Protein for increasing expression yield of exogenous protein and use thereof

Abstract

Provided are a protein for increasing the expression yield of an exogenous protein and the use thereof, wherein an ERV29 protein and a related protein, and a P180 protein and a related protein can be used as molecular chaperones to improve the expression yield of exogenous proteins such as brazzein, the ERV29 protein has an amino acid sequence as shown in SEQ ID No. 4, and the P180 protein has an amino acid sequence as shown in SEQ ID No. 6.

Description

Proteins for increasing the expression yield of exogenous proteins and their applications Technical Field

[0001] This invention relates to the field of functional genes, and in particular to proteins used to increase the expression yield of exogenous proteins. Background Technology

[0002] 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 successively banned the use of certain products containing these sweeteners. Therefore, finding safe, non-toxic sweeteners with a pure sweet taste is of paramount importance.

[0003] Brazzein (sometimes translated as Brazzein in Chinese) is the main sweet protein in the fruit of Oubli (Pentadiplandra brazzeana Baillon) in West Africa. 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 was 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. The relative molecular mass of Brazzein is approximately 6500. Under neutral conditions, it is 2000 times sweeter than an equal mass of sucrose, and under acidic conditions, it is 800 to 1000 times sweeter. 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 10 hours, exhibiting good thermal stability and low pH stability. Fermentation to produce Brazil protein, which can replace naturally extracted products, can effectively reduce damage to African rainforests, ensure a stable supply, and promote the healthy use of natural sugar substitutes.

[0004] Pichia pastoris (aka Komagataella phaffii) is a relatively simple eukaryotic expression system capable of correctly folding and glycosylating exogenous recombinant proteins. Using the strong AOX1 promoter and methanol as a carbon source, it achieves efficient expression of various exogenous proteins, thus gaining widespread application in the field of exogenous protein expression. Compared to bacteria, yeast cells themselves possess certain safety advantages, do not produce endotoxins, and offer advantages such as post-translational modification, secretory expression, and ease of genetic manipulation, significantly reducing purification costs. Currently, Pichia pastoris has been recognized by the US Food and Drug Administration (FDA) as a "Generally Regarded As Safe" (GRAS) microorganism. In recent years, increasing research has also shown that Pichia pastoris is an ideal host for metabolic engineering research and has enormous potential for industrial applications. Currently, over 5000 exogenous proteins can be successfully expressed using Pichia pastoris. However, with the increasing number of exogenous proteins being secreted and expressed in Pichia pastoris, many studies have found that many exogenous proteins suffer from low yields due to intracellular aggregation and other factors during secretory expression.

[0005] Molecular chaperones, also known as accessory proteins, are a class of intracellular protein molecules that play crucial roles in protein translation, transport, folding, and modification. Numerous studies have shown that molecular chaperones are significant for the secretory expression of exogenous proteins. Their main function is to assist in the correct folding of proteins, reducing degradation caused by misfolding and thus enhancing secretory protein expression. Molecular chaperones can originate from mammalian, prokaryotic, and eukaryotic cells, with a few originating from plants. They are distributed primarily in the endoplasmic reticulum, cytoplasm, nucleus, cytoplasm, mitochondria, and chloroplasts. Molecular chaperones have wide applications in genetically engineered bacteria. Different proteins exhibit selective expression of molecular chaperones in Pichia pastoris. There is still no definitive and unified answer as to which specific molecular chaperone is effective for the expression of which exogenous protein. Furthermore, the mechanisms of action of many chaperone molecules remain unclear; and it is also uncertain whether many gene products can be used as chaperone molecules. Summary of the Invention

[0006] One aspect of the present invention provides the application of a first protein and / or a second protein as molecular chaperones to enhance the expression yield of exogenous proteins in microorganisms; wherein the first protein is an ERV29 protein, the amino acid sequence of which is shown in SEQ ID No. 4; the second protein is a P180 protein or a P180-QNT protein, the amino acid sequence of which is shown in SEQ ID No. 6, and the amino acid sequence of which is shown in SEQ ID No. 8.

[0007] The second invention provides the application of a third protein and / or a fourth protein as molecular chaperones to enhance the expression yield of exogenous proteins in microorganisms; wherein the amino acid sequence of the third protein has a similarity of more than 85% to the amino acid sequence shown in SEQ ID No. 4, and the third protein has the same function as the first protein in the application described in the first invention; the amino acid sequence of the fourth protein has a similarity of more than 85% to the amino acid sequence shown in SEQ ID No. 6 or SEQ ID No. 8, and the fourth protein has the same function as the second protein in the application described in the first invention.

[0008] In one specific embodiment, the amino acid sequence of the third protein has a similarity of more than 90% to the amino acid sequence shown in SEQ ID No. 4; and the amino acid sequence of the fourth protein has a similarity of more than 90% to the amino acid sequence shown in SEQ ID No. 6 or SEQ ID No. 8.

[0009] In one specific embodiment, the amino acid sequence of the third protein has a similarity of more than 98% to the amino acid sequence shown in SEQ ID No. 4; and the amino acid sequence of the fourth protein has a similarity of more than 98% to the amino acid sequence shown in SEQ ID No. 6 or SEQ ID No. 8.

[0010] The third invention provides the application of a fifth protein and / or a sixth protein as molecular chaperones to enhance the expression yield of exogenous proteins in microorganisms; wherein the fifth protein is derived from the first protein in the application described in the first invention, and the fifth protein has the same function as the first protein in the application described in the first invention; the sixth protein is derived from the second protein in the application described in the first invention, and the sixth protein has the same function as the second protein in the application described in the first invention.

[0011] In one specific embodiment, the fifth protein is obtained by performing a circular mutation on the first protein in one of the applications of the present invention, and the fifth protein has the same function as the first protein in one of the applications of the present invention; the sixth protein is obtained by performing a circular mutation on the second protein in one of the applications of the present invention, and the sixth protein has the same function as the second protein in one of the applications of the present invention.

[0012] In one specific embodiment, the exogenous protein in the application described in one, two, or three of the present invention is brassinosteroids.

[0013] In one specific embodiment, the amino acid sequence of the Brazil sweet protein is shown in SEQ ID No. 2.

[0014] The fourth invention provides a method for increasing the expression yield of a foreign protein in a recipient microorganism. The method includes transferring into the recipient microorganism a gene encoding the foreign protein as described in the application of the first invention, as well as a gene encoding the first protein and / or a gene encoding the second protein, to co-express the foreign protein and the molecular chaperone; or transferring into the recipient microorganism a gene encoding the foreign protein as described in any one of the applications of the second invention, as well as a gene encoding the third protein and / or a gene encoding the fourth protein, to co-express the foreign protein and the molecular chaperone; or transferring into the recipient microorganism a gene encoding the foreign protein as described in the application of the third invention, as well as a gene encoding the fifth protein and / or a gene encoding the sixth protein, to co-express the foreign protein and the molecular chaperone.

[0015] In one specific embodiment, the exogenous protein is brassinoprotein.

[0016] In one specific embodiment, the nucleic acid sequence of the gene encoding the Brazilian sweet protein is shown in SEQ ID No. 1.

[0017] In one specific embodiment, the nucleic acid sequence of the gene encoding the first protein is shown in SEQ ID No. 3; and the nucleic acid sequence of the gene encoding the second protein is shown in SEQ ID No. 5 or SEQ ID No. 7.

[0018] In one specific embodiment, the recipient microorganism is yeast.

[0019] In one specific embodiment, the yeast is Pichia Pastoris (aka Komagataella phaffii).

[0020] In one specific embodiment, the Pichia pastoris is one of the following strains: Pichia pastoris GS115, MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169.

[0021] 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.

[0022] Encoding gene: When used herein, the term "encoding gene" refers to a polynucleotide sequence that directly specifies the amino acid sequence of its protein product. The boundaries of a encoding gene are typically defined by an open reading frame (OPF), which usually begins with the ATG start codon or alternative start codons such as GTG and TTG, and ends with a stop codon such as TAA, TAG, and TGA. Encoding genes can be DNA, cDNA, RNA, synthetic, or recombinant nucleotide sequences.

[0023] 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.

[0024] Expression vectors are linear or circular nucleic acid molecular constructs containing polynucleotides encoding proteins such as the brassinolide protein 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.

[0025] 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 terminal orientation (i.e., N→C).

[0026] Primer design

[0027] Use Snapgene software to design the corresponding primers for the constructed plasmids. When designing primers, pay attention to the GC content, Tm value, hairpin structure, primer length, primer dimer, primer mismatch, and the introduction of restriction enzyme sites.

[0028] The terms “comprising,” “including,” “having,” and their combination mean “including but not limited to,” but also refer to situations consisting only of the listed elements.

[0029] 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.

[0030] 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 further contributes to the low sweetness of the resulting Brazil sweeteners. Therefore, genetic engineering methods for preparing Brazil sweeteners cannot meet the low-cost requirements of industrial production. This invention addresses these problems by proposing a method for preparing high-sweetness Brazil sweeteners. This invention involves knocking out the gene encoding a specific proteolytic enzyme in the host cell, fusing the signal peptide sequence with the Brazil sweetener protein sequence, and using a *Pichia pastoris* strain with a chaperone molecule-co-protease deficiency to express the fusion sequence. The resulting Brazil sweetener protein can undergo high-density fermentation, facilitating downstream extraction and purification, and saving production costs. Furthermore, this invention has demonstrated that the prepared Brazil sweetener can be effectively used as a sweetener to replace sucrose in yogurt beverages and sparkling water.

[0031] 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.

[0032] Beneficial effects of this invention: This invention is the first to discover that ERV29 or P180 can act as molecular chaperones to increase the expression yield of brassinosteroids. Taking the combination of ERV29 and P180 molecular chaperones as an example, compared with the case without co-expression with these two molecular chaperones, the expression level of brassinosteroids increased by 1.6 times, the yield in shake flasks reached more than 1 g / L, and the yield in a 5L fermenter reached more than 5 g / L.

[0033] The brazzein expressed in this invention is ideally suited for use as a natural, high-intensity, calorie-free sweetener. Studies have shown that brazzein 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, in many food and beverage applications, brazzein can be used not only as a standalone sweetener but also as a taste modifier in combination with other sweeteners and flavorings, with a wide range of potential applications and significant implications.

[0034] Furthermore, Pichia pastoris, the expression host of Brazilin, is recognized by the FDA as a GRAS (Generally Recognized As Safe) food-grade strain and is also a safe strain for the production of health food products under Chinese law. Secondly, Pichia pastoris possesses the unique ability to grow under conditions where methanol is the sole carbon and energy source. Pichia pastoris also exhibits the ability to perform high-density fermentation using inexpensive culture media, has a tightly regulated and very strong promoter, excellent post-translational modification and secretion capabilities, and is easily manipulated genetically. These characteristics have made this yeast a highly successful heterologous protein expression system, widely used in recombinant protein production, particularly for vaccine production (currently, over 5000 proteins have been successfully expressed). Attached Figure Description

[0035] Figure 1 shows the ELISA detection data of MF001-143-Bra4-KRx3, MF001-143-Bra-KRx3-ERV29, MF001-143-Bra-KRx3-P180, MF001-143-Bra-KRx3-P180-QNT, and MF001-143-Bra-KRx3-P180-ERV29 in the bottle at 96 hours.

[0036] Figure 2 shows the OD analysis data of MF001-143-Bra4-KRx3, MF001-143-Bra-KRx3-ERV29, MF001-143-Bra-KRx3-P180, MF001-143-Bra-KRx3-P180-QNT, and MF001-143-Bra-KRx3-P180-ERV29 in shake flasks at 96H units.

[0037] Figure 3 shows the HPLC peak chromatogram of Brazzein standard (1 g / L).

[0038] Figure 4 shows the HPLC peak chromatogram of MF001-143-Bra4-KRx3 at 96H in a shake flask.

[0039] Figure 5 shows the HPLC peak chromatogram of MF001-143-Bra-KRx3-ERV29 at 96H in a shake flask.

[0040] Figure 6 shows the HPLC peak chromatogram of MF001-143-Bra-KRx3-P180 at 96H in a shake flask.

[0041] Figure 7 shows the HPLC peak chromatogram of MF001-143-Bra-KRx3-P180-QNT in a shake flask at 96H.

[0042] Figure 8 shows the HPLC peak chromatogram of MF001-143-Bra-KRx3-P180-ERV29 at 96H in a shake flask.

[0043] Figure 9 shows the growth curves of each expression strain in the fermenter.

[0044] Figure 10 shows the wet weight curves of each expression strain in the fermenter.

[0045] Figure 11 shows the SDS-PAGE analysis of Brazil protein in the fermenter. In Figure A, M represents the Prestained Protein Marker (3.3kD-31.0kD); 1 represents the first 22 hours of fermentation in the MF001-143-Bra4-KRx3-ERV29 fermenter; 2 represents the last 40 hours after fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter; 3 represents the last 66 hours after fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter; 4 represents the last 88 hours after fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter; and 5 represents the last 5 hours after fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter. 135H; 6 represents Brazzein standard product; 7 represents 22H before fermentation induction in fermenter MF001-143-Bra4-KRx3-P180; 8 represents 40H after fermentation induction in fermenter MF001-143-Bra4-KRx3-P180; 9 represents 66H after fermentation induction in fermenter MF001-143-Bra4-KRx3-P180; 10 represents 88H after fermentation induction in fermenter MF001-143-Bra4-KRx3-P180; 11 represents 135H after fermentation induction in fermenter MF001-143-Bra4-KRx3-P180.M: Prestained Protein Marker (3.3kD-31.0kD); In Figure B, 1 represents 22 hours before fermentation induction in the MF001-143-Bra4-KRx3-P180-ERV29 fermenter; 2 represents 40 hours after fermentation induction in the MF001-143-Bra4-KRx3-P180-ERV29 fermenter; 3 represents 66 hours after fermentation induction in the MF001-143-Bra4-KRx3-P180-ERV29 fermenter; 4 represents 88 hours after fermentation induction in the MF001-143-Bra4-KRx3-P180-ERV29 fermenter; 5 represents 135 hours after fermentation induction in the MF001-143-Bra4-KRx3-P180-ERV29 fermenter; 6 represents Brazzein standard; In Figure C, M: Prestained Protein Marker (3.3kD-31.0kD); 1 represents 12 hours before fermentation induction in the MF001-143-Bra4-KRx3 fermenter; 2 represents 24 hours after fermentation induction in the MF001-143-Bra4-KRx3 fermenter; 3 represents 48 hours after fermentation induction in the MF001-143-Bra4-KRx3 fermenter; 4 represents 72 hours after fermentation induction in the MF001-143-Bra4-KRx3 fermenter; 5 represents 96 hours after fermentation induction in the MF001-143-Bra4-KRx3 fermenter; 6 represents Brazzein standard; In Figure D, M: Prestained Protein Marker (3.3kD-31.0kD); 1 represents Brazzein standard product; 2 represents 22 hours before fermentation induction in GS115 fermenter; 3 represents 40 hours after fermentation induction in GS115 fermenter; 4 represents 66 hours after fermentation induction in GS115 fermenter; 5 represents 88 hours after fermentation induction in GS115 fermenter; 6 represents 135 hours after fermentation induction in GS115 fermenter.

[0046] Figure 12 shows the HPLC peak chromatogram of Brazzein standard (1 g / L).

[0047] Figure 13 shows the HPLC peak chromatogram of MF001-143-Bra4-KRx3 at 135H in the fermenter.

[0048] Figure 14 shows the HPLC peak chromatogram of MF001-143-Bra-KRx3-ERV29 at 135H in the fermenter.

[0049] Figure 15 shows the HPLC peak chromatogram of MF001-143-Bra-KRx3-P180 at 135H in the fermenter.

[0050] Figure 16 shows the HPLC peak chromatogram of MF001-143-Bra-KRx3-P180-ERV29 at 135H in the fermenter.

[0051] Figure 17 shows the yield curves of MF001-143-Bra4-KRx3, MF001-143-Bra-KRx3-ERV29, MF001-143-Bra-KRx3-P180, and MF001-143-Bra-KRx3-P180-ERV29 in the fermenter at 44H, 62H, 88H, 115H, and 135H.

[0052] Figure 18 shows the ELISA quantitative detection data of MF001-143-Bra4-KRx3, MF001-143-Bra-KRx3-ERV29, MF001-143-Bra-KRx3-P180, and MF001-143-Bra-KRx3-P180-ERV29 in the fermenter at 86H and 135H.

[0053] Figure 19 shows the detection data of unit OD of MF001-143-Bra4-KRx3, MF001-143-Bra-KRx3-ERV29, MF001-143-Bra-KRx3-P180, and MF001-143-Bra-KRx3-P180-ERV29 in the fermenter after ELISA quantification at 86H and 135H. Detailed Implementation

[0054] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0055] Unless otherwise specified, the strains, plasmids and reagents used in the embodiments of the present invention can be purchased commercially.

[0056] 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.

[0057] 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 the volume was adjusted to 100mL. The plates were then autoclaved at 115℃ for 20min. After the culture medium temperature dropped to approximately 50-55℃, 25mg / mL kanamycin aqueous solution was added to the LB medium in a clean bench to a final antibiotic concentration of 100µg / mL. The plates were then shaken well and poured onto agar plates.

[0058] LBLZ 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 the volume was adjusted to 100mL. The plates were then autoclaved at 115℃ for 20min. After the culture medium temperature dropped to approximately 50-55℃, 25mg / mL bleomycin aqueous solution was added to the LB medium in a clean bench to a final antibiotic concentration of 25µg / mL. The plates were shaken well and then poured into plates.

[0059] MD solid plate medium: 2g glucose (2% w / v), 1.34g YNB (amino acid-free, i.e., amino-free yeast nitrogen source base) (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.

[0060] 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. Once the medium temperature has cooled to approximately 50-55℃, add 25mg / mL bleomycin (Zeocin or phleomycin D1) aqueous solution to the medium in a clean bench until the final antibiotic concentration is 100µg / mL. Shake well and pour into plates.

[0061] 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.

[0062] 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).

[0063] 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.

[0064] The Pichia pastoris genome extraction was performed according to the specific operating procedures in the Yeast DNAiso Kit (Takara Code: D9082) instruction manual.

[0065] The preparation of the Brazilian sweet protein rabbit-derived antibody was commissioned to Wuhan Huamei Biotechnology Co., Ltd., with a purity of 98% and a titer of 1:128000.

[0066] The Brazil sweet protein gene used in the following embodiments of the present invention was optimized according to the codon preference of red yeast, and its nucleic acid sequence is shown in SEQ ID No. 1, and its amino acid sequence is shown in SEQ ID No. 2.

[0067] The ERV29 gene used in the following embodiments of the present invention was optimized according to the codon preference of Pichia pastoris, and its nucleic acid sequence is shown in SEQ ID No. 3, and its amino acid sequence is shown in SEQ ID No. 4.

[0068] The P180 gene used in the following embodiments of the present invention is optimized according to the codon preference of Pichia pastoris, and its nucleic acid sequence is shown in SEQ ID No. 5, and the amino acid sequence of the P180 protein is shown in SEQ ID No. 6.

[0069] The P180-QNT gene used in the following embodiments of the present invention was optimized according to the codon preference of red yeast, and its nucleic acid sequence is shown in SEQ ID No. 7, and its amino acid sequence is shown in SEQ ID No. 8.

[0070] Example 1

[0071] Starting with GS115, the PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 genes were knocked out simultaneously, resulting in a gene-deficient strain with all three genes knocked out. The strain number is MF001-29.

[0072] Starting with GS115, the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013 genes were knocked out in two rounds, and finally a gene-deficient strain with all six genes knocked out was obtained, which was numbered MF001-94.

[0073] Starting with GS115, the following genes were knocked out in four rounds: 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. Finally, a gene-deficient strain with all 11 genes knocked out was obtained, and its strain number is MF001-136.

[0074] Starting with GS115, the following genes were knocked out in five rounds: 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. Finally, a gene-deficient strain with all 12 genes knocked out was obtained, and its strain number was MF001-143.

[0075] Starting with GS115, the following genes were knocked out in five rounds: 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. Finally, a gene-deficient strain with all 13 genes knocked out was obtained, and its strain number is MF001-249.

[0076] Starting with GS115, the following genes were knocked out in six rounds: 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. Finally, a gene-deficient strain with all 15 genes knocked out was obtained, and its strain number is MF001-251.

[0077] Starting with GS115, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, and PRC1 were knocked out in seven rounds. The genes 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, and PAS_chr1-1_0226 were knocked out, resulting in a gene-deficient strain with all 16 genes knocked out. This strain is designated MF001-256.

[0078] Starting with GS115, the PAS_chr3_0689 gene was knocked out, resulting in a gene-deficient strain with the gene knocked out simultaneously. The strain number is MF001-169.

[0079] For details of the gene knockout procedures described above, please refer to CN2024116716299. All strains obtained through the gene knockout procedures described above can be purchased from Suzhou Shuohong Biotechnology Co., Ltd.

[0080] Example 2

[0081] The brassinolide gene in this invention was cloned into the plasmid PHKA vector (Thermo Fisher, Pichia pastoris expression kit, catalog number K171001). Specifically, the α-factor secretion signal, the brassinolide gene, the spacer sequence (as shown in SEQ ID No. 9), the brassinolide gene, the spacer sequence (as shown in SEQ ID No. 9), the brassinolide gene, the spacer sequence (as shown in SEQ ID No. 9), and the brassinolide gene were tandemly linked from 5' to 3' and then ligated into the PHKA vector, finally obtaining the positive plasmid PHKA-Bra4-KRx3.

[0082] The ERV29 gene in this invention was cloned into the pGAP (Thermo Fisher, Pichia pastoris expression kit, catalog number V20020) vector, ultimately obtaining the positive plasmid PGAP-ERV29. This was accomplished by Nanjing Genscript Biotech Co., Ltd.

[0083] In this invention, the P180 gene was cloned into the pGAP plasmid vector, ultimately obtaining the positive plasmid PGAP-P180. This was accomplished by Nanjing Genscript Biotech Co., Ltd.

[0084] The P180-QNT gene in this invention was cloned into the pGAP plasmid vector, ultimately obtaining the positive plasmid PGAP-P180-QNT. This was accomplished by Nanjing Genscript Biotech Co., Ltd.

[0085] Example 3

[0086] Plasmid PHKA-Bra4-KRx3 was transformed into strains MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169, respectively. The strains were screened using YPDS plates containing 100 μg / mL bleomycin, and free plasmids were eliminated to obtain MF001-29-Br... The expression strains are a4-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, and MF001-169-Bra4-KRx3.

[0087] Example 4

[0088] PGAP-ERV29, PGAP-P180, and PGAP-P180-QNT were transformed into strains 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, and MF001-169-Bra4-KRx3, respectively, and treated with 200 μg / mL of hygromycin YPD. S-plate screening and elimination of free plasmids yielded the following plasmids: MF001-29-Bra4-KRx3-ERV29, MF001-94-Bra4-KRx3-ERV29, MF001-136-Bra4-KRx3-ERV29, MF001-143-Bra4-KRx3-ERV29, MF001-249-Bra4-KRx3-ERV29, MF001-251-Bra4-KRx3-ERV29, MF001-256-Bra4-KRx3-ERV29, and MF001-169-Bra4-KRx3-E RV29, MF001-29-Bra4-KRx3-P180, MF001-94-Bra4-KRx3-P180, MF001-136-Bra4-KRx3-P180, MF001-143-Bra4-KRx3-P180, MF001- 249-Bra4-KRx3-P180, MF001-251-Bra4-KRx3-P180, MF001-256-Bra4-KRx3-P180, MF001-169-Bra4-KRx3-P180, MF001-29-Bra4-K Rx3-P180-QNT, MF001-94-Bra4-KRx3-P180-QNT, MF001-136-Bra4-KRx3-P180-QNT, MF001-143-Bra4-KRx3-P180-QNT, MF001-249- Bra4-KRx3-P180-QNT, MF001-251-Bra4-KRx3-P180-QNT, MF001-256-Bra4-KRx3-P180-QNT and MF001-169-Bra4-KRx3-P180-QNT expression strains.

[0089] PGAP-ERV29 and PGAP-P180 were simultaneously transformed into strains 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, and MF001-169-Bra4-KRx3, respectively. Screening was performed using YPDS plates containing 200 μg / mL hygromycin, and free plasmids were eliminated to obtain strains MF001-29-Bra4. The expression strains are -KRx3-P180-ERV29, MF001-94-Bra4-KRx3-P180-ERV29, MF001-136-Bra4-KRx3-P180-ERV29, MF001-143-Bra4-KRx3-P180-ERV29, MF001-249-Bra4-KRx3-P180-ERV29, MF001-251-Bra4-KRx3-P180-ERV29, MF001-256-Bra4-KRx3-P180-ERV29, and MF001-169-Bra4-KRx3-P180-ERV29.

[0090] Example 5

[0091] Single yeast colonies with good growth from the expression strains of Examples 3 and 4 were picked from solid plates and inoculated into 50 mL Erlenmeyer flasks containing 5 mL of BMGY medium. The flasks were then incubated at 30°C and 250 rpm for approximately 24 hours to obtain seed culture. The OD of the seed culture was then measured. 600 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. 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. Add 1% methanol every 24 h and incubate for 5 days.

[0092] The supernatant samples from the above cultures were analyzed by SDS-PAGE using FuturePAGE™ precast protein gels (4%-20% ACE). 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), showing that Brazilian sweet protein accumulated at different concentrations.

[0093] Using standard references, the expression levels of brassinolide in the above cultures at 96H were quantitatively detected by ELISA. The expression levels of brassinolide in the following strains at 96H are shown in Figure 1. As shown in Figure 1, the values ​​of MF001-143-Bra4-KRx3, MF001-143-Bra4-KRx3-ERV29, MF001-143-Bra4-KRx3-P180, MF001-143-Bra4-KRx3-P180-QNT, and MF001-143-Bra4-KRx3-P180-ERV29 were 1.45±0.02 g / L, 1.51±0.03 g / L, 1.54±0.02 g / L, 1.52±0.02 g / L, and 1.82±0.02 g / L, respectively. These results indicate that both ERV29 and P180 alone can increase the secretory expression of brassinosteroids, and that co-expression of ERV29 and P180 further synergistically increases the secretory expression of brassinosteroids. Compared to strain MF001-143-Bra4-KRx3, strain MF001-143-Bra4-KRx3-P180-ERV29 showed a 26.03% increase in the expression level of Brazilian sweet protein in shake flasks.

[0094] To eliminate the influence of bacterial concentration, the ELISA quantitative detection results of 96H brassinolide from the above cultures were divided by the respective bacterial concentrations of 96H to obtain the expression level per unit OD, as shown in Figure 2. According to Figure 2, the values ​​of MF001-143-Bra4-KRx3, MF001-143-Bra4-KRx3-ERV29, MF001-143-Bra4-KRx3-P180, MF001-143-Bra4-KRx3-P180-QNT, and MF001-143-Bra4-KRx3-P180-ERV29 were 0.19 + / - 0.01 mg / L / OD, respectively. 600 0.21 + / - 0.01 mg / L / OD 600 0.21 + / - 0.01 mg / L / OD 600 0.21 + / - 0.01 mg / L / OD 600 and 0.27+ / -0.02mg / L / OD 600 Similarly, the results showed that co-expression of ERV29 and P180 further synergistically increased the secretory expression level of brassinolide. Specifically, compared to strain MF001-143-Bra4-KRx3, strain MF001-143-Bra4-KRx3 showed a 24.1% increase in brassinolide expression in shake flasks. This result is largely consistent with the quantitative detection results obtained by ELISA.

[0095] HPLC was used to quantitatively analyze the fermentation broth of brassinolide expressed by various expression strains at 96H. The yield of brassinolide expressed by each strain was determined by comparison with brassinolide standards. Before analysis, the fermentation broth was centrifuged at high speed, treated with a 0.22-micron membrane, filtered, and then analyzed. Specific procedures were as follows: A Thermo Scientific Vanquish HPLC system was used, equipped with… 5μm 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. The results are shown in Figures 3 to 8.

[0096] Table 1

[0097] As shown in Figures 3 to 8, the peak elution time of the standard sample was 6.9 min, and the peak elution time of the filtered sample of the fermentation broth after 96 hours of shake-flask fermentation was also 6.9 min. Observing the peak shape changes, it can be clearly seen in Figure 8 that the fermentation broth of strain MF001-143-Bra4-KRx3-P180-ERV29 showed a significant improvement compared to the fermentation broths of other strains.

[0098] Example 6

[0099] Expression and characterization of recombinant brassinoprotein with different molecular chaperones in protease-deficient Pichia pastoris strains in a 5L fermenter, and the effect of their combination on brassinoprotein yield.

[0100] (1) Preparation of fermentation seed liquid

[0101] Select a single yeast colony that has been streaked and activated on a YPD plate and inoculate it into a 50 mL container of sterilized YPD. Incubate the colony overnight at 250 rpm in a shaker at 30°C to activate the yeast. Transfer the activated yeast seed culture to a 500 mL Erlenmeyer flask containing 100 mL of YPD liquid culture medium at a ratio of 4%. Incubate the flask overnight at 250 rpm in a shaker at 30°C to obtain the seed culture for fermentation in a 5 L fermenter.

[0102] (2) High-density culture of recombinant yeast strain in a 5L fermenter

[0103] 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°C for 30 min). During cell growth: pH was maintained at 5.5 using 25% concentrated ammonia, dissolved oxygen was maintained at (30-60)%, temperature was controlled at 30°C, 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 concentration reaches 400, the glycerol feeding is stopped. When dissolved oxygen (DO) rebounds to above 80%, methanol-feed induction fermentation begins. During the methanol feeding stage: the temperature is controlled at 30℃, the pH is maintained at 5.5 using 25% concentrated ammonia, the rotation speed is controlled at 800 r / min, the aeration rate is controlled at 4 L / min, the pressure is controlled at 0.05 MPa, and the aeration rate is adjusted to 2 vvm. A variable-speed feeding method is used for methanol feeding, maintaining dissolved oxygen at 15%-25%. The BLBIO B-type control system software is used for automatic process control and related data acquisition. The fermentation status is recorded, and growth curves are generated (see Figure 9). Wet weight analysis of cell yield during fermentation is performed (see Figure 10).

[0104] The accumulation of Brazil glycoprotein at various time points was detected by SDS-PAGE, and the results are shown in Figure 11.

[0105] The HPLC analysis of brassinoprotein fermented for 135 hours was performed according to Example 5. The results are shown in Figures 12 to 16, where Figure 12 shows the detection of the brassinoprotein standard.

[0106] After the fermentation process was completed by taking samples at regular intervals, the supernatant of the fermentation broth was obtained, filtered through a 0.22-micron nylon membrane, and 10 microliters of the supernatant was directly injected into HPLC. Based on the signal strength, the yield of Brazil gluten at each fermentation time was obtained by using the standard curve prepared from the Brazil gluten standard. The results are shown in Figure 17.

[0107] Using standard references, the expression levels of brassinolide at 86H and 135H in the above cultures were quantitatively detected by ELISA. The expression levels of brassinolide in the expression strains MF001-143-Bra4-KRx3, MF001-143-Bra4-KRx3-ERV29, MF001-143-Bra4-KRx3-P180 and MF001-143-Bra4-KRx3-P180-ERV29 at 86H and 135H are shown in Figure 18. As shown in Figure 18, at 135H, the values ​​of MF001-143-Bra4-KRx3, MF001-143-Bra4-KRx3-ERV29, MF001-143-Bra4-KRx3-P180, and MF001-143-Bra4-KRx3-P180-ERV29 were 0.10+ / -0.01g / L, 0.12+ / -0.01g / L, 0.37+ / -0.02g / L, and 0.50+ / -0.04g / L, respectively. This result indicates that both ERV29 and P180 alone can increase the secretory expression of brassinosteroids, and when ERV29 and P180 are co-expressed, they further synergistically increase the secretory expression of brassinosteroids. Compared to strain MF001-143-Bra4-KRx3, strain MF001-143-Bra4-KRx3-P180-ERV29 showed a 3.8-fold increase in the expression of Brazilian sweet protein in a 135H fermenter.

[0108] To eliminate the influence of bacterial concentration, the ELISA quantitative results of 86H and 135H brassinolide from the above cultures were divided by their respective bacterial concentrations to obtain the expression level per unit OD, as shown in Figure 19. According to Figure 19, the values ​​of MF001-143-Bra4-KRx3, MF001-143-Bra4-KRx3-ERV29, MF001-143-Bra4-KRx3-P180, and MF001-143-Bra4-KRx3-P180-ERV29 were 0.25 ± 0.01 mg / L / OD. 600 0.28±0.01mg / L / OD 600 0.98±0.01mg / L / OD 600 and 1.14±0.02 mg / L / OD 600 Similarly, the results showed that co-expression of ERV29 and P180 further synergistically increased the secretory expression level of brassinosteroids. Specifically, the expression level of brassinosteroids in the 135H fermenter was 3.5-fold higher in strain MF001-143-Bra4-KRx3-P180-ERV29 compared to strain MF001-143-Bra4-KRx3. This result is largely consistent with the quantitative detection results obtained by ELISA.

Claims

1. Use of a first protein and / or a second protein as a chaperone for increasing the expression yield of a foreign protein in a microorganism; wherein, The first protein is ERV29 protein, and its amino acid sequence is shown in SEQ ID No. 4; the second protein is P180 protein or P180-QNT protein, the amino acid sequence of the P180 protein is shown in SEQ ID No. 6, and the amino acid sequence of the P180-QNT protein is shown in SEQ ID No.

8.

2. The application of third and / or fourth proteins as molecular chaperones to enhance the expression yield of exogenous proteins in microorganisms; wherein, The amino acid sequence of the third protein is more than 85% similar to the amino acid sequence shown in SEQ ID No. 4, and the third protein has the same function as the first protein in the application of claim 1; the amino acid sequence of the fourth protein is more than 85% similar to the amino acid sequence shown in SEQ ID No. 6 or SEQ ID No. 8, and the fourth protein has the same function as the second protein in the application of claim 1.

3. The application according to claim 2, characterized in that, The amino acid sequence of the third protein is more than 90% similar to the amino acid sequence shown in SEQ ID No. 4; the amino acid sequence of the fourth protein is more than 90% similar to the amino acid sequence shown in SEQ ID No. 6 or SEQ ID No.

8.

4. The application of the fifth and / or sixth proteins as molecular chaperones to enhance the expression yield of exogenous proteins in microorganisms; among which, The fifth protein is derived from the first protein in the application of claim 1 by mutation, and the fifth protein has the same function as the first protein in the application of claim 1; the sixth protein is derived from the second protein in the application of claim 1 by mutation, and the sixth protein has the same function as the second protein in the application of claim 1. Preferably, the fifth protein is obtained by cyclically mutagenesis of the first protein in the application of claim 1, and the fifth protein has the same function as the first protein in the application of claim 1; the sixth protein is obtained by cyclically mutagenesis of the second protein in the application of claim 1, and the sixth protein has the same function as the second protein in the application of claim 1.

5. The application according to any one of claims 1 to 4, characterized in that, The exogenous protein is brassinoprotein; Preferably, the amino acid sequence of the Brazilian sweet protein is shown in SEQ ID No.

2.

6. A method for increasing the expression yield of exogenous proteins in recipient microorganisms, the method comprising: The gene encoding the exogenous protein as described in claim 1 or 5, as well as the gene encoding the first protein and / or the gene encoding the second protein, are transferred into the recipient microorganism to co-express the exogenous protein and the molecular chaperone. or The recipient microorganism is introduced with the gene encoding the exogenous protein as described in any one of claims 2, 3 and 5, as well as the gene encoding the third protein and / or the gene encoding the fourth protein, to co-express the exogenous protein and the molecular chaperone. or The gene encoding the foreign protein as described in claim 4 or 5, as well as the gene encoding the fifth protein and / or the gene encoding the sixth protein, are transferred into the recipient microorganism to co-express the foreign protein and the molecular chaperone.

7. The method according to claim 6, characterized in that, The exogenous protein is brassinoprotein; Preferably, the nucleic acid sequence of the gene encoding the Brazilian sweet protein is shown in SEQ ID No.

1.

8. The method according to claim 6, characterized in that, The nucleic acid sequence of the gene encoding the first protein is shown in SEQ ID No. 3; the nucleic acid sequence of the gene encoding the second protein is shown in SEQ ID No. 5 or SEQ ID No.

7.

9. The method according to any one of claims 6 to 8, characterized in that, The recipient microorganism is yeast; Preferably, the yeast is Pichia Pastoris (aka Komagataella phaffii).

10. The method according to claim 9, characterized in that, The *Pichia pastoris* strain is one of the following: *Pichia pastoris* GS115, MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169.

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