Protein for improving expression yield of foreign protein and application thereof

Abstract

The invention relates to a protein for improving the expression yield of a foreign protein and application of the protein. Wherein the ERV29 protein and the related protein as well as the P180 protein and the related protein can be used as molecular chaperones for improving the expression yield of foreign proteins such as Brazilian sweet and the like; the amino acid sequence of the ERV29 protein is as shown in SEQ ID No. 4; the amino acid sequence of the P180 protein is as shown in SEQ ID No. 6.

Description

Technical Field

[0001] The present invention relates to the field of functional genes, in particular to a protein used for increasing the expression yield of foreign proteins. Background Art

[0002] Sweeteners are food additives that give food or feed a sweet taste. According to their sources, they can be divided into natural sweeteners and artificial sweeteners. With the development of organic chemistry, artificial sweeteners have gradually become dominant, but because some chemically synthesized sweeteners are suspected of having safety issues, many countries in the world have successively banned the use of certain products of these sweeteners. In view of this, it is extremely important to find safe, non-toxic, and pure sweet sweeteners.

[0003] Brazzein (sometimes translated as Brazzein in Chinese) is the main sweet protein of the West African Oubli fruit (Pentadiplandra brazzeana Baillon). It is present in the extracellular pulp tissue surrounding the seeds. For thousands of years, locals have used it as a sweetener. Since this juice is also used to help infants wean, Brazzein has been proven to have a long history of safe use for both infants and adults. Brazzein is a single-chain polypeptide composed of 54 amino acid residues, containing 8 cysteines, forming 4 pairs of intramolecular disulfide bonds. The relative molecular mass of brazzein is about 6500, and its sweetness is 2000 times that of sucrose of the same mass under neutral conditions, and 800 to 1000 times that of sucrose under acidic conditions. Compared with other sweet proteins, brazzein has the smallest molecular weight and the best water solubility. Its aqueous solution still retains sweetness after 10 hours of heat treatment at 80 degrees Celsius, and has good thermal stability and low pH stability. Fermenting and producing Brazilian sweet protein to replace natural extract products can effectively reduce the damage to African rainforests, ensure a stable supply, and promote the healthy use of natural sweeteners.

[0004] Pichia Pastoris (aka Komagataella phaffii) is a relatively simple eukaryotic expression system that can correctly fold and process exogenous recombinant proteins, glycosylate them, etc. It uses the strong AOX1 promoter and methanol as a carbon source to achieve efficient expression of a variety of exogenous proteins, and is therefore widely used in the field of exogenous protein expression. Compared with bacteria, yeast cells themselves have certain safety, do not produce endotoxins, have advantages such as post-translational modification, secretory expression, and easy genetic manipulation, which greatly reduces the purification cost. At present, Pichia has been recognized by the US Food and Drug Administration (FDA) as a "Generally Regarded As Safe" (GRAS) level microorganism. In recent years, more and more studies have shown that Pichia is an ideal host for metabolic engineering research and has great potential in industrial applications. At present, more than 5,000 exogenous proteins can be successfully expressed using Pichia. As more and more exogenous proteins are secreted and expressed in Pichia, many studies have found that many exogenous proteins have problems such as low yields due to intracellular aggregation and other reasons during the secretory expression process.

[0005] Molecular chaperones, also known as auxiliary proteins, are a class of protein molecules that play an important role in protein translation, transport, folding and modification in cells. A large number of studies have shown that molecular chaperones are of great significance to the secretion and expression of exogenous proteins. Their main function is to assist the correct folding of proteins, reduce the degradation caused by misfolding of proteins, and thus improve the secretion and expression of proteins. Molecular chaperones can be derived from mammals, prokaryotes and eukaryotic cells, and a few are derived from plants. The organs in which they are distributed mainly include the endoplasmic reticulum, cytoplasm, nucleus, cytoplasm, mitochondria and chloroplasts. Molecular chaperones are widely used in genetically engineered bacteria. Different proteins are selective for molecular chaperones when expressed in Pichia pastoris. There is no definite and unified answer to which specific molecular chaperone has an effect on the expression of which exogenous protein. In addition, the mechanism of action of many chaperone molecules is still vague; and it is not clear whether many gene products can be used as chaperone molecules. Summary of the invention

[0006] One of the present inventions provides the use of a first protein and / or a second protein as a molecular chaperone for increasing the expression yield of an exogenous protein in a microorganism; wherein the first protein is an ERV29 protein, and its amino acid sequence is shown in SEQ ID No.4; the second protein is a P180 protein or a P180-QNT protein, and 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.

[0007] The second aspect of the present invention provides the use of a third protein and / or a fourth protein as a molecular chaperone for increasing 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 described in one of the present invention; 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 described in one of the present invention.

[0008] In a specific embodiment, 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.

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

[0010] The third aspect of the present invention provides the use of a fifth protein and / or a sixth protein as a molecular chaperone for increasing the expression yield of foreign proteins in microorganisms; wherein the fifth protein is mutated from the first protein in the application described in one of the present inventions, and the fifth protein has the same function as the first protein in the application described in one of the present inventions; the sixth protein is mutated from the second protein in the application described in one of the present inventions, and the sixth protein has the same function as the second protein in the application described in one of the present inventions.

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

[0012] In a specific embodiment, the exogenous protein in the application of the first, second or third invention is brazilian tamarind.

[0013] In a specific embodiment, the amino acid sequence of the brazilian tamanin is shown as SEQ ID No.2.

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

[0015] In a specific embodiment, the exogenous protein is brazilian tamarind.

[0016] In a specific embodiment, the nucleic acid sequence of the gene encoding brasilin is shown as SEQ ID No.1.

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

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

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

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

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

[0022] Coding gene: When used herein, the term "coding gene" means a polynucleotide sequence that directly specifies the amino acid sequence of its protein product. The boundaries of a coding gene are usually determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG, and ends with a stop codon such as TAA, TAG and TGA. A coding gene can be a DNA, cDNA, RNA, synthetic or recombinant nucleotide sequence.

[0023] Expression: in the context of the present invention includes any step involved in the production of the sweet protein of the present invention including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0024] Expression vector: refers to a linear or circular nucleic acid molecule construct, which comprises a polynucleotide of a coding protein such as the Brazilian sweet protein provided herein, and the polynucleotide is operably connected to the additional nucleotides provided for its expression. This additional nucleotide sequence includes, for example, a promoter, a suitable transcription initiation and termination sequence. "Promoter" is a DNA sequence that RNA polymerase recognizes, binds to and begins to transcribe, and it contains a conserved sequence required for RNA polymerase specific binding and transcription initiation, most of which are located upstream of the transcription start point, and the promoter itself is not transcribed. In addition, the expression vector has the host internal replication ability usually given by the replication origin, and / or carries a selection gene that helps to identify the transformant. Usually, the expression vector utilized in the recombinant DNA technique is often in the form of a "plasmid", i.e., refers to a circular double-stranded DNA ring. 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. The conventional one-letter code or the three-letter code for amino acid residues is used, with the amino acid sequence presented in the standard amino to carboxyl terminal orientation (ie, N→C).

[0026] Primer design

[0027] Snapgene software was used to design the corresponding primers for the constructed plasmid. When designing primers, attention should be paid to the GC content, Tm value, hairpin structure, primer length, primer dimer, primer mismatch, and the introduction of restriction sites.

[0028] The terms "comprising," "including," "having," and combinations thereof mean "including but not limited to," but also consist of only the listed elements.

[0029] The definitions provided herein are intended to facilitate understanding of certain terms frequently used herein and are not meant to limit the scope of the present disclosure.

[0030] Due to the structural complexity of Brazilian sweet, especially the presence of numerous cysteine ​​residues, Brazilian sweet expressed in traditional systems such as Escherichia coli and Pichia pastoris cannot fold into the correct conformation. Coupled with the degradation of its own proteases, the prepared Brazilian sweet has low sweetness. Ultimately, the method of preparing Brazilian sweet by genetic engineering cannot meet the demand for low cost of industrial production. In view of the above problems, the present invention proposes a method for preparing Brazilian sweet with high sweetness. The present invention knocks out the gene encoding a specific proteolytic enzyme in the host cell, fuses the signal peptide sequence and the Brazilian sweet protein sequence, and uses a chaperone molecule to cooperate with a protease-deficient Pichia pastoris strain host to express the fusion sequence. The Brazilian sweet protein finally prepared can be fermented at high density, which is convenient for downstream extraction and purification, saving production costs. At the same time, the present invention also applies the prepared Brazilian sweet to scenes such as yogurt drinks and sparkling water, proving that it can well replace sucrose as a sugar substitute sweetener.

[0031] The disclosure below provides many different embodiments or examples to realize different implementations of the present invention. In order to simplify the disclosure of the present invention, specific embodiments or examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the examples of various specific processes and materials provided by the present invention, those of ordinary skill in the art can be aware of the applicability of other processes and / or the use of other materials. Unless otherwise stated, the implementation of the present invention will adopt the traditional techniques in the fields of chemistry, molecular biology, etc. within the capabilities of those skilled in the art. In addition, unless otherwise stated, in this article, nucleic acids are written from left to right in the direction of 5' to 3', and amino acid sequences are written from left to right in the direction of amino terminal to carboxyl terminal.

[0032] Beneficial effects of the present invention: The present invention first discovered that ERV29 or P180 can be used as a molecular chaperone to increase the expression yield of brazilian thauma protein. Taking the combination of ERV29 and P180 molecular chaperones as an example, compared with the case where the two molecular chaperones are not co-expressed, the expression amount of brazilian thauma protein is increased by 1.6 times, the yield in the shake flask reaches more than 1g / L, and the yield in the 5L fermenter reaches more than 5g / L.

[0033] The Brazilian sweet protein Brazzein expressed by the present invention is very suitable for use as a natural high-intensity non-caloric sugar substitute sweetener. Studies have shown that Brazilian sweet protein Brazzein can block the bitterness of other natural and artificial sweeteners. When used in combination with other sweeteners, it can significantly improve the taste, intensity and duration of other sweeteners. Therefore, in many food and beverage applications, Brazilian sweet protein Brazzein can not only be used as an independent sweetener, but also as a taste modifier together with other sweeteners and flavorings. The potential application range is wide and the significance is significant.

[0034] In addition, the expression host of Brazilian sweet protein, Pichia pastoris, has been recognized by the FDA as a food safety grade GRAS strain, and is also a safe strain for health food production as stipulated by Chinese law; secondly, Pichia pastoris has the unique ability to grow under conditions where methanol is the only carbon source and energy source. Pichia pastoris also has the ability to use cheap culture media for high-density fermentation, strict regulation and very strong promoters, good post-translational modification and secretion capabilities, and easy genetic manipulation. This yeast has been used as a very successful heterologous protein expression system and is widely used in recombinant protein production, especially for vaccine production (currently more than 5,000 proteins have been successfully expressed). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The graph 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 96H.

[0036] Figure 2 A graph showing the 96H unit 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.

[0037] Figure 3 The HPLC peak profile of Brazzein standard (1 g / L) is shown.

[0038] Figure 4 The HPLC peak profile of MF001-143-Bra4-KRx3 at 96H in a shake flask is shown.

[0039] Figure 5 The HPLC peak profile of MF001-143-Bra-KRx3-ERV29 at 96 h in a shake flask is shown.

[0040] Figure 6 The HPLC peak profile of MF001-143-Bra-KRx3-P180 at 96H in a shake flask is shown.

[0041] Figure 7 The HPLC peak profile of MF001-143-Bra-KRx3-P180-QNT at 96H in a shake flask is shown.

[0042] Figure 8 The HPLC peak profile of MF001-143-Bra-KRx3-P180-ERV29 at 96 h in a shake flask is shown.

[0043] Fig. 9 A graph showing the growth curve of each expression strain in a fermenter is shown.

[0044] Fig.10 A graph of the wet weight of each expression strain in the fermentor is shown.

[0045] Fig.11The SDS-PAGE analysis of brazilian sweet protein in the fermenter is shown. In Figure A, M: Prestained Protein Marker (3.3kD-31.0kD); 1 represents 22 hours before fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter; 2 represents 40 hours after fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter; 3 represents 66 hours after fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter; 4 represents 88 hours after fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter; 5 represents 66 hours after fermentation induction in the MF001-143-Bra4-KRx3-ERV29 fermenter; 135H; 6 represents Brazzein standard; 7 represents 22H before fermentation induction in MF001-143-Bra4-KRx3-P180 fermenter; 8 represents 40H after fermentation induction in MF001-143-Bra4-KRx3-P180 fermenter; 9 represents 66H after fermentation induction in MF001-143-Bra4-KRx3-P180 fermenter; 10 represents 88H after fermentation induction in MF001-143-Bra4-KRx3-P180 fermenter; 11 represents 135H after fermentation induction in MF001-143-Bra4-KRx3-P180 fermenter.M: Prestained Protein Marker (3.3kD-31.0kD); In Figure B, 1 represents 22 hours before induction of MF001-143-Bra4-KRx3-P180-ERV29 fermenter fermentation; 2 represents 40 hours after induction of MF001-143-Bra4-KRx3-P180-ERV29 fermenter fermentation; 3 represents 66 hours after induction of MF001-143-Bra4-KRx3-P180-ERV29 fermenter fermentation; 4 represents 88 hours after induction of MF001-143-Bra4-KRx3-P180-ERV29 fermenter fermentation; 5 represents 135 hours after induction of MF001-143-Bra4-KRx3-P180-ERV29 fermenter fermentation; 6 represents Brazzein standard; In Figure C, M: Prestained Protein Marker (3.3kD-31.0kD); 1 represents 12 hours before fermentation induction in MF001-143-Bra4-KRx3 fermenter; 2 represents 24 hours after fermentation induction in MF001-143-Bra4-KRx3 fermenter; 3 represents 48 hours after fermentation induction in MF001-143-Bra4-KRx3 fermenter; 4 represents 72 hours after fermentation induction in MF001-143-Bra4-KRx3 fermenter; 5 represents 96 hours after fermentation induction in MF001-143-Bra4-KRx3 fermenter; 6 represents Brazzein standard product; In Figure D, M: Prestained Protein Marker (3.3kD-31.0kD); 1 represents Brazzein standard; 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] Fig.12 The HPLC peak profile of Brazzein standard (1 g / L) is shown.

[0047] Fig.13 The HPLC peak profile of MF001-143-Bra4-KRx3 at 135H in the fermenter is shown.

[0048] Fig.14 The HPLC peak profile of MF001-143-Bra-KRx3-ERV29 at 135H in the fermenter is shown.

[0049] Fig.15 The HPLC peak profile of MF001-143-Bra-KRx3-P180 at 135H in the fermenter is shown.

[0050] Fig.16 The HPLC peak profile of MF001-143-Bra-KRx3-P180-ERV29 in the fermenter at 135H is shown.

[0051] Fig.17 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 are shown.

[0052] Fig.18 The graph 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] Fig.19 The graph 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 DESCRIPTION

[0054] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

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

[0056] LB liquid culture medium: 0.5 g (0.5% w / v) yeast extract, 1 g (1% w / v) tryptone, 1 g (1% w / v) NaCl, add distilled water and make up to a total volume of 100 mL, sterilize under high pressure at 115°C for 20 min before use.

[0057] LBK resistance screening plate: 0.5g yeast extract (0.5% w / v), 1g tryptone (1% w / v), 1g NaCl (1% w / v), 2g agar powder (2% w / v) were added to distilled water and the total volume was adjusted to 100mL, and then sterilized by high pressure at 115℃ for 20min. When the temperature of the culture medium dropped to about 50 to 55℃, 25mg / mL kanamycin aqueous solution was added to the LB culture medium in a clean bench to a final concentration of 100 micrograms / mL of antibiotics, and the plate was poured after shaking evenly.

[0058] LBLZ resistance screening plate: 0.5g yeast extract (0.5% w / v), 1g tryptone (1% w / v), 1g NaCl (1% w / v), 2g agar powder (2% w / v) were added to distilled water and the total volume was adjusted to 100mL, and then sterilized by high pressure at 115°C for 20min. When the temperature of the culture medium dropped to about 50 to 55°C, 25mg / mL bleomycin aqueous solution was added to the LB culture medium in a clean bench to a final concentration of 25 micrograms / mL of antibiotics, and the plate was poured after shaking evenly.

[0059] MD solid plate medium: 2 g (2% w / v) of glucose, 1.34 g (1.34% w / v) of YNB (without amino acids, i.e., amino-free yeast nitrogen base), 2 g (2% w / v) of agar powder, add distilled water and make up to a total volume of 100 mL, sterilize at 115°C for 20 min, and then pour into the plate.

[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 dilute to a total volume of 100mL, 115℃, 20min autoclave. When the temperature of the culture medium drops to about 50 to 55℃, add 25mg / mL bleomycin (Zeocin or phleomycin D1) aqueous solution to the culture medium in a clean bench to a final concentration of 100 micrograms / mL of antibiotics, shake evenly and pour into the plate.

[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 with a certain amount of distilled water and made up to the final volume, sterilized at 115°C for 20 min.

[0062] BMMY liquid culture medium: 1% (w / v) yeast extract, 2% (w / v) tryptone, 1.34% (w / v) YNB, 10% (v / v) 1M pH 6.0 phosphate buffer, dissolved with a certain amount of distilled water and made up to the final volume, autoclaved at 115°C for 20 min, and then added with methanol at a ratio of 1% (v / v).

[0063] 1M phosphate buffer (pH 6.0): Mix 868mL of 1M KH2PO4 and 132mL of 1M K2HPO4 evenly, adjust the pH to 6.0 with phosphoric acid, sterilize at 121℃ for 20min, and place at room temperature for use.

[0064] The Pichia pastoris genome was extracted according to the specific steps in the instruction manual of Yeast DNAiso Kit (Takara Code: D9082).

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

[0066] The brasilin gene used in the following examples of the present invention is optimized according to the codon preference of Erythroplastes spp., 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 examples 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.3, and its amino acid sequence is shown in SEQ ID No.4.

[0068] The P180 gene used in the following examples 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 examples of the present invention is optimized according to the codon preference of Erythrorhizium truncatum, 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] Using GS115 as the starting strain, the PAS_chr3_0934, PAS_chr4_0913 and PAS_chr1-4_0048 genes were knocked out at the same time, and finally a gene-deficient strain in which these three genes were knocked out was obtained, and its strain number was MF001-29.

[0072] Taking GS115 as the starting strain, 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 were knocked out in two rounds, and finally a gene-deficient strain with these six genes knocked out was obtained, and its strain number was MF001-94.

[0073] Taking GS115 as the starting strain, 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 were knocked out in four rounds, and finally a gene-deficient strain with these 11 genes knocked out was obtained, and its strain number was MF001-136.

[0074] Taking GS115 as the starting strain, 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 were knocked out in five rounds, and finally a gene-deficient strain with these 12 genes knocked out was obtained, and its strain number was MF001-143.

[0075] Taking GS115 as the starting strain, the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, and PAS_chr1-1_0194 genes were knocked out in five rounds, and finally a gene-deficient strain with these 13 genes knocked out was obtained, and its strain number was MF001-249.

[0076] Taking GS115 as the starting strain, the PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 PAS_chr1-4_0013, PAS_chr3_0979, PAS_chr4_0113, PAS_chr1-1_0174, PAS_chr3_0953, PAS_chr2-2_0380, PAS_chr1-4_0611, PAS_chr1-1_0194, PAS_chr4_0584, and PAS_chr3_1087 genes were knocked out in six rounds, and finally a gene-deficient strain with these 15 genes knocked out was obtained, and its strain number was MF001-251.

[0077] Using GS115 as the starting strain, PAS_chr3_0934, PAS_chr4_0913, PAS_chr1-4_0048, PAS_chr2-1_0652, PAS_chr3_0633, PRC1 were knocked out in seven rounds. 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 genes were knocked out, and finally a gene-deficient strain in which these 16 genes were knocked out was obtained, and its strain number was MF001-256.

[0078] Using GS115 as the starting strain, the PAS_chr3_0689 gene was knocked out, and finally a gene-deficient strain in which this gene was also knocked out was obtained, and the strain number was MF001-169.

[0079] For details of the above gene knockout operations, please refer to CN2024116716299, and the strains obtained by the above gene knockout can be purchased from Suzhou Shuohong Biotechnology Co., Ltd.

[0080] Example 2

[0081] The brazilian tadalafil gene of the present invention is cloned on a plasmid PHKA vector (Thermo Fisher, Pichia pastoris expression kit, item number K171001). Among them, the α factor secretion signal, the brazilian tadalafil gene, the spacer sequence (as shown in SEQ ID No. 9) encoding gene, the brazilian tadalafil gene, the spacer sequence (as shown in SEQ ID No. 9) encoding gene, the brazilian tadalafil gene, the spacer sequence (as shown in SEQ ID No. 9) encoding gene, and the brazilian tadalafil gene are connected in series from 5' to 3', and then connected to the PHKA vector, and finally the positive plasmid PHKA-Bra4-KRx3 is obtained.

[0082] The ERV29 gene in the present invention was cloned into a plasmid pGAP (Thermo Fisher, Pichia pastoris expression kit, catalog number V20020) vector, and finally a positive plasmid PGAP-ERV29 was obtained. The result was completed by Nanjing GenScript Biotechnology Co., Ltd.

[0083] The P180 gene in the present invention is cloned into the plasmid pGAP vector, and finally the positive plasmid PGAP-P180 is obtained. It is completed by Nanjing GenScript Biotechnology Co., Ltd.

[0084] The P180-QNT gene in the present invention is cloned into the plasmid pGAP vector, and finally the positive plasmid PGAP-P180-QNT is obtained. It is completed by Nanjing GenScript Biotechnology Co., Ltd.

[0085] Example 3

[0086] The plasmid PHKA-Bra4-KRx3 was transferred into MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256 and MF001-169 strains, respectively, and screened on YPDS plates containing 100 μg / ml bleomycin to eliminate free plasmids, thereby obtaining MF001-29-Br 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 expression strains.

[0087] Example 4

[0088] PGAP-ERV29, PGAP-P180, and PGAP-P180-QNT were transformed into 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 strains, respectively, and cultured in YPD medium containing 200 μg / ml hygromycin. S plate screening, and elimination of free plasmids, to obtain 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, MF001-169-Bra4-KRx3-ERV29, respectively. 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 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 strains, respectively, and selected on YPDS plates containing 200 μg / ml hygromycin to eliminate free plasmids, thereby obtaining MF001-29-Bra4-KRx3, respectively. -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 expression strains.

[0090] Example 5

[0091] The yeast single colonies with relatively good growth of the expression strains of Example 3 and Example 4 were picked from the solid plate and inoculated into a 50 mL Erlenmeyer flask containing 5 mL of BMGY medium, and cultured in a shaking incubator at 30 degrees Celsius and 250 rpm for about 24 hours to obtain seed solution, and the OD value of the seed solution was measured. 600 value, centrifuge at 6000rpm for 2min at room temperature, discard the supernatant and collect the bacteria, then wash the bacteria 2 to 3 times with 800μl of BMMY liquid culture medium, then transfer all the washed bacteria to a 250mL Erlenmeyer flask with 25mL of BMMY, place it in a shaker at 30 degrees Celsius and 250rpm for culture, add 1% methanol every 24h, and culture for 5 days.

[0092] The culture supernatant samples were analyzed by SDS-PAGE, and the SDS gel used was FuturePAGE TMProtein precast gel 4%-20% (ACE). Apply 10 μl of Marker: (pre-stained SDS-PAGE standard, GenScript Bio, #M00624-250). In the matching special electrophoresis buffer: MOPS-SDS Running Buffer (Cat. No.: BR0001-02), the gel was electrophoresed at a constant voltage of 160V for 80 min. The protein band was stained with Bio-Safe Coomassie dye (Bio-Rad), and the results showed that brazilian sweet protein had different concentrations of accumulation.

[0093] The expression of brazilian tadalafil in the above culture at 96H was quantitatively detected by ELISA using the standard as the benchmark. Among them, the expression of brazilian tadalafil in 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 expression strains at 96H was as follows: Figure 1 .according to Figure 1 It can be seen that 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±0.02g / L, 1.51±0.03g / L, 1.54±0.02g / L, 1.52±0.02g / L and 1.82±0.02g / L, respectively; the results indicate that ERV29 alone and P180 alone can increase the secretion expression level of brazilian sweet protein, and when ERV29 and P180 are co-expressed, the two further synergistically increase the secretion expression level of brazilian sweet protein. Among them, the expression level of brazilian thauma protein in MF001-143-Bra4-KRx3-P180-ERV29 in shake flasks increased by 26.03% compared with that of MF001-143-Bra4-KRx3 strain.

[0094] In order to eliminate the influence of bacterial concentration, the ELISA quantitative detection results of 96H brazilian sweet protein in the above culture were divided by the bacterial concentration of 96H to obtain the expression level per unit OD. The results are shown in Figure 2 .according to Figure 2It can be seen that 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+ / -0.01mg / L / OD 600 、0.21+ / -0.01mg / L / OD 600 、0.21+ / -0.01mg / L / OD 600 、0.21+ / -0.01mg / L / OD 600 and 0.27+ / -0.02mg / L / OD 600 Similarly, the results showed that when ERV29 and P180 were co-expressed, the two further synergistically increased the secretion expression of brazilian tamanin. Among them, the expression of brazilian tamanin in the shake flask of MF001-143-Bra4-KRx3-P180-ERV29 was increased by 24.1% compared with that of MF001-143-Bra4-KRx3 strain. This result is consistent with the results of quantitative detection by ELISA mentioned above.

[0095] HPLC was used to quantitatively analyze the fermentation broth of brasilin expressed by each expression strain at 96 hours. The yield of brasilin expressed by each expression strain was detected by comparing it with the brasilin standard. Before the detection, the fermentation broth was centrifuged at high speed, treated with a 0.22 micron membrane, and the sample was filtered for detection. The specific operation was as follows: A Thermo Scientific Vanquish HPLC system was used, equipped with 5μm C18(2) 250×4.6mm chromatographic column. Use stationary phase buffer A (water + 0.1% TFA) and mobile phase buffer B (ACN + 0.1% TFA). UV detector monitors absorbance at 220nm. Column temperature is set to 37 degrees Celsius, injection volume is 10 microliters. 15-minute gradient program settings are shown in Table 1. Results are shown in Figures 3 to 8 .

[0096] Table 1

[0097] time Flow rate (mL / min) ％A ％B 0.000 0.5 70 30 0.100 0.5 70 30 10.000 0.5 10 90 10.100 0.5 70 30 15.000 0.5 70 30

[0098] according to Figures 3 to 8 The results show that the peak time of the standard sample is 6.9min, and the peak time of the fermentation broth filtered sample after 96 hours of shake flask fermentation is 6.9min. By observing the change in peak shape, it can be clearly seen Figure 8That is, the fermentation broth of MF001-143-Bra4-KRx3-P180-ERV29 strain is significantly improved compared with the fermentation broth of other strains.

[0099] Example 6

[0100] Expression characterization of recombinant brasiliensis proteins with different molecular chaperones in a protease-deficient Pichia pastoris strain in a 5-L fermenter and the effect of their combination on brasiliensis protein production.

[0101] (1) Preparation of fermentation seed solution

[0102] Pick a single yeast colony that was streaked, activated and growing well on the YPD plate, inoculate it into 50 mL of sterilized YPD, and place it in a 30°C shaker at 250 rpm for overnight culture to activate the strain. Transfer the YPD-activated yeast seed culture liquid at a ratio of 4% to a 500 mL Erlenmeyer flask containing 100 mL of YPD liquid culture medium, place it in a 30°C shaker at 250 rpm for overnight culture, and use it as the seed liquid for fermentation in a 5 L fermenter.

[0103] (2) High-density culture of recombinant yeast strains in 5L fermenters

[0104] The cultured yeast seed liquid was inoculated into a 5L fully automatic mechanical stirring and ventilated fermentation tank (sterilized) at an inoculation rate of 8%, and glycerol was added to start bacterial growth (the volume of the initial culture medium was 2L, sterilized at 121 degrees Celsius for 30 minutes). Bacterial growth stage: Use 25% concentrated ammonia water to control its pH at 5.5, maintain dissolved oxygen at (30-60)%, control the temperature at 30 degrees Celsius, set the upper limit of the stirring speed to 200r / min, the lower limit to 120r / min, and the ventilation volume to 2vvm. Initial cell density OD 600 After reaching 400, stop adding glycerol. When the dissolved oxygen (DO) rebounds to more than 80% again, start methanol feeding and induced fermentation. Methanol feeding stage: the temperature is controlled at 30°C, and the pH is controlled at 5.5 using 25% concentrated ammonia water. The speed is controlled at 800r / min, the ventilation volume is controlled at 4L / min, the pressure is controlled at 0.05Mpa, the ventilation volume is adjusted to 2vvm, and the methanol is added by variable speed feeding, and the dissolved oxygen is maintained at 15%-25%. The Bailun BLBIO B control system software is used to automatically control the fermentation process and collect related data, record the fermentation status, and make a growth curve. The results are shown in Fig. 9 ; The wet weight of the bacterial yield during the fermentation process was tested, and the results are shown in Fig.10 .

[0105] The accumulation of brazilian tamarind protein at different time points was detected by SDS-PAGE. Fig.11 .

[0106] HPLC of brazilian sweet protein fermented for 135 hours was carried out according to Example 5, and the results are shown in Figures 12 to 16 ,in, Fig.12 For the detection of Brazilian sweet protein standards.

[0107] After the scheduled sampling during the fermentation process, the supernatant of the fermentation broth was obtained, filtered through a 0.22 μm nylon membrane, and 10 μl of the supernatant was directly injected into HPLC. According to the signal size, the standard curve prepared by the brasilien standard was used to obtain the yield of brasilien at each fermentation time. The results are shown in Fig.17 .

[0108] The expression levels of brazilian tadalafil in the above-mentioned cultures at 86H and 135H were quantitatively detected by ELISA using the standard sample as the benchmark. Among them, the expression levels of brazilian tadalafil 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 were as follows: Fig.18 .according to Fig.18 It can be seen that 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; the results indicate that ERV29 alone and P180 alone can increase the secretion expression level of brazilian sweet protein, and when ERV29 and P180 are co-expressed, the two further synergistically increase the secretion expression level of brazilian sweet protein. Among them, the expression level of Brazilian sweet protein in MF001-143-Bra4-KRx3-P180-ERV29 in the 135H fermentation tank was increased by 3.8 times compared with that of MF001-143-Bra4-KRx3 strain.

[0109] In order to eliminate the influence of bacterial concentration, the ELISA quantitative results of 86H and 135H brasilienin in the above culture were divided by their respective bacterial concentrations to obtain the expression level per unit OD. The results are shown in Fig.19 .according to Fig.19It can be seen that 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±0.01mg / L / OD 600 , 0.28±0.01mg / L / OD 600 , 0.98±0.01mg / L / OD 600 and 1.14±0.02mg / L / OD 600 Similarly, the results showed that when ERV29 and P180 were co-expressed, the two further synergistically increased the secretion expression of brazilian tamanin. Among them, the expression of brazilian tamanin in the 135H fermenter was increased by 3.5 times compared with the MF001-143-Bra4-KRx3 strain. This result is consistent with the results of quantitative detection by the above ELISA.

Claims

1. Use of the first protein and / or the second protein as a molecular chaperone for increasing the expression yield of an exogenous 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, and 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. Use of the third protein and / or the fourth protein as a molecular chaperone for increasing the expression yield of foreign 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 of claim 1; 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 of claim 1.

3. The application according to claim 2, characterized in that: 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; 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.

4. Use of the fifth protein and / or the sixth protein as a molecular chaperone for increasing the expression yield of foreign proteins in microorganisms; wherein, The fifth protein is derived from the mutation 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 derived from the mutation 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; Preferably, the fifth protein is obtained by cyclically mutagenizing 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 mutagenizing 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 use according to any one of claims 1 to 4, characterized in that: The exogenous protein is brazilian thauma protein; Preferably, the amino acid sequence of the brazilian thauma protein is as shown in SEQ ID No.

2.

6. A method for increasing the expression yield of a foreign protein in a recipient microorganism, the method comprising: Transferring the gene encoding the exogenous protein in the use according to claim 1 or 5, as well as the gene encoding the first protein and / or the gene encoding the second protein into the recipient microorganism to co-express the exogenous protein and the molecular chaperone; or Transferring the gene encoding the exogenous protein in the use according to any one of claims 2, 3 and 5, and the gene encoding the third protein and / or the gene encoding the fourth protein into the recipient microorganism to co-express the exogenous protein and the molecular chaperone; or The gene encoding the exogenous protein in the application 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 exogenous protein and the molecular chaperone.

7. The method according to claim 6, characterized in that The exogenous protein is brazilian thauma protein; Preferably, the nucleic acid sequence of the gene encoding brasilin is as 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 as SEQ ID No.3; the nucleic acid sequence of the gene encoding the second protein is shown as 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 Komagataellaphaffii).

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

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