Protein combination affecting yeast growth rate and exogenous protein expression level

Abstract

The present invention relates to a protein combination affecting a yeast growth rate and an exogenous protein expression level. Knocking out gene combinations from the peptidase A1 family, peptidase M1 family, peptidase M18 family, peptidase M28 family, peptidase S54 family, peptidase S10 family, peptidase S16 family, peptidase S14 family, and peptidase S8 family, or knocking out transcriptional activators of yeast alone, can increase the yeast growth rate and increase the exogenous protein expression level.

Description

Protein combinations that affect yeast growth rate and exogenous protein expression levels Technical Field

[0001] This invention relates to the field of proteins, and particularly to a combination of proteins that affect yeast growth rate and the expression level of exogenous proteins. Background Technology

[0002] Yeast expression systems offer advantages such as high fermentation density, strong secretion capacity, and low glycosylation levels. However, exogenous proteins are susceptible to degradation by yeast proteolytic enzymes during expression, which is one of the main factors affecting the efficient expression of exogenous proteins in Pichia pastoris. Previous studies have indicated that the amounts of various intracellular proteolytic enzymes in yeast change with variations in nutritional conditions during culture, such as starvation stress and changes in carbon and nitrogen sources. Especially during high-density fermentation, higher levels of proteases are often detected in the culture medium.

[0003] During fermentation, when cells are under stress, proteases located in yeast vacuoles are overexpressed or released into the extracellular space through cell autolysis, thereby degrading recombinant proteins secreted into the extracellular space.

[0004] Sweeteners are food additives that impart a sweet taste to food or feed. Based on their source, they can be divided into natural sweeteners and artificial sweeteners. With the development of organic chemistry, artificial sweeteners have gradually become dominant. However, due to concerns about the safety of some chemically synthesized sweeteners, many countries around the world have discouraged or even banned their use. Therefore, finding safe, non-toxic, and purely sweet natural sweeteners is of paramount importance.

[0005] Using yeast-expressed exogenous proteins, brazzein is an example. Brazzein (sometimes translated as Brazzein in Chinese) is the main sweet protein in the West African fruit Oubli (Pentadiplandra brazzeana Baillon). It is found in the extracellular pulp tissue surrounding the seeds. For thousands of years, locals have used it as a sweetener. Because the juice is also used to help wean infants, Brazzein has 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 cysteine ​​residues forming 4 pairs of intramolecular disulfide bonds. Brazzein has a relative molecular mass of 6500 and is 2000 times sweeter than an equal mass of sucrose. Compared to other sweet proteins, brazzein has the smallest molecular weight, the best water solubility, and its aqueous solution retains its sweetness even after heat treatment at 80 degrees Celsius for 4 hours, exhibiting good thermal and pH stability.

[0006] Salt is the most common seasoning, and NaCl is one of its main components. However, excessive salt intake leads to an increase in sodium ion concentration. This can result in increased osmotic pressure, increased vascular tone and cardiac output, ultimately increasing 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) released the "Definition and Requirements for a Healthy Diet," recommending that adults consume no more than 5 grams of salt per day. However, the vast majority of countries in the world exceed this limit. Generally, the industry uses food additives to reduce the sodium ion concentration in table salt, with potassium chloride being the most widely used. Although potassium ions, which also have a salty taste, can reduce the risk of high blood pressure while maintaining a salty flavor, increased potassium levels are detrimental to patients with kidney disease and may interact with medications used to treat cardiovascular diseases, potentially worsening their condition and posing certain health risks. Furthermore, at concentrations exceeding 15%, potassium ions have a bitter taste in the mouth. In addition to metallic salt substitutes, flavor enhancers can be added to activate taste receptors located on the human mouth and tongue, compensating for the taste differences caused by reduced sodium ion concentration. Among these, umami and saltiness are most closely related. Currently, the most widely produced and used food flavor enhancer in my country is monosodium glutamate (MSG). Although it is inexpensive and has a prominent umami flavor, and can economically and effectively reduce the amount of salt added, MSG still contains sodium ions, which cannot meet the true meaning of "reduced sodium salt".

[0008] The concept of salty peptides was proposed in 1984 by Tada et al., who discovered Orn-Tau-HCl and Lys-Tau-HCl during the synthesis of N-terminal analogs of casein hydrolysates. These two peptides do not contain sodium. + However, it has a similar or even higher salinity to NaCl. Summary of the Invention

[0009] One of the present inventions 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, and PRC1. The application of at least two proteins selected from PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226, and PAS_chr3_0689, or the use of PAS_chr3_0689 protein alone, in influencing yeast growth rate and / or the expression level of exogenous proteins; 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, and the amino acid sequence of the PAS_chr4_0913 protein is shown in SEQ ID No. 8. As 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; and the amino acid sequence of the PAS_chr3_0979 protein is shown in SEQ ID No. 14. As shown in 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.As shown in Figure 34. The foreign protein can be obtained by introducing a gene encoding the foreign protein into the yeast, and then having the yeast express the introduced gene.

[0010] In one specific implementation, 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 to before the expression of at least two of 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 was silenced, this represents a reduction in the expression of PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_0913, and PAS_chr1-4_0013, respectively. Silencing the expression of at least two proteins selected from 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 PAS_chr3_0689 alone, to increase the growth rate of the yeast and / or the expression level of the exogenous protein in the yeast.

[0011] In one 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, and the nucleotide sequence of the PAS_chr1-1_0194 gene is shown in SEQ ID No. 15. As shown in 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.

[0012] In one specific 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, and PRC1 from the yeast cells... The expression of the corresponding protein is silenced by at least two of the following genes: PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226, and PAS_chr3_0689, or by the PAS_chr3_0689 gene alone.

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

[0014] In one specific embodiment, 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 in the yeast are simultaneously knocked out.

[0015] In one specific embodiment, 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 in the yeast are simultaneously knocked out.

[0016] In one specific embodiment, 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 in the yeast are simultaneously knocked out.

[0017] In one specific embodiment, the following genes in the yeast are simultaneously knocked 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, PAS_chr1-1_0226, and PAS_chr3_1087.

[0018] In one specific embodiment, the following genes in the yeast are simultaneously knocked 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.

[0019] In one specific embodiment, the following genes in the yeast are simultaneously knocked 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.

[0020] In one specific embodiment, the exogenous protein is at least one selected from brassinoprotein, reduced-salt protein, smegglutinin, casein, and whey protein.

[0021] In one specific embodiment, the amino acid sequence of the Brazil sweet protein is shown in SEQ ID No. 36.

[0022] In one specific embodiment, the gene encoding the brassinolide is introduced into the yeast to cause the yeast to express the brassinolide.

[0023] In one specific embodiment, the nucleotide sequence of the gene encoding the Brazilian sweet protein is shown in SEQ ID No. 35.

[0024] In one specific embodiment, the amino acid sequence of the desalination protein is shown in SEQ ID No. 38.

[0025] In one specific embodiment, a gene encoding the salt-reducing protein is introduced into the yeast to cause the yeast to express the salt-reducing protein.

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

[0027] In one specific embodiment, the amino acid sequence of the smegglutinin is shown in SEQ ID No. 40.

[0028] In one specific embodiment, the gene encoding the smegglutinin is transferred into the yeast to enable the yeast to express the smegglutinin.

[0029] In one specific embodiment, the nucleotide sequence of the gene encoding the smegglutinin is shown in SEQ ID No. 39.

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

[0031] In one specific embodiment, the starting strain of the yeast is either Komagataella pastoris GS115 or SMD1163.

[0032] The second aspect of this invention provides an engineered yeast strain obtained by knocking out the gene in the yeast strain described in any of the applications described in the first aspect of this invention. These engineered strains can have various commercial applications, including but not limited to providing a testing and comparison platform for interested individuals or companies, or using this platform to express different exogenous and endogenous protein and peptide products.

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

[0034] In one specific embodiment, the engineered yeast strain is a yeast strain obtained by 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 of PAS_chr3_0913 as the starting strain of PAS_chr1_0045.

[0035] In one specific embodiment, the engineered yeast strain is a yeast strain obtained by using Komagataella pastoris GS115 as the starting strain and simultaneously 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.

[0036] In one specific embodiment, the engineered yeast strain is a yeast strain obtained by 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 of PAS_chr1-4_0611, using PAS_chr1 as the starting strain.

[0037] In one specific embodiment, the engineered yeast strain is Pichia pastoris (Komagataella). Yeast engineered strains were obtained by using strain GS115 (pastoris) as the starting strain and simultaneously knocking out the following genes: 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.

[0038] In one specific embodiment, the engineered yeast strain is a yeast strain obtained by using Komagataella pastoris GS115 as the starting strain and simultaneously 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, PAS_chr1-4_0611, and PAS_chr1-1_0194 genes.

[0039] In one specific embodiment, the engineered yeast strain is a yeast strain obtained by knocking out the following genes: 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, from the starting strain PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, and PAS_chr3_1087.

[0040] In one specific embodiment, the engineered yeast strain is a yeast strain obtained by knocking out the PAS_chr3_0689 gene from the Pichia pastoris strain GS115.

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

[0042] In one specific embodiment, the exogenous protein is at least one selected from brassinoprotein, reduced-salt protein, smegglutinin, casein, and whey protein.

[0043] In one specific embodiment, the amino acid sequence of the Brazil sweet protein is shown in SEQ ID No. 36.

[0044] In one specific embodiment, the gene encoding the brassinolide is introduced into the yeast to cause the yeast to express the brassinolide.

[0045] In one specific embodiment, the nucleotide sequence of the gene encoding the Brazilian sweet protein is shown in SEQ ID No. 35.

[0046] In one specific embodiment, the amino acid sequence of the desalination protein is shown in SEQ ID No. 38.

[0047] In one specific embodiment, a gene encoding the salt-reducing protein is introduced into the yeast to cause the yeast to express the salt-reducing protein.

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

[0049] In one specific embodiment, the amino acid sequence of the smegglutinin is shown in SEQ ID No. 40.

[0050] In one specific embodiment, the gene encoding the smegglutinin is transferred into the yeast to enable the yeast to express the smegglutinin.

[0051] In one specific embodiment, the nucleotide sequence of the gene encoding the smegglutinin is shown in 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, beverage, and / or pharmaceutical products intended for human consumption. A sweetener may comprise a single active ingredient, i.e., a single substance having a sweet taste, or may comprise a blend of several such active ingredients, i.e., substances contributing to sweetness. The sweetener may be an active ingredient in its substantially pure form, such as a sweet protein isolated from its producing cells and currently in a form applicable to products intended for human consumption. Alternatively, the sweetener may contain other substances in addition to the active ingredient, such as fillers (e.g., lactose). The sweetener may be further blended with other substances before application to food or beverage products, or before being sold to the end consumer for home use, such as for sweetening tea or coffee.

[0053] Brazil sweetener, also known as Brazilian sweet protein, brazzeana protein, or brazzeanain, refers to a form extracted from the fruit of the West African climbing plant *Pen tadiplandra brazzeana* (Baillon), or a recombinant form thereof. In nature, Brazil sweetener occurs in three different forms, with or without glutamine residues or pyroglutamic acid at its N-terminus. In the context of this invention, the wild-type Brazil sweetener sequence preferably comprises the amino acid sequence of SEQ ID NO:36, without glutamine residues or pyroglutamic acid at its N-terminus.

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

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

[0056] Expression vectors are linear or circular nucleic acid molecular constructs containing polynucleotides encoding proteins such as Brazil nuts as described herein, and said polynucleotides are operatively linked to additional nucleotides provided for their expression. These additional nucleotide sequences include, for example, a promoter, suitable transcription initiation and termination sequences. A “promoter” is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for RNA polymerase-specific binding and transcription initiation, and is mostly located upstream of the transcription start site; the promoter itself is not transcribed. Additionally, expression vectors possess the host replication capacity typically conferred by the origin of replication, and / or carry selection genes that aid in the recognition of transformants. Typically, expression vectors used in recombinant DNA technology are often in the form of “plasmids,” i.e., circular double-stranded DNA loops. Obviously, vectors derived from viruses such as retroviruses and adenoviruses can also be used.

[0057] Amino acid sequence: Synonymous with the terms "polypeptide," "protein," and "peptide," and used interchangeably. A standard single-letter or three-letter code is used for the amino acid residues, where the amino acid sequence is presented with a standard amino-to-carboxyl terminus orientation (i.e., N→C).

[0058] Signal peptide: A short peptide chain (5 to 30 amino acids in length) that guides the transmembrane transport of newly synthesized proteins into the secretory pathway. It typically refers to the N-terminal amino acid sequence of a newly synthesized polypeptide chain that guides the transmembrane transport (localization) of the protein.

[0059] Primer design: Primers for the constructed plasmids were designed using Snapgene software. When designing primers, attention should be paid to factors such as GC content, Tm value, hairpin structure, primer length, primer dimers, primer mismatches, and the introduction of restriction enzyme sites.

[0060] The terms “comprising,” “including,” “having,” and combinations thereof mean “including but not limited to,” but also refer to situations consisting only of the listed elements.

[0061] The definitions provided herein are for the convenience of understanding certain terms frequently used herein and are not intended to limit the scope of this disclosure.

[0062] Because exogenous proteins are susceptible to degradation by yeast proteolytic enzymes during expression, their efficient expression in Pichia pastoris is affected. The levels of various proteolytic enzymes in yeast cells change with nutritional conditions during culture, especially in high-density fermentation, where high levels of proteases are often detected in the culture medium. Optimizing culture conditions can reduce the degradation of recombinant proteins to some extent. Key measures include: 1) altering the pH, temperature, or time of culture to reduce protease activity; 2) adding specific amino acids or casein hydrolysates as competitive substrates for proteases or adding specific protease inhibitors. However, these methods are usually limited in effectiveness and not applicable to all recombinant proteins. Knocking out the genes encoding specific proteolytic enzymes in host cells using genetic engineering is an effective way to fundamentally solve the problem of recombinant protein degradation. This method has been widely used in various expression systems, including E. coli and yeast.

[0063] Due to the structural complexity of Brazil sweeteners, particularly the presence of numerous cysteine ​​residues, traditional systems such as *E. coli* and *Pichia pastoris* cannot fold into the correct conformation. Furthermore, degradation by proteases results in low sweetness, making genetic engineering methods unsuitable for low-cost industrial production. This invention addresses these issues by proposing a method for preparing high-sweetness Brazil sweeteners. The invention involves knocking out a specific proteolytic enzyme encoding gene within the host cell, fusing a signal peptide sequence with a Brazil sweetener protein sequence, and using a protease-deficient *Pichia pastoris* strain as the host to express the fusion sequence. The resulting Brazil sweetener protein can undergo high-density fermentation, facilitating downstream extraction and purification, and reducing production costs. Furthermore, this invention demonstrates that the prepared Brazil sweetener can be effectively used as a sweetener to replace sucrose in yogurt beverages and sparkling water.

[0064] Beneficial effects of the present invention: The present invention has found that knocking out 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, and PRC1... Knocking out at least two of the genes PAS_chr1-4_0013, PAS_chr1-1_0174, PAS_chr3_0979, PAS_chr1-1_0226, and PAS_chr3_0689, or knocking out PAS_chr3_0689 alone, can improve the growth rate of Pichia pastoris and increase the expression yield of exogenous proteins.

[0065] To meet the growing demand for low-sugar and sugar-reduced foods, the inventors of this invention have used a gene-deficient strain to express high-sweetness Brazilin, resulting in higher yields and significantly higher sweetness than Brazilin prepared using existing technologies. Furthermore, the expressed protein exhibits no significant delay in sweetness, making its taste closer to sucrose. Simultaneously, this preparation method slows down the fermentation degradation of Brazilin protein, meeting the requirements for industrial production. Due to its significantly enhanced sweetness, its production costs are also significantly reduced.

[0066] This invention artificially synthesized a DNA sequence and cloned it into the expression plasmid PHKA, then recombinantly expressed using a protease-deficient Pichia pastoris strain. The protein was then purified by ion exchange chromatography and affinity chromatography to obtain a purity higher than 90%. Growth curve experiments using the protease-deficient Pichia pastoris strain demonstrated that this strain effectively promotes cell growth rate. Shake-flask experiments showed that, compared to the control group, this protease-deficient Pichia pastoris strain significantly enhanced cell growth rate. Therefore, the protease-deficient Pichia pastoris strain obtained by this invention can, to some extent, mitigate protein degradation during high-density fermentation, while also reducing fermentation time and costs, thereby lowering production costs, facilitating downstream extraction, and providing high-purity protein.

[0067] The carbazin expressed in this invention is ideally suited for use as a natural, high-intensity, calorie-free sweetener. Furthermore, studies have shown that carbazin can block the bitterness of other natural and artificial sweeteners. When used in combination with other sweeteners, it can significantly improve the taste, strength, and duration of those sweeteners. Therefore, carbazin has broad potential applications and significant implications in many food and beverage applications, not only as a standalone sweetener but also as a taste modifier in combination with other sweeteners and flavorings.

[0068] The salt-reducing protein expressed in this invention is a salty peptide derived from food itself. It is a type of salty oligopeptide among flavor-enhancing polypeptides. Adding an appropriate amount of salty peptide can reduce the salt content from 0.9% to 0.3% while maintaining palatability. Salty peptides are a healthy and safe salt-enhancing product with umami flavor; they are composed of amino acids and are harmless to the human body. The salt-reducing protein of this invention can be mass-produced through microbial fermentation. As a natural salt-enhancing agent with certain saltiness and umami-enhancing effects, salty peptides respond to the WHO's recommendations on low-sodium salt reduction while also meeting consumers' demand for natural products. Therefore, the salt-reducing protein developed in this invention is of great significance in responding to the global demand for low-sodium salt reduction. Attached Figure Description

[0069] Figure 1 shows the growth curves of each gene knockout strain.

[0070] Figure 2 shows the protein expression 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 time points. In this diagram, the M protein molecular weight marker (3.3kD-31.0kD) is defined as follows: lane 1 represents the sample before induction; lane 2 represents the sample after 6 hours of induction; lane 3 represents the sample after 24 hours of induction; lane 4 represents the sample after 48 hours of induction; lane 5 represents the sample after 72 hours of induction; lane 6 represents the sample after 96 hours of induction; lane 2 represents the sample after 6 hours of induction; lane 7 represents the Brazilian sweet protein standard; lane 8 represents the sample before induction; lane 9 represents the sample after 6 hours of induction; lane 10 represents the sample after 24 hours of induction; lane 11 represents the sample after 48 hours of induction; lane 12 represents the sample after 72 hours of induction; and lane 13 represents the sample after 96 hours of induction.

[0071] Figure 3 shows the protein expression 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 time points. In this diagram, the M protein molecular weight marker (3.3kD-31.0kD) is defined as follows: 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; and lane 13 represents the sample after 96 hours of induction.

[0072] Figure 4 shows the HPLC peak time and area of ​​the Brazilian sweet protein standard and MF001-29-Bra4-KRx3 fermented for 72 hours.

[0073] Figure 5 shows the 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.

[0074] Figure 6 shows 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.

[0075] Figure 7 shows the growth curve and biomass curve of MF001-94-PF.

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

[0077] Figure 9 shows the growth curve of MF001-94-P29-SUMO.

[0078] Figure 10 shows the SDS-PAGE electrophoresis results of the supernatant protein of MF001-94-P29-SUMO. In the figure, 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; and lane 7 represents the sample 70 hours after MF001-94-PF fermentation induction. Detailed Implementation

[0079] The following disclosure provides numerous different embodiments or examples for implementing various ways of carrying out the invention. To simplify the disclosure, specific embodiments or examples are described below. Of course, these are merely examples and are not intended to limit the invention. Furthermore, the examples of various specific processes and materials provided by the invention will allow those skilled in the art to recognize the applicability of other processes and / or the use of other materials. Unless otherwise stated, the implementation of the invention will employ conventional techniques in fields such as chemistry and molecular biology, which are within the capabilities of those skilled in the art. Additionally, unless otherwise stated, nucleic acids are written from left to right in a 5' to 3' orientation, and amino acid sequences are written from left to right in a direction from the amino terminus to the carboxyl terminus.

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

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

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

[0083] The PAS_chr3_1087 gene of the Peptidase A1 family has the nucleic acid sequence shown in SEQ ID No. 1 and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 2; the PAS_chr4_0584 gene has the nucleic acid sequence shown in SEQ ID No. 3 and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 4.

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

[0085] The PAS_chr4_0913 gene in the Peptidase M18 family has the nucleic acid sequence shown in SEQ ID No. 11 and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 12.

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

[0087] The PAS_chr1-4_0048 gene, belonging to the Peptidase S54 family, has the nucleic acid sequence shown in SEQ ID No. 19, and the amino acid sequence of its encoded protein is shown in SEQ ID No. 20; the PAS_chr3_0934 gene has the nucleic acid sequence shown in SEQ ID No. 21, and the amino acid sequence of its encoded protein is shown in SEQ ID No. 22.

[0088] The PAS_chr3_0633 gene of the Peptidase S10 family has the nucleic acid sequence shown in SEQ ID No. 23, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 24; the PRC1 PAS_chr1-4_0013 gene has the nucleic acid sequence shown in SEQ ID No. 25, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 26.

[0089] The PAS_chr1-1_0174 gene, belonging to the Peptidase S16 family, has the nucleic acid sequence shown in SEQ ID No. 27, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 28.

[0090] The PAS_chr3_0979 gene in the Peptidase S14 family has the nucleic acid sequence shown in SEQ ID No. 29 and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 30.

[0091] The PAS_chr1-1_0226 gene in the Peptidase S8 family has the nucleic acid sequence shown in SEQ ID No. 31 and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 32.

[0092] The protein defect-related gene used in the following embodiments 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.

[0093] The nucleic acid sequence of the Brazil sweet protein gene used in the following embodiments 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.

[0094] The nucleic acid sequence of the salt-reducing protein gene used in the following embodiments 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.

[0095] The nucleic acid sequence of the smegglutinin gene described in this invention is shown in SEQ ID No. 39, and the amino acid sequence of the protein is shown in SEQ ID No. 40.

[0096] Culture medium, reagents, and experimental methods:

[0097] LB liquid 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 bring the total volume to 100 mL. Autoclave at 115 °C for 20 min before use.

[0098] LBK resistance screening plates: Yeast extract 0.5g (0.5% w / v), tryptone 1g (1% w / v), NaCl 1g (1% w / v), agar powder 2g (2% w / v) were added to distilled water and brought to a total volume of 100mL. The plates were autoclaved at 115℃ for 20min. After the culture medium temperature cooled to approximately 50-55℃, kanamycin (25mg / mL) was added in a clean bench to a final antibiotic concentration of 100μg / mL. The plates were then shaken well and poured onto agar plates.

[0099] LBLZ resistance screening plates: Yeast extract 0.5g (0.5% w / v), NaCl 1g (1% w / v), tryptone 1g (1% w / v), agar powder 2g (2% w / v) were added to distilled water and brought to a total volume of 100mL. The plates were then autoclaved at 115℃ for 20min and poured onto plates. After the culture medium cooled to 50-55℃, bleomycin (25mg / mL) was added in a clean bench to a final antibiotic concentration of 25μg / mL. The plates were shaken well and then poured onto plates.

[0100] MD solid plate medium: 2g glucose (2% w / v), 1.34g YNB (amino acid-free) (1.34% w / v), 2g agar powder (2% w / v) are added to distilled water and the volume is adjusted to 100mL. The plates are then autoclaved at 115℃ for 20min and poured into plates.

[0101] YPD liquid medium: 1g (1% w / v) yeast extract, 2g (2% w / v) glucose, 2g (2% w / v) tryptone, add distilled water and bring to a total volume of 100mL, autoclave at 115℃ for 20min.

[0102] 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 bring the total volume to 100mL. Autoclave at 115℃ for 20min and pour into plates. Once the medium temperature has cooled to approximately 50-55℃, add bleomycin (25mg / mL) to a final antibiotic concentration of 100μg / mL in a clean bench, shake well, and then pour into plates.

[0103] 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 in a certain amount of distilled water and brought to a final volume. Sterilize at 115°C for 20 min.

[0104] BMMY liquid medium: 1% (w / v) yeast extract, 2% (w / v) tryptone, 1.34% (w / v) YNB, 10% (v / v) 1M pH6.0 phosphate buffer, dissolved in a certain amount of distilled water and brought to a final volume, autoclaved at 115°C for 20 min, and then methanol was added at a ratio of 1% (v / v).

[0105] 1M phosphate buffer (pH 6.0): Mix 868 mL of 1M KH2PO4 and 132 mL of 1M K2HPO4 thoroughly to adjust the pH to 6.0 with phosphate, sterilize at 121°C for 20 min, and then store at room temperature for later use.

[0106] The Pichia pastoris genome extraction was performed according to the instructions of the Yeast DNAiso Kit (Takara Code: D9082).

[0107] The DNA polymerase used in the PCR amplification reaction was Phanta Max high-fidelity DNA polymerase.

[0108] Recombinant plasmid linearization and electroporation transformation of yeast

[0109] 1. Linearization of recombinant vectors

[0110] The plasmid to be transformed was digested using an enzyme digestion linearization system. The linearized product was then recovered, and the concentration of the recovered linearized DNA product was measured and stored at -20°C for later use.

[0111] 2. Preparation of GS115 electrocompetent cells

[0112] 1) Pick a single colony of GS115 from the streak-activated YPD solid plate and inoculate it into 5 mL of YPD liquid medium. Incubate overnight at 30°C and 250 rpm.

[0113] 2) Inoculate 1 mL of bacterial culture into 100 mL of YPD liquid medium and incubate at 30°C and 250 rpm for 6 to 8 hours until OD (Organic Depth) is reached. 600 It ranges from 0.6 to 0.8;

[0114] 4) Transfer the bacterial culture to a 50mL sterile centrifuge tube, centrifuge at 4℃ and 5000rpm for 10min, discard the supernatant and collect the bacterial cells;

[0115] 5) Add sterile deionized water to 50 mL, wash the bacterial cells twice, centrifuge at 4℃ and 5000 rpm for 10 min, discard the supernatant, and collect the bacterial cells;

[0116] 6) Add 25 mL of LDST solution, let stand at room temperature for 30 min, centrifuge at 4℃ and 5000 rpm for 10 min, discard the supernatant and collect the bacterial cells;

[0117] 7) Add 50 mL of 1 M pre-cooled sorbitol to wash, gently mix, resuspend the bacterial cells, centrifuge at 4°C and 5000 rpm for 10 min, discard the supernatant, and collect the bacterial cells;

[0118] 8) Add 25 mL of 1 M pre-cooled sorbitol to wash, gently mix, resuspend the bacterial cells, centrifuge at 4°C and 5000 rpm for 10 min, discard the supernatant, and collect the bacterial cells;

[0119] 9) Then resuspend the cells in 0.5 mL of 1 M pre-chilled sorbitol and store at 4°C;

[0120] 10) Electrotransfer competent cells are best used immediately. If you want to freeze competent cells, aliquot 80 μL into sterile 1.5 mL centrifuge tubes and store at -80°C.

[0121] 3. Electrotransformation of linearized plasmids into competent Pichia pastoris cells

[0122] 1) Add approximately 0.5 to 1 μg of linearized DNA to the prepared yeast competent cells, gently mix the linearized DNA and yeast competent cells evenly, and transfer to an electroporation cup that has been preheated to an ice bath, and then place it on ice for 5 min;

[0123] 2) Set the voltage of the stun gun to 1.5Kv, the resistance to 200Ω, the capacitance to 25mF, and the stun mode to fungal mode, then stun the mixture.

[0124] 3) Add 1M ice-chilled sorbitol to the electroporation vessel, and transfer the mixed bacterial solution to a sterile 1.5mL EP tube using a pipette. Incubate at 30℃ for 1.5h, then centrifuge at 6000rpm for 1 to 2min, discarding a portion of the supernatant and leaving approximately 200μL of liquid. Resuspend the bacterial cells and spread them on the appropriate selection plates. MD solid plates are used to screen histidine auxotrophic strains, and YPDZ solid plates are used for bleomycin resistance screening. Incubate the plates upside down at 30℃ for 2 to 4 days.

[0125] Shake-flask fermentation culture of recombinant yeast engineered strains

[0126] Pick a solid agar plate, pick a single yeast colony with good growth, and inoculate it into a 50mL Erlenmeyer flask containing 5mL of BMGY medium. Incubate the flask at 30℃ and 250rpm for about 24 hours, and then measure the OD of the seed culture. 600 Value, calculate and extract its actual OD 600 Transfer 1 to 25 mL of BMMY medium, centrifuge at 6000 rpm for 2 min at room temperature, discard the supernatant and collect the bacterial cells, then wash the bacterial cells 2 to 3 times with 800 μL of BMMY liquid medium, and then transfer all the washed bacterial cells to a 250 mL Erlenmeyer flask containing 25 mL of BMMY medium, and incubate in a shaker at 30 °C and 250 rpm, adding 1% methanol every 24 h.

[0127] SDS-PAGE

[0128] Culture supernatant or purified samples were analyzed by SDS-PAGE using FuturePAGE gel. TM Protein precast gels were prepared at 4% to 20% (ACE). On the gel, 20 μl of sample was loaded (10 μl of each sample mixed with 10 μl of loading buffer), and 10 μl of marker was applied (pre-stained SDS-PAGE standard, GenScript Biotech, #M00624-250). Electrophoresis was performed at a constant voltage of 160 V for 80 min in the accompanying dedicated electrophoresis buffer: MOPS-SDS Running Buffer (catalog number: BR0001-02). Protein bands were stained with Bio-Safe Coomassie dye (Bio-Rad Laboratories).

[0129] HPLC analysis of brassinolide

[0130] HPLC was performed as described: using a Thermo Scientific Vanquish HPLC system equipped with... 5um C18(2) A 250 × 4.6 mm column was used. Stationary phase buffer A (water + 0.1% TFA) and mobile phase buffer B (ACN + 0.1% TFA) were employed. The absorbance at 220 nm was monitored using a UV detector. The column temperature was set to 37 °C, and the injection volume was 10 μL. The 15-minute gradient program settings are shown in Table 1.

[0131] Table 1

[0132] The content of brassinolide was analyzed by calculating the content of brassinolide during shake-flask fermentation based on standard samples.

[0133] Knockout of protease genes in Pichia pastoris

[0134] Example 1

[0135] Also, knock out PAS_chr3_0934, PAS_chr4_0913, and PAS_chr1-4_0048.

[0136] Construction of plasmids containing CrRNA fragments

[0137] Two PAM sites were selected from the PAS_chr3_0934 gene, and two CrRNA fragments corresponding to the selected PAM sites were identified. One CrRNA fragment was ligated into 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, whose CrRNA fragment base sequence is shown in SEQ ID No. 41; the other CrRNA fragment was ligated into the pCrRNA plasmid to obtain pCrRNA-0934-2, whose CrRNA fragment base sequence is shown in SEQ ID No. 42.

[0138] Similarly, two PAM sites were selected from the PAS_chr4_0913 gene, and two CrRNA fragments corresponding to the selected PAM sites were identified. The base sequence of one CrRNA fragment is shown in SEQ ID No. 43, and it was ligated into the pCrRNA plasmid to obtain pCrRNA-0913-1; the base sequence of the other CrRNA fragment is shown in SEQ ID No. 44, and it was ligated into the pCrRNA plasmid to obtain pCrRNA-0913-2.

[0139] Similarly, two PAM sites were selected from the PAS_chr1-4_0048 gene, and two CrRNA fragments corresponding to the selected PAM sites were identified. The base sequence of one CrRNA fragment is shown in SEQ ID No. 45, and it was ligated into the pCrRNA plasmid to obtain pCrRNA-0048-1; the base sequence of the other CrRNA fragment is shown in SEQ ID No. 46, and it was ligated into the pCrRNA plasmid to obtain pCrRNA-0048-2.

[0140] Preparation of PAS_chr3_0934 gene donor DNA

[0141] Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0934-LF (as shown in SEQ ID No. 47) and 0934-D-RF (as shown in SEQ ID No. 48) to obtain the 0934 left-wing PCR product. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0934-D-LF (as shown in SEQ ID No. 49) and 0934-RF (as shown in SEQ ID No. 50) to obtain the 0934 right-wing PCR product. Using a mixture of the 0934 left-wing and 0934 right-wing PCR products as a template, PCR amplification was performed using primer pairs 0934-LF and 0934-RF to obtain the 0934 donor DNA fragment.

[0142] Preparation of PAS_chr4_0913 gene donor DNA

[0143] Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0913-LF (as shown in SEQ ID No. 51) and 0913-D-RF (as shown in SEQ ID No. 52) to obtain the 0913 left-wing PCR product. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0913-D-LF (as shown in SEQ ID No. 53) and 0913-RF (as shown in SEQ ID No. 54) to obtain the 0913 right-wing PCR product. Using a mixture of the 0913 left-wing and 0913 right-wing PCR products as a template, PCR amplification was performed using primer pairs 0913-LF and 0913-RF to obtain the 0913 donor DNA fragment.

[0144] Preparation of PAS_chr1-4_0048 gene donor DNA

[0145] Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0048-LF (as shown in SEQ ID No. 55) and 0048-D-RF (as shown in SEQ ID No. 56) to obtain the 0048 left-wing PCR product. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0048-D-LF (as shown in SEQ ID No. 57) and 0048-RF (as shown in SEQ ID No. 58) to obtain the 0048 right-wing PCR product. Using a mixture of the 0048 left-wing and 0048 right-wing PCR products as a template, PCR amplification was performed using primer pairs 0048-LF and 0048-RF to obtain the 0048 donor DNA fragment.

[0146] Gene knockout

[0147] First, the CRISPR CAS12 gene editing helper 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.

[0148] The linearized plasmid pGAP-Cpf1 was electroporated into Pichia pastoris GS115 competent cells, and the positive strain GS115-Cpf1 was obtained after culture and identification.

[0149] 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. The cells were cultured on YPDS plates containing 100 μg / mL bleomycin, and then identified by PCR and sequencing as cells with simultaneous knockout of three genes. Positive transformants were further cultured on antibiotic-free YPD agar plates until single colonies grew. Single colonies were picked from the antibiotic-free YPD agar plates and cultured again on bleomycin-treated YPD agar plates. Colonies that could not grow on the bleomycin-treated plates were the strains with plasmids eliminated. These strains were then verified by colony PCR and sequencing. Finally, a defective strain with simultaneous knockout of the PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 genes was obtained, numbered MF001-29.

[0150] Example 2

[0151] Simultaneously, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013 are knocked out.

[0152] The base sequence of one CrRNA fragment from the PAS_chr2-1_0652 gene that was knocked out is shown in SEQ ID No. 59. It was ligated into the pCrRNA plasmid to obtain pCrRNA-0652-1; the base sequence of the other CrRNA fragment is shown in SEQ ID No. 60. It was ligated into the pCrRNA plasmid to obtain pCrRNA-0652-2.

[0153] The base sequence of one CrRNA fragment from the PAS_chr3_0633 gene that was knocked out is shown in SEQ ID No. 61. It was ligated into the pCrRNA plasmid to obtain pCrRNA-0633-1; the base sequence of the other CrRNA fragment is shown in SEQ ID No. 62. It was ligated into the pCrRNA plasmid to obtain pCrRNA-0633-2.

[0154] The base sequence of one CrRNA fragment from the PRC1 PAS_chr1-4_0013 gene that was knocked out is shown in SEQ ID No. 63. It was ligated into the pCrRNA plasmid to obtain pCrRNA-0013-1; the base sequence of the other CrRNA fragment is shown in SEQ ID No. 64. It was ligated into the pCrRNA plasmid to obtain pCrRNA-0013-2.

[0155] Preparation of PAS_chr2-1_0652 gene donor DNA

[0156] Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0652-LF (as shown in SEQ ID No. 65) and 0652-D-RF (as shown in SEQ ID No. 66) to obtain the 0652 left-wing PCR product. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0652-D-LF (as shown in SEQ ID No. 67) and 0652-RF (as shown in SEQ ID No. 68) to obtain the 0652 right-wing PCR product. Using a mixture of the 0652 left-wing and 0652 right-wing PCR products as a template, PCR amplification was performed using primer pairs 0652-LF and 0652-RF to obtain the 0652 donor DNA fragment.

[0157] Preparation of PAS_chr3_0633 gene donor DNA

[0158] Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0633-LF (as shown in SEQ ID No. 69) and 0633-D-RF (as shown in SEQ ID No. 70) to obtain the 0633 left-wing PCR product. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0633-D-LF (as shown in SEQ ID No. 71) and 0633-RF (as shown in SEQ ID No. 72) to obtain the 0633 right-wing PCR product. Using a mixture of the 0633 left-wing and 0633 right-wing PCR products as a template, PCR amplification was performed using primer pairs 0633-LF and 0633-RF to obtain the 0633 donor DNA fragment.

[0159] Preparation of PRC1 PAS_chr1-4_0013 gene donor DNA

[0160] Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0013-LF (as shown in SEQ ID No. 73) and 0013-D-RF (as shown in SEQ ID No. 74) to obtain the 0013 left-wing PCR product. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs 0013-D-LF (as shown in SEQ ID No. 75) and 0013-RF (as shown in SEQ ID No. 76) to obtain the 0013 right-wing PCR product. Using a mixture of the 0013 left-wing and 0013 right-wing PCR products as a template, PCR amplification was performed using primer pairs 0013-LF and 0013-RF to obtain the 0013 donor DNA fragment.

[0161] Linearized plasmid pGAP-Cpf1 was electroporated into Pichia pastoris MF001-29 competent cells, and positive strain MF001-29-Cpf1 was obtained after culture and identification. Further, 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 co-transformed into Pichia pastoris MF001-29-Cpf1 competent cells. Positive transformants were obtained by culturing on bleomycin-resistant plates, followed by culture on antibiotic-free plates to eliminate the plasmids, ultimately yielding a gene-deficient strain with simultaneous knockout of 6 genes, designated MF001-94.

[0162] Example 3

[0163] 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.

[0164] The base sequence of one CrRNA fragment from the PAS_chr3_0979 gene knocked out 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 (as shown in SEQ ID No. 79), 0979-D-RF (as shown in SEQ ID No. 80), 0979-D-LF (as shown in SEQ ID No. 81), and 0979-RF (as shown in SEQ ID No. 82).

[0165] The base sequence of one CrRNA fragment from the PAS_chr4_0113 gene knocked out 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 (as shown in SEQ ID No. 85), 0113-D-RF (as shown in SEQ ID No. 86), 0113-D-LF (as shown in SEQ ID No. 87), and 0113-RF (as shown in SEQ ID No. 88).

[0166] The base sequence of one CrRNA fragment from the PAS_chr1-1_0174 gene knocked out 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 (as shown in SEQ ID No. 91), 0174-D-RF (as shown in SEQ ID No. 92), 0174-D-LF (as shown in SEQ ID No. 93), and 0174-RF (as shown in SEQ ID No. 94).

[0167] The base sequence of one CrRNA fragment from the PAS_chr3_0953 gene knocked out 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 (as shown in SEQ ID No. 97), 0953-D-RF (as shown in SEQ ID No. 98), 0953-D-LF (as shown in SEQ ID No. 99), and 0953-RF (as shown in SEQ ID No. 100).

[0168] The base sequence of one CrRNA fragment from the PAS_chr2-2_0380 gene knocked out 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 (as shown in SEQ ID No. 103), 0380-D-RF (as shown in SEQ ID No. 104), 0380-D-LF (as shown in SEQ ID No. 105), and 0380-RF (as shown in SEQ ID No. 106).

[0169] 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 group was knocked out using MF001-94 as the starting strain. Then, the gene knockout strain obtained was used as the starting strain to knock out the other group. Finally, a gene-deficient strain with 11 genes knocked out was obtained, which was numbered MF001-136.

[0170] Everything else is the same as in Example 1.

[0171] Example 4

[0172] 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.

[0173] The construction of plasmids containing CrRNA fragments of the genes 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 and the preparation of donor DNA were the same as in Example 3.

[0174] The base sequence of one CrRNA fragment from the PAS_chr1-4_0611 gene knocked out 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 (as shown in SEQ ID No. 109), 0611-D-RF (as shown in SEQ ID No. 110), 0611-D-LF (as shown in SEQ ID No. 111), and 0611-RF (as shown in SEQ ID No. 112).

[0175] Starting with strain MF001-136, the PAS_chr1-4_0611 gene was knocked out, and a gene-deficient strain with 12 genes knocked out was finally obtained, which was numbered MF001-143.

[0176] Everything else is the same as in Example 1.

[0177] Example 5

[0178] 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.

[0179] The construction of the plasmid containing the CrRNA fragment of the PAS_chr1-4_0611 gene and the preparation of the donor DNA were the same as in Example 4.

[0180] The base sequence of one CrRNA fragment from the PAS_chr1-1_0194 gene knocked out 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 (as shown in SEQ ID No. 115), 0194-D-RF (as shown in SEQ ID No. 116), 0194-D-LF (as shown in SEQ ID No. 117), and 0194-RF (as shown in SEQ ID No. 118).

[0181] Starting with strain MF001-136, the PAS_chr1-4_0611 and PAS_chr1-1_0194 genes were simultaneously knocked out, resulting in a gene-deficient strain with 13 genes knocked out simultaneously, which was designated as MF001-249.

[0182] Everything else is the same as in Example 1.

[0183] Example 6

[0184] 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.

[0185] The base sequence of one CrRNA fragment from the PAS_chr4_0584 gene knocked out 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 (as shown in SEQ ID No. 121), 0584-D-RF (as shown in SEQ ID No. 122), 0584-D-LF (as shown in SEQ ID No. 123), and 0584-RF (as shown in SEQ ID No. 124).

[0186] The base sequence of one CrRNA fragment from the PAS_chr3_1087 gene knocked out 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 (as shown in SEQ ID No. 127), 1087-D-RF (as shown in SEQ ID No. 128), 1087-D-LF (as shown in SEQ ID No. 129), and 1087-RF (as shown in SEQ ID No. 130).

[0187] Starting with strain MF001-249, the PAS_chr4_0584 and PAS_chr3_1087 genes were simultaneously knocked out, ultimately resulting in a gene-deficient strain with 15 genes knocked out simultaneously, which was designated as MF001-251.

[0188] Everything else is the same as in Example 1.

[0189] Example 7

[0190] 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.

[0191] The plasmid construction of the CrRNA fragments of the PAS_chr4_0584 and PAS_chr3_1087 genes and the preparation of donor DNA are the same as in Example 6.

[0192] The base sequence of one CrRNA from which the PAS_chr1-1_0226 gene was knocked out is shown in SEQ ID No. 131, and the base sequence of the other CrRNA is shown in SEQ ID No. 132; (see SEQ ID No. 136 for the preparation of its donor DNA).

[0193] Starting with strain MF001-249, the PAS_chr4_0584, PAS_chr3_1087, and PAS_chr1-1_0226 genes were simultaneously knocked out, resulting in a gene-deficient strain with 16 genes knocked out simultaneously. The strain was numbered MF001-256.

[0194] Everything else is the same as in Example 1.

[0195] Example 8

[0196] Knock out PAS_chr3_0689.

[0197] The base sequence of one CrRNA fragment from the PAS_chr3_0689 gene knocked out 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 (as shown in SEQ ID No. 139), 0689-D-RF (as shown in SEQ ID No. 140), 0689-D-LF (as shown in SEQ ID No. 141), and 0689-RF (as shown in SEQ ID No. 142).

[0198] Everything else is the same as in Example 1.

[0199] The final genetically defective strain was numbered MF001-169.

[0200] Growth rate determination of gene knockout strains

[0201] A growth curve is the population growth pattern exhibited by single-celled microorganisms under specific environmental conditions during liquid culture. In measurement, a certain number of pure microbial cultures are inoculated into a specific volume of sterilized, suitable, fresh culture medium, cultured at a suitable temperature, and samples are taken periodically to determine the number of bacteria in the culture medium, expressed as the OD (Organic Deposition Rate) of the bacterial culture. 600 (OD is the optical density of a bacterial suspension measured using a photoelectric colorimeter, based on the relationship between the number of bacterial cells in the culture medium and turbidity, and inversely proportional to transmittance.) A growth curve is plotted with the growth time on the x-axis. The growth curve is generally divided into four phases: the lag phase, the logarithmic phase, the stationary phase, and the death phase. Measuring the growth curve of microorganisms under specific culture conditions has certain guiding significance for actual production.

[0202] MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169 were activated and inoculated into 50 mL of YPD liquid culture medium at a volume ratio of 1%. The starting strain GS115 was used as control-1, and SMD1163 was used as control-2. OD values ​​were measured at 0, 6, 9, 24, 30, 48, and 54 h. 600 OD of each strain 600 The results are shown in Table 2; then, the sampling time was plotted on the x-axis, and the OD values ​​of each strain were plotted on the y-axis. 600 The values ​​are plotted on the ordinate to create growth curves, as shown in Figure 1, for comparison of growth rates.

[0203] Table 2

[0204] The data analysis above shows that the growth rates of strains MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169 are all higher than or have no impact on the growth rate of their originating strain GS115.

[0205] Construction of expression plasmids for different copy numbers of brassinoprotein

[0206] Example 9

[0207] In yeast, including *Pseudomonas pastoris*, α-mating factors are expressed as tandem repeats. These α-mating factors are separated by amino acid sequences of the order KR(EA)x. In *Pichia pastoris*, this gene has 10 repeats of the mature α-mating factor, with spacer sequences ranging from 1 to 9 EA repeats. By mimicking the tandem repeat expression of the yeast α-mating factor as a peptide sweetener, the production of Brazilin by *P. pastoris* can be increased.

[0208] The brassin in this invention is synthesized according to the codon preference of Pichia pastoris and cloned into the plasmid PHKA vector. The elements are tandemly linked to the PHKA vector in the following order:

[0209] 1) For the PHKA-Bra2-KR plasmid: α factor secretion signal, brassinoprotein gene, KR spacer sequence and brassinoprotein gene are tandem.

[0210] 2) For the PHKA-Bra2-KRx plasmid: the α factor secretion signal, the brassin gene, the spacer sequence 1 (as shown in SEQ ID No. 143) and the brassin gene are tandemly linked.

[0211] 3) For the PHKA-Bra2-KRx2 plasmid: the α factor secretion signal, the brassin gene, spacer sequence 2 (as shown in SEQ ID No. 144) and the brassin gene are tandemly linked.

[0212] 4) For the PHKA-Bra2-KRx3 plasmid: the α factor secretion signal, the brassin gene, spacer sequence 3 (as shown in SEQ ID No. 145) and the brassin gene are tandemly linked.

[0213] 5) For the PHKA-Bra2-KRx4 plasmid: the α factor secretion signal, the brassin gene, spacer sequence 4 (as shown in SEQ ID No. 146) and the brassin gene are tandemly linked.

[0214] 6) For the PHKA-Bra2-KRx5 plasmid: α factor secretion signal, brassinoprotein gene, spacer sequence 5 (as shown in SEQ ID No. 147) and brassinoprotein gene are tandemly linked.

[0215] 7) For the PHKA-Bra4-KR plasmid: tandem the α factor secretion signal, brassinoprotein gene, KR spacer sequence, brassinoprotein gene, KR spacer sequence, brassinoprotein gene, KR spacer sequence, and brassinoprotein gene.

[0216] 8) For the PHKA-Bra4-KRx plasmid: α factor secretion signal, Brazil glycoprotein gene, spacer sequence 1 (as shown in SEQ ID No. 143), Brazil glycoprotein gene, spacer sequence 1 (as shown in SEQ ID No. 143), Brazil glycoprotein gene, spacer sequence 1 (as shown in SEQ ID No. 143), Brazil glycoprotein gene tandem.

[0217] 9) For the PHKA-Bra4-KRx2 plasmid: α factor secretion signal, Brazil glycoprotein gene, spacer sequence 2 (as shown in SEQ ID No. 144), Brazil glycoprotein gene, spacer sequence 2 (as shown in SEQ ID No. 144), Brazil glycoprotein gene, spacer sequence 2 (as shown in SEQ ID No. 144), Brazil glycoprotein gene tandem.

[0218] 10) For the PHKA-Bra4-KRx3 plasmid: α factor secretion signal, Brazil glycoprotein gene, spacer sequence 3 (as shown in SEQ ID No. 145), Brazil glycoprotein gene, spacer sequence 3 (as shown in SEQ ID No. 145), Brazil glycoprotein gene, spacer sequence 3 (as shown in SEQ ID No. 145), Brazil glycoprotein gene tandem.

[0219] 11) For the PHKA-Bra4-KRx4 plasmid: α factor secretion signal, Brazil glycoprotein gene, spacer sequence 4 (as shown in SEQ ID No. 146), Brazil glycoprotein gene, spacer sequence 4 (as shown in SEQ ID No. 146), Brazil glycoprotein gene, spacer sequence 4 (as shown in SEQ ID No. 146), Brazil glycoprotein gene tandem.

[0220] 12) For the PHKA-Bra4-KRx5 plasmid: α factor secretion signal, brassinoprotein gene, spacer sequence 5 (as shown in SEQ ID No. 147), brassinoprotein gene, spacer sequence 5 (as shown in SEQ ID No. 147), brassinoprotein gene, spacer sequence 5 (as shown in SEQ ID No. 147), and brassinoprotein gene are tandem.

[0221] Expression of Brazilin in Pichia pastoris with missing protease gene

[0222] Example 10

[0223] 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 transformed into the genodeficient 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.

[0224] Example 11

[0225] 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 transformed into the genotypic 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.

[0226] Example 12

[0227] 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 transformed into the genodeficient 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.

[0228] Example 13

[0229] 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 transformed into the genotype-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.

[0230] Example 14

[0231] 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 transformed into the genodeficient 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.

[0232] Example 15

[0233] 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 transformed into the genodeficient 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.

[0234] Example 16

[0235] 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 transformed into the genodeficient 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.

[0236] Example 17

[0237] 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 transformed into the genodeficient 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.

[0238] Comparative Example 1

[0239] 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 transformed into the genotypic strain GS115, and then eliminated. 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.

[0240] Comparative Example 2

[0241] Plasmids PHKA-Bra2-KRx3 and PHKA-Bra4-KRx3 were transformed 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.

[0242] Determination of Brazil sweet protein expression yield

[0243] Single clones of the strain were selected for shake-flask fermentation, inoculated into 250 mL partitioned shake flasks containing 100 mL of BMGY (Buffered Glycerol-complex Medium). The mixture was grown at 30°C and 250-300 rpm on a shaker until the OD reached [value missing]. 600 =2 to 6 (approximately 16 to 18 hours) Centrifuge at 1500 to 3000g for 5 minutes at room temperature to collect cells, remove the supernatant, and resuspend the cells (approximately 10-20 ml) in 1 / 5 to 1 / 10 of the original culture medium volume of BMMY (Buffered Minimal Methanol YP Medium). Place the cells in a 100 ml septum shake flask, cover the flask opening with two layers of sterile gauze or cheesecloth, and place it on a shaker to continue incubation. Every 24 hours, add methanol to a final concentration of 1% to continue induction. At 6H, 12H, 24H, 48H, 72H, and 96H, transfer 1 ml of culture medium to 1 to 5 ml centrifuge tubes for OD analysis at each time point. 600The expression of protein in the fermentation broth supernatant was detected by SDS-PAGE. After confirming the expression of carbapenem, the carbapenem was purified by ion-exchange chromatography-gel chromatography. The purified protein was then lyophilized under low temperature vacuum to obtain lyophilized powder. The purity of the lyophilized powder was confirmed to be above 90% using carbapenem standard (purchased from Nanjing Wobo Biotechnology Co., Ltd., catalog number: Wb2641-1mg). The SDS-PAGE results of proteins expressed by some strains at various time points are shown in Figures 2 and 3. The results show that some strains, such as the protease-deficient strain MF001-136-Bra4-KRx3, showed a 114% increase in carbapenem protein yield at 72 hours compared to the control strain GS115-Bra4-KRx3.

[0244] HPLC was used to quantitatively analyze the carbapenem standard and the lyophilized powder of carbapenem expressed by various expression strains. By comparing with the carbapenem standard, the yield of carbapenem expressed by each expression strain was detected. It was found that the peak time of the standard at a concentration of 1 g / L was 6.650 min and the peak area was 19.3746. The sample of MF001-29-Bra4-KRx3 fermented in shake flask for 72 hours was first centrifuged, treated with a 0.22 μm membrane, filtered, and then analyzed. The peak time of the sample was 6.643 min and the peak area was 18.1872. After conversion, the yield of MF001-29-Bra4-KRx3 fermented for 72 hours reached 0.939 g / L. The HPLC peak time and area are shown in 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 for x3, 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.

[0245] The above data show that the gene knockout strain of the present invention can significantly increase the expression level of exogenous proteins.

[0246] Table 3

[0247] Growth rate determination of various strains expressing brassinolide

[0248] The strains from Examples 10 to 17, as well as Comparative Examples 1 and 2, were activated and inoculated into 50 mL of YPD liquid culture medium at a volume ratio of 1%. OD values ​​were measured at 0, 6, 9, 24 h, 30 h, 48 h, and 54 h. 600 Then, with sampling time as the x-axis, the OD values ​​of each strain were plotted. 600 The values ​​are plotted on the ordinate as growth curves. The growth curves for 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. 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. The results in Figures 5 and 6 indicate that the insertion of the linearized plasmids PHKA-Bra2-KRx3 and PHKA-Bra4-KRx3 did not affect the growth of the strains.

[0249] Construction of salt-reduced protein expression plasmid

[0250] Example 18

[0251] For the PHKA-PF plasmid: the α-factor secretion signal and the salt-reducing protein gene were tandemly linked 16 times. The linearized plasmid PHKA-PF was then transformed into each gene-deficient strain, and the free plasmid was eliminated to obtain gene-deficient strains capable of expressing the salt-reducing protein. Among them, the expression strain after transformation into MF001-94 was MF001-94-PF.

[0252] OD values ​​of each expression strain were determined at various time points using high-density fermentation. 600 Biomass and the production of salt-reduced protein.

[0253] The following example of high-density fermentation of recombinant yeast engineered salt-reducing protein strain MF001-94-PF illustrates that the gene-deficient strains of this invention can produce higher yields of salt-reducing protein compared to their original strain GS115.

[0254] 1) Preparation of fermentation seed liquid

[0255] Select single colonies of MF001-94-PF strain, streak them on YPD plates to activate and culture them, and inoculate them into 50 mL of sterile YPD liquid medium. Incubate overnight at 30°C and 250 rpm to activate the strain. Transfer the YPD-activated yeast seed culture at a ratio of 4% to 100 mL of YPD liquid medium in a 500 mL Erlenmeyer flask and incubate overnight at 30°C and 250 rpm to obtain the seed culture for fermentation in a 5L fermenter.

[0256] 2) High-density culture of recombinant yeast strains in a 5L fermenter

[0257] The cultured yeast seed culture was inoculated at an 8% inoculation rate into a 5L fully automated mechanically stirred and aerated fermenter (sterilized). Glycerol supplementation was initiated for cell growth (initial culture medium volume 2L, sterilized at 121℃ for 30 min). During cell growth: pH was maintained at 5.5 using 25% concentrated ammonia, dissolved oxygen was maintained between 30% and 60%, temperature was controlled at 30℃, stirring speed was set to an upper limit of 200 rpm and a lower limit of 120 rpm, and aeration rate was set to 2 vvm. Initial cell density OD... 600 Once the dissolved oxygen (DO) reaches 400, stop the glycerol feeding. When the DO rebounds to above 80%, begin methanol-feed induction fermentation. During the methanol feeding stage: temperature is controlled at 30℃, pH is maintained at 5.5 using 25% concentrated ammonia, rotation speed is controlled at 800 rpm, aeration rate is controlled at 4 L / min, pressure is controlled at 0.05 MPa, aeration rate is adjusted to 2 vvm, and a variable-speed feed method is used to maintain dissolved oxygen between 15% and 25%. The BLBIO B-type control system software is used for automatic process control and data acquisition during fermentation.

[0258] The relationship between the collection time of MF001-94-PF desalinated protein and OD and WCW (biomass) is shown in Figure 7.

[0259] Samples were taken from the fermenter at 22, 46, 70, and 94 hours after methanol fermentation induction, centrifuged, and the supernatant was subjected to SDS-PAGE electrophoresis. The results are shown in Figure 8. From the fermentation electrophoresis of the MF001-94-PF desalting protein strain in Figure 8, it can be seen that the desalting protein gradually accumulates with fermentation time. The SDS gel is a FuturePAGE gel. 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) and apply 10 μl of marker label: (pre-stained SDS-PAGE standard, GenScript Biotech, #M00624-250).

[0260] The product fermented for 117 hours was then centrifuged, and the supernatant was purified using oligofructose gel. HPLC analysis revealed that the yield of the reduced-salt protein reached 12 g / L.

[0261] Construction of smegglutinin expression plasmid

[0262] Example 19

[0263] For the PHKA-P29-SUMO plasmid: the α factor secretion signal and smegglutinin gene were tandemly linked twice, and the linearized plasmid PHKA-P29-SUMO was transformed into the gene-deficient strain MF001-94. The free plasmid was then eliminated to obtain MF001-94-P29-SUMO.

[0264] Single-clone strain MF001-94-P29-SUMO was selected and cultured in shake flasks, inoculated into 250mL septum shake flasks containing 100mL of BMGY. The cells were grown at 30°C and 250-300rpm on a shaker until OD600 = 2-6 (approximately 16-18 hours). The cells were then collected by centrifugation at 1500-3000g for 5 minutes at room temperature. The supernatant was removed, and the cells were resuspended in 1 / 5-1 / 10 of the original culture medium volume of BMGY (approximately 10-20mL). The cells were placed in a 100mL septum shake flask, and the flask opening was covered with two layers of sterile gauze or cheesecloth. The flask was then placed in a shaker for further culture. Every 24 hours, methanol was added to a final concentration of 1% to continue induction. 1mL of culture medium was periodically transferred to 1-5mL centrifuge tubes for analysis of OD at various time points. 600 The expression of proteins in the fermentation broth supernatant was detected by SDS-PAGE. The growth curve is shown in Figure 9, and the SDS-PAGE electrophoresis results of the supernatant proteins are shown in Figure 10.

[0265] Taking the shake-flask fermentation of the recombinant yeast engineered smegglutinin MF001-94-P29-SUMO strain as an example, this invention illustrates that the various gene-deficient strains can produce higher yields of smegglutinin compared to their original 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 The application of at least two combinations of proteins from r3_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 protein alone, in influencing yeast growth rate and / or the expression level of exogenous proteins; wherein, The amino acid sequences of the PAS_chr3_1087 protein are shown in SEQ ID No. 2, the PAS_chr4_0584 protein is shown in SEQ ID No. 4, the PAS_chr2-1_0652 protein is shown in SEQ ID No. 6, the PAS_chr4_0113 protein is shown in SEQ ID No. 8, the PAS_chr2-2_0380 protein is shown in SEQ ID No. 10, the PAS_chr4_0913 protein is shown in SEQ ID No. 12, the PAS_chr1-4_0611 protein is shown in SEQ ID No. 14, the PAS_chr3_0953 protein is shown in SEQ ID No. 16, and the PAS_chr1-1_0194 protein is shown in SEQ ID No.

16. As shown in 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 application 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, PAS_chr4_0913, PA S_chr1-4_0611, PAS_chr3_0953, PAS_chr1-1_0194, PAS_chr1-4_0048, PAS_chr3_0934, PAS_chr3_0633, PRC1 Compared to before the expression of at least two of 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 was silenced, this represents a reduction in the expression of PAS_chr3_1087, PAS_chr4_0584, PAS_chr2-1_0652, PAS_chr4_0113, PAS_chr2-2_0380, PAS_chr4_0913, and PAS_chr1-4_0013, respectively. Silencing the expression of at least two proteins selected from 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 PAS_chr3_0689 alone, to increase the growth rate of the yeast and / or the expression level of the exogenous protein in the yeast.

3. The application according to claim 2, characterized in that, The nucleotide sequences of the PAS_chr3_1087 gene are shown in SEQ ID No. 1, the PAS_chr4_0584 gene in SEQ ID No. 3, the PAS_chr2-1_0652 gene in SEQ ID No. 5, the PAS_chr4_0113 gene in SEQ ID No. 7, the PAS_chr2-2_0380 gene in SEQ ID No. 9, the PAS_chr4_0913 gene in SEQ ID No. 11, the PAS_chr1-4_0611 gene in SEQ ID No. 13, the PAS_chr3_0953 gene in SEQ ID No. 15, and the PAS_chr1-1_0194 gene in SEQ ID No.

15. As shown in 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 application 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, and PAS_chr1 from the yeast cells... -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 application according to claim 4, characterized in that, Simultaneously knock out the PAS_chr3_0934, PAS_chr4_0913, and PAS_chr1-4_0048 genes in the yeast; or Simultaneously knock 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 in the yeast; or Simultaneously knock 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 in the yeast; or Simultaneously knock 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 in the yeast; or Simultaneously knock out the following genes in the yeast: 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; or Simultaneously knock out the following genes in the yeast: 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; or Simultaneously, the following genes in the yeast were knocked 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, and PAS_chr3_1087.

6. The application according to claim 1, characterized in that, The exogenous protein is at least one of brassinolide, reduced-salt protein, smegglutinin, casein, and whey protein; Preferably, the amino acid sequence of the Brazilian sweet protein is shown in SEQ ID No. 36; Preferably, the yeast is introduced with a gene encoding the brassinolide to enable the yeast to express the brassinolide; preferably, the nucleotide sequence of the gene encoding the brassinolide is shown in SEQ ID No.

35. Preferably, the amino acid sequence of the salt-reducing protein is shown in SEQ ID No. 38; Preferably, the yeast is introduced with a gene encoding the salt-reducing protein to enable the yeast to express the salt-reducing protein. Preferably, the nucleotide sequence of the gene encoding the salt-reducing protein is shown in SEQ ID No.

37. Preferably, the amino acid sequence of the smegglutinin is shown in SEQ ID No. 40; Preferably, the yeast is introduced with a gene encoding the smegglutinin to enable the yeast to express the smegglutinin. Preferably, the nucleotide sequence of the gene encoding the smegglutinin is as shown in SEQ ID No.

39.

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

8. An engineered yeast strain obtained by knocking out the gene in the yeast strain used in any one of claims 1 to 6.

9. The engineered yeast strain according to claim 8, characterized in that, The engineered yeast strain is a yeast strain obtained by knocking out the PAS_chr3_0934, PAS_chr4_0913, and PAS_chr1-4_0048 genes from the Pichia pastoris strain GS115; or The engineered yeast strain is a yeast strain obtained by 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 of Pichia pastoris GS115 as the starting strain; or The engineered yeast strain is a yeast strain obtained by knocking out the following genes: 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, from the *Komagataella pastoris* GS115 strain; or The engineered yeast strain is a yeast strain obtained by knocking out the following genes: 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, using *P. spp.* as the starting strain; or The engineered yeast strain was based on Komagataella pastoris GS115, with PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, and PRC1 knocked out. Engineered yeast strains derived from the 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 engineered yeast strain is a yeast strain obtained by knocking out the following genes: 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, from the *Komagataella pastoris* GS115 strain; or The engineered yeast strain was obtained by knocking out the following genes: 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, from the starting strain PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, and PAS_chr3_1087.

10. The engineered yeast strain according to claim 8, characterized in that, The engineered yeast strain is a yeast strain obtained by knocking out the PAS_chr3_0689 gene from the Pichia pastoris strain GS115.

11. The engineered yeast strain according to claims 8 to 10, characterized in that, The yeast engineer was introduced with a gene expressing a foreign protein to express the foreign protein.

12. The engineered yeast strain according to claim 11, characterized in that, The exogenous protein is at least one of brassinolide, reduced-salt protein, smegglutinin, casein, and whey protein; Preferably, the amino acid sequence of the Brazilian sweet protein is shown in SEQ ID No. 36; Preferably, the yeast is introduced with a gene encoding the brassinolide to enable the yeast to express the brassinolide; preferably, the nucleotide sequence of the gene encoding the brassinolide is shown in SEQ ID No.

35. Preferably, the amino acid sequence of the salt-reducing protein is shown in SEQ ID No. 38; Preferably, the yeast is introduced with a gene encoding the salt-reducing protein to enable the yeast to express the salt-reducing protein. Preferably, the nucleotide sequence of the gene encoding the salt-reducing protein is shown in SEQ ID No.

37. Preferably, the amino acid sequence of the smegglutinin is shown in SEQ ID No. 40; Preferably, the yeast is introduced with a gene encoding the smegglutinin to enable the yeast to express the smegglutinin. Preferably, the nucleotide sequence of the gene encoding the smegglutinin is shown in SEQ ID No. 39.

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