel 4% to 20% (ACE). On the gel, load 20 μl of sample (10 μL of sample mixed with 10 μl of loading buffer), apply 10 μl of Marker: (pre-stained SDS-PAGE standard, GenScript Bio, #M00624-250).

[0264] Subsequently, the product of fermentation 117H was centrifuged, and the supernatant was purified with oligofructose gel. After HPLC identification, it was found that the production of salt-reducing protein reached 12g / L.

[0265] Construction of semaglutide expression plasmid

[0266] Embodiment 19

[0267] For the PHKA-P29-SUMO plasmid: the α-factor secretion signal and semaglutide gene were concatenated twice, the linearized plasmid PHKA-P29-SUMO was transferred into the gene-deficient strain MF001-94, and the free plasmid was eliminated to obtain MF001-94-P29-SUMO.

[0268] Pick the monoclonal strain MF001-94-P29-SUMO for shake flask fermentation culture, and inoculate it into a 250mL baffled shake flask containing 100ml BMGY. Grow in a shaker at 30 degrees, 250 to 300rpm to OD600 = 2-6 (about 16-18 hours). Collect the cells by centrifugation at 1500 to 3000g for 5 minutes at room temperature, remove the supernatant, resuspend the cells with 1 / 5-1 / 10 of the original culture medium volume of BMMY (about 10-20ml) and place them in a 100ml baffled shake flask. Cover the bottle mouth with 2 layers of sterile gauze or cheesecloth, and continue to culture in a shaker. Every 24 hours, add methanol to a final concentration of 1% to continue induction. Take 1ml of culture medium regularly to a 1-5ml centrifuge tube for analysis of OD at each time point. 600 And SDS-PAGE was used to detect the expression of protein in the fermentation broth supernatant. Fig. 9 The results of SDS-PAGE electrophoresis of supernatant proteins are shown in Fig.10 .

[0269] The shake flask fermentation of the recombinant yeast engineered semaglutide MF001-94-P29-SUMO strain was used as an example to illustrate that the gene-deficient strains of the present invention can produce a higher yield of semaglutide than the starting strain GS115.

Claims

1. PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS _chr4_0913, PAS_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_ch Use of a combination of at least two proteins selected from PAS_chr3_0934, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 or a single PAS_chr3_0689 protein in affecting yeast growth rate and / or exogenous protein expression; wherein, The amino acid sequence of the PAS_chr3_1087 protein is shown in SEQ ID No.2, the amino acid sequence of the PAS_chr4_0584 protein is shown in SEQ ID No.4, the amino acid sequence of the PAS_chr2-1_0652 protein is shown in SEQ ID No.6, the amino acid sequence of the PAS_chr4_0113 protein is shown in SEQ ID No.8, the amino acid sequence of the PAS_chr2-2_0380 protein is shown in SEQ ID No.10, the amino acid sequence of the PAS_chr4_0913 protein is shown in SEQ ID No.12, the amino acid sequence of the PAS_chr1-4_0611 protein is shown in SEQ ID No.14, the amino acid sequence of the PAS_chr3_0953 protein is shown in SEQ ID No.16, the amino acid sequence of the PAS_chr1-1_0194 protein is shown in SEQ ID No.18, the amino acid sequence of the PAS_chr1-4_0048 protein is shown in SEQ ID No.20, the amino acid sequence of the PAS_chr3_0934 protein is shown in SEQ ID No.22, the amino acid sequence of the PAS_chr3_0633 protein is shown in SEQ ID No.24, the amino acid sequence of the PRC1 PAS_chr1-4_0013 protein is shown in SEQ ID No.26, the amino acid sequence of the PAS_chr1-1_0174 protein is shown in SEQ ID No.28, the amino acid sequence of the PAS_chr3_0979 protein is shown in SEQ ID No.30, the amino acid sequence of the PAS_chr1-1_0226 protein is shown in SEQ ID No.32, and the amino acid sequence of the PAS_chr3_0689 protein is shown in SEQ ID No.

34.

2. The use according to claim 1, characterized in that: With PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PA S_chr4_0913, PAS_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_ chr3_0934, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689, or the PAS_chr3_0689 gene alone, compared to before the expression of the PAS_chr3_0934, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 gene in yeast is silenced. _1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_091 3. PAS_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_chr3_0934, The expression of at least two proteins among PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 or the expression of PAS_chr3_0689 protein alone is silenced to increase the growth rate of the yeast and / or the expression amount of the exogenous protein in the yeast.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the PAS_chr3_1087 gene is shown in SEQ ID No.1, the nucleotide sequence of the PAS_chr4_0584 gene is shown in SEQ ID No.3, the nucleotide sequence of the PAS_chr2-1_0652 gene is shown in SEQ ID No.5, the nucleotide sequence of the PAS_chr4_0113 gene is shown in SEQ ID No.7, the nucleotide sequence of the PAS_chr2-2_0380 gene is shown in SEQ ID No.9, the nucleotide sequence of the PAS_chr4_0913 gene is shown in SEQ ID No.11, the nucleotide sequence of the PAS_chr1-4_0611 gene is shown in SEQ ID No.13, the nucleotide sequence of the PAS_chr3_0953 gene is shown in SEQ ID No.15, the nucleotide sequence of the PAS_chr1-1_0194 gene is shown in SEQ ID The nucleotide sequence of the PAS_chr1-4_0048 gene is shown in SEQ ID No.17, the nucleotide sequence of the PAS_chr1-4_0048 gene is shown in SEQ ID No.19, the nucleotide sequence of the PAS_chr3_0934 gene is shown in SEQ ID No.21, the nucleotide sequence of the PAS_chr3_0633 gene is shown in SEQ ID No.23, the nucleotide sequence of the PRC1 PAS_chr1-4_0013 gene is shown in SEQ ID No.25, the nucleotide sequence of the PAS_chr1-1_0174 gene is shown in SEQ ID No.27, the nucleotide sequence of the PAS_chr3_0979 gene is shown in SEQ ID No.29, the nucleotide sequence of the PAS_chr1-1_0226 gene is shown in SEQ ID No.31, and the nucleotide sequence of the PAS_chr3_0689 gene is shown in SEQ ID No.

33.

4. The use according to claim 2 or 3, characterized in that: By knocking out PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_0913, PAS_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1 -4_0048, PAS_chr3_0934, PAS_chr3_0633, PRC1PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226 and PAS_chr3_0689 or the PAS_chr3_0689 gene alone to silence the expression of the corresponding protein.

5. The use according to claim 4, characterized in that: Knock out the PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 genes in the yeast simultaneously; or Knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633 and PRC1 PAS_chr1-4_0013 genes in the yeast simultaneously; or Knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953 and PAS_chr2-2_0380 genes in the yeast at the same time; or Knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380 and PAS_chr1-4_0611 genes in the yeast at the same time; or Knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, PAS_chr1-1_0226 and PAS_chr3_1087 genes in the yeast at the same time; or Knock out PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611 and PAS_chr1-1_0194 genes in the yeast at the same time; or At the same time, the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584 and PAS_chr3_1087 genes in the yeast were knocked out.

6. The use according to claim 1, characterized in that: The exogenous protein is at least one of brazilian tamarind, reduced salt protein, semaglutide, casein and whey protein; Preferably, the amino acid sequence of the brazilian tamarind protein is as shown in SEQ ID No. 36; Preferably, the gene encoding the brazilian tamarind protein is introduced into the yeast to allow the yeast to express the brazilian tamarind protein. Preferably, the nucleotide sequence of the gene encoding the brazilian tamarind protein is as shown in SEQ ID No.

35. Preferably, the amino acid sequence of the salt-reducing protein is as shown in SEQ ID No.38; Preferably, a gene encoding the salt-reducing protein is transferred into the yeast so that the yeast expresses the salt-reducing protein. Preferably, the nucleotide sequence of the gene encoding the salt-reducing protein is as shown in SEQ ID No. 37; Preferably, the amino acid sequence of semaglutide is shown as SEQ ID No.40; Preferably, the gene encoding the semaglutide is transferred into the yeast so that the yeast expresses the semaglutide. Preferably, the nucleotide sequence of the gene encoding the semaglutide is as shown in SEQ ID No.

39.

7. The use according to claim 1, characterized in that: The yeast is Pichia pastoris; Preferably, the starting strain of the yeast is Pichia pastoris GS115 strain or SMD1163 strain.

8. An engineered yeast obtained by knocking out the gene in the yeast for use according to any one of claims 1 to 6.

9. The yeast engineering bacteria according to claim 8, characterized in that: The yeast engineered bacteria is a yeast engineered bacteria obtained by taking the Pichia pastoris GS115 strain as the starting strain and knocking out the PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 genes therein; or The yeast engineering bacteria is a yeast engineering bacteria obtained by taking the Pichia pastoris GS115 strain as the starting strain and knocking out the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633 and PRC1 PAS_chr1-4_0013 genes therein; or The yeast engineering bacteria is a yeast engineering bacteria obtained by taking the Pichia pastoris GS115 strain as the starting strain and knocking out the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953 and PAS_chr2-2_0380 genes therein; or The yeast engineering bacteria is a yeast engineering bacteria obtained by taking the Pichia pastoris GS115 strain as the starting strain and knocking out the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380 and PAS_chr1-4_0611 genes therein; or The yeast engineering bacteria is based on the Komagataella pastoris GS115 strain as the starting strain, and PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 Yeast engineered bacteria obtained by using PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, PAS_chr1-1_0226 and PAS_chr3_1087 genes; or The yeast engineering bacteria is a yeast engineering bacteria obtained by taking the Pichia pastoris GS115 strain as the starting strain and knocking out the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611 and PAS_chr1-1_0194 genes therein; or The yeast engineering bacteria is a yeast engineering bacteria obtained by taking the Pichia pastoris GS115 strain as a starting strain and knocking out the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584 and PAS_chr3_1087 genes therein.

10. The yeast engineering bacteria according to claim 8, characterized in that: The yeast engineering bacteria is a yeast engineering bacteria obtained by taking the Pichia pastoris GS115 strain as a starting strain and knocking out the PAS_chr3_0689 gene therein.

11. The engineered yeast according to claims 8 to 10, characterized in that: A gene for expressing a foreign protein is transferred into the yeast engineering bacteria to express the foreign protein.

12. The yeast engineering bacteria according to claim 11, characterized in that: The exogenous protein is at least one of brazilian tamarind, reduced salt protein, semaglutide, casein and whey protein; Preferably, the amino acid sequence of the brazilian tamarind protein is as shown in SEQ ID No. 36; Preferably, the gene encoding the brazilian tamarind protein is introduced into the yeast to allow the yeast to express the brazilian tamarind protein. Preferably, the nucleotide sequence of the gene encoding the brazilian tamarind protein is as shown in SEQ ID No.

35. Preferably, the amino acid sequence of the salt-reducing protein is as shown in SEQ ID No.38; Preferably, a gene encoding the salt-reducing protein is transferred into the yeast so that the yeast expresses the salt-reducing protein. Preferably, the nucleotide sequence of the gene encoding the salt-reducing protein is as shown in SEQ ID No. 37; Preferably, the amino acid sequence of semaglutide is shown as SEQ ID No.40; Preferably, the gene encoding the semaglutide is transferred into the yeast so that the yeast expresses the semaglutide. Preferably, the nucleotide sequence of the gene encoding the semaglutide is as shown in SEQ ID No.39.

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