Highly efficient expression of beta-lactoglobulin strain and its construction method and application

Abstract

The present disclosure belongs to the field of genetic engineering technology, and specifically relates to a strain that efficiently expresses β-lactoglobulin, and its construction method and application. Using the β-lactoglobulin expression vector disclosed in the present disclosure, β-lactoglobulin is secreted outside the host cell under the guidance of an exogenous signal peptide, and the efficient secretory expression of β-lactoglobulin in the host cell is achieved by integrating multiple copies of the expression frame in the genome and co-expressing the ATPase Ssa4 involved in protein folding and coping with stress, and the secretory expression of β-lactoglobulin does not contain redundant amino acids at the amino and carboxyl ends, and does not contain animal-derived components. At the same time, the present disclosure provides a construction method and a fermentation culture method for engineering bacteria. Compared with single-copy genetically engineered bacteria, the expression amount of the genetically engineered strain disclosed in the present disclosure in shake flask fermentation is increased by 2.47 times, and it has good industrial application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of gene engineering technology, and specifically relates to a strain for efficiently expressing β-lactoglobulin, a construction method and application thereof, and a method for efficiently producing β-lactoglobulin. Background Art

[0002] Beta lactoglobulin (βLg) is a nutrient synthesized and secreted by mammary epithelial cells. It is a globular protein that exists in the milk of most mammals, especially in the milk of ruminants (such as cows and buffaloes), but is not found in human milk. βLg is the main component of milk whey protein, with a content of about 4 mg / mL in cow's milk, accounting for about 50% of the total whey protein and 10% to 12% of the total protein. The molecular weight of β-lactoglobulin in cow's milk is about 18.4 kDa, containing 162 amino acid residues. It is rich in lysine, which cannot be synthesized by the human body itself, and is a protein that is of great significance to human nutrition.

[0003] At present, β-lactoglobulin is mainly extracted from milk by separation and filtration. After acidification, heating or the action of specific enzymes, the protein in the whey is precipitated, and then purified and separated by column chromatography or electrophoresis to obtain purified β-lactoglobulin. β-lactoglobulin is generally considered safe, non-toxic and harmless, but some people may be allergic to milk protein. In recent years, the use of technical means such as genetic engineering and cell factories to efficiently express various milk protein components in natural animal milk has become a hot spot in the current research and development of biosynthesis. Cell factories can produce products with nutritional flavors comparable to natural milk, and can selectively remove undesirable factors such as lactose, cholesterol and allergens. Among them, the prokaryotic expression system has cumbersome separation and purification steps, lacks the necessary protein post-translational modification function and potential risk factors such as endotoxins, and the use of eukaryotic expression systems has advantages in the recombinant expression of β-lactoglobulin, but there are still some technical bottlenecks in production.

[0004] At the current technical level in this field, the ability of heterologous hosts to express β-lactoglobulin is still insufficient to meet production needs. The high cost due to the high technical difficulty is the main reason limiting the expansion of production and application. At the same time, purification of the product is another major difficulty. Existing studies have shown that the direct expression of lactoglobulin using animal cells requires high separation, extraction and purification technology. Therefore, this field urgently needs to improve the expression amount and production efficiency of β-lactoglobulin and reduce industrial production costs. Summary of the invention

[0005] To solve the above problems, the purpose of the present disclosure is to provide a strain that efficiently expresses β-lactoglobulin, which improves the expression level of β-lactoglobulin and reduces the difficulty of separation and purification of β-lactoglobulin, thereby reducing production costs.

[0006] In order to achieve the above technical objectives, the present disclosure proposes the following technical solutions:

[0007] In a first aspect, the present disclosure provides a β-lactoglobulin expression vector, which comprises a plurality of β-lactoglobulin expression cassettes, wherein the expression cassettes comprise a promoter, an α signal peptide coding sequence, a β-lactoglobulin coding sequence and a terminator.

[0008] In a second aspect, the present disclosure provides a vector combination, which includes the β-lactoglobulin expression vector and the Ssa4 expression vector described in the first aspect.

[0009] In a third aspect, the present disclosure provides a host cell, which comprises the β-lactoglobulin expression vector described in the first aspect or the vector combination described in the second aspect.

[0010] In a fourth aspect, the present disclosure provides a method for constructing a host cell expressing β-lactoglobulin, comprising:

[0011] Transform host cells simultaneously with the β-lactoglobulin expression vector and the Ssa4 expression vector described in the first aspect; or

[0012] The β-lactoglobulin expression vector and the Ssa4 expression vector described in the first aspect are used to transform host cells successively.

[0013] In a fifth aspect, the present disclosure provides a method for producing β-lactoglobulin, comprising:

[0014] The host cell of the third aspect is cultured to obtain a culture containing the β-lactoglobulin.

[0015] By using the β-lactoglobulin expression vector disclosed in the present invention, β-lactoglobulin is secreted outside the host cell under the guidance of an exogenous signal peptide, and the β-lactoglobulin is efficiently secreted and expressed in the host cell by integrating multiple copies of the expression frame in the genome and co-expressing the ATPase Ssa4 involved in protein folding and coping with stress, and the amino and carboxyl ends of the secreted β-lactoglobulin do not contain redundant amino acids and animal-derived components. At the same time, the present invention provides a construction method and a fermentation culture method for an engineered bacterium. Compared with a single-copy genetically engineered bacterium, the expression amount of the genetically engineered strain disclosed in the present invention in a shake flask fermentation is increased by 2.47 times, and has good industrial application prospects.

[0016] Biological Deposit Description

[0017] Recombinant Pichia pastoris ( Komagataella phaffii , also known as Shepherd's pie ) The genetically engineered bacteria were deposited in the China Center for Type Culture Collection (CCTCC) at Wuhan University, with the deposit number CCTCC M20242480, the deposit date November 7, 2024, and the culture name Komagataella phaffii CJ-PP-BG1. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a map of the three-copy recombinant expression plasmid pPIC9K-α1-3BLG constructed in Example 2.

[0019] Figure 2 The results of SDS PAGE of the fermentation broth of the recombinant strain in Example 6. 1: 0.2 g / L β-lactoglobulin standard (Sigma, L3908); 2: recombinant single copy bovine β-lactoglobulin fermentation broth supernatant; 3: recombinant multi-copy bovine β-lactoglobulin fermentation broth supernatant; 4: co-expression Ssa4 fermentation broth supernatant; M: 10-170kD protein marker.

[0020] FIG3 is the result of detecting the β-lactoglobulin production in the supernatant of the fermentation broth of the recombinant strain using high performance liquid chromatography, wherein Figure 3A This is the HPLC peak diagram of β-lactoglobulin in the supernatant of the fermentation broth of the recombinant expression strain containing the pPIC9K-3BLG-Ssa4 plasmid. Figure 3B The expression level of β-lactoglobulin was obtained by high performance liquid chromatography and standard analysis. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0022] The present disclosure may be implemented in other specific forms without departing from the basic attributes of the present disclosure. It should be understood that, without conflict, any and all embodiments of the present disclosure may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present disclosure includes other embodiments obtained by such combinations.

[0023] All publications and patents mentioned in this disclosure are hereby incorporated by reference into this disclosure in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0024] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0025] (I) Definitions or terms

[0026] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," "the," "said," and similar referents include plural referents unless the content clearly dictates otherwise.

[0028] As used herein, the conjunction term "and / or" between various elements is intended to include both the meanings of "and" and "or", for example, the phrase "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0029] As used herein, the terms "comprises," "comprising," "having," and "containing," and any variations thereof, are intended to cover a non-exclusive inclusion. The terms are intended to be open ended, to specify the presence of any stated features, elements, integers, steps, or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of."

[0030] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0031] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0032] In the description of this document, reference is made to "some embodiments", "some embodiments" or "some embodiments", which describes a subset of all possible embodiments, but it can be understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0033] As used herein, the terms "optional," "optional," "either," "any," or "either" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not occur.

[0034] As used herein, the terms "polypeptide", "protein" interchangeably refer to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), which may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. A polypeptide may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0035] As used herein, the term "amino acid" refers to the 20 naturally occurring amino acids that can be translated from gene coding and that constitute protein chains, including L-amino acids and D-amino acids, unless otherwise specified, and also refers to chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are usually not bound to proteins such as norleucine, and chemically synthesized compounds having amino acid properties known in the art. For example, analogs or mimetics of Phe or Pro that can make the conformational constraints of peptide compounds the same as those of natural phenylalanine or proline are included in the definition of "amino acid" and are known to those skilled in the art. The analogs and mimetics referred to in this article refer to "functional equivalents" of amino acids. Other examples of amino acids and amino acid analogs have been given by Roberts and Vellaccio (The Peptides: Andlysis, Synthesis, Biology, Eds. Gross and Meienhofer, Vol. 5, P. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference).

[0036] As used herein, the term "amino acid substitution" refers to replacing an amino acid residue present in a parent sequence with another amino acid residue. The amino acid in the parent sequence can be substituted, for example, via chemical peptide synthesis or by recombinant methods known in the art. Therefore, reference to "substitution at position X" refers to replacing the amino acid present in position X with an alternative amino acid residue. In some embodiments, the substitution type can be described according to the pattern AXY, wherein A is a single letter code corresponding to an amino acid naturally present in position X, and Y is a substituted amino acid residue. In other aspects, the substitution type can be described according to the pattern XY, wherein Y is a single letter code corresponding to replacing an amino acid residue naturally present in position X.

[0037] As used herein, the term "nucleic acid molecule" or "nucleic acid" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA, cDNA or RNA fragments. When applied to nucleic acids or polynucleotides, the term "isolated" refers to a nucleic acid molecule (DNA or RNA) that has been removed from its natural environment, such as for the purposes of this disclosure, a recombinant polynucleotide encoding an antigen-binding protein contained in a vector is considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or recombinant polynucleotides purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides disclosed herein. Polynucleotides or nucleic acids according to the separation of the present disclosure further include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may include regulatory elements, such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0038] As used herein, the term "polynucleotide" or "nucleotide" is intended to encompass a single nucleic acid as well as a plurality of nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA).

[0039] As used herein, the term "homology" has a meaning recognized in the art and is a central concept in comparative biology. The basic meaning of homology is that the two samples being compared (e.g., an amino acid sequence or a nucleotide sequence) have a common ancestor. Generally speaking, if two traits (states) in two species meet any of the following two conditions, the two traits can be called a pair of homologous traits: 1. They are the same as a trait found in the ancestral group of these species; 2. They are different traits with an ancestor-descendant relationship. Amino acid sequence homology can be determined by methods known to itself. For example, amino acid sequence homology (%) can be determined using programs commonly used in the field (e.g., BLAST, FASTA, etc.) according to initial settings. On the other hand, homology (%) can be determined using any algorithm known in the field, such as the algorithm of Needleman et al. (1970) (J. Mol. Biol. 48:444-453), Myers and Miller (CABIOS, 1988, 4: 11-17), etc. The algorithm of Needleman et al. is integrated into the GAP program of the GCG software package (available at www.gcg.com), and homology (%) can be determined, for example, using a BLOSUM 62 matrix or a PAM250 matrix, and gap weights: 16, 14, 12, 10, 8, 6, or 4 and any one of length weights: 1, 2, 3, 4, 5, or 6. In addition, the algorithm of Myers and Miller is integrated into the ALIGN program that is part of the GCG sequence alignment software package. In the case of utilizing the ALIGN program for comparing amino acid sequences, for example, a PAM120 weight residue table, a gap length penalty, and a gap penalty can be used.

[0040] Although the present disclosure provides some specific amino acid sequences or nucleotide sequences, such as the amino acid sequences or nucleotide sequences shown in the sequence listing, it should be understood that a specific amino acid sequence or nucleotide sequence includes conservatively modified variants thereof, such as sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology thereto, as long as the biological function or activity of the specific amino acid sequence or nucleotide sequence is not lost.

[0041] The term "conservatively modified variant" is used to refer to amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids encoding the same or conservatively modified amino acid sequence variants. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, it is well known in the art that degenerate codons (and therefore corresponding DNA codons, T replacing U) can be used interchangeably to encode each specific amino acid, and a degenerate codon refers to a different three-letter codon used to specify the same amino acid, for example, the codons GCA, GCC, GCG and GCU all encode alanine. Therefore, at any position where alanine is specified by a codon, the codon can be changed to any corresponding codon without changing the encoded polypeptide. This nucleic acid variation is a "silent variation", which is a conservatively modified variation. The skilled person will realize that each codon in the nucleic acid (except AUG and UGG, AUG is usually only a codon for methionine, and UGG is only a codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of the nucleic acid encoding the polypeptide of the present disclosure is undoubted in each of the described polypeptide sequences, which is included in the scope of the present disclosure.

[0042] For amino acid sequences, the skilled artisan will recognize that each substitution, deletion or addition of a nucleic acid, peptide, polypeptide or protein sequence that changes, adds or deletes a single amino acid or a small number of amino acids in the coding sequence is a "conservatively modified variant", wherein the change results in the replacement of an amino acid with a chemically similar amino acid. Thus, an amino acid residue selected from any integer between 1-15 may be altered. For example, 1, 2, 3, 4, 5, 7 or 10 changes may be produced. Conservatively modified variants typically provide biological activity similar to that of the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzymatic activity or ligand / receptor binding is typically at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of that of a native protein with its native substrate. A conservative substitution table of amino acids providing similar functions is well known to those skilled in the art. For example, modifications may be introduced into the sequence listing described herein by standard techniques known in the art (e.g., gene synthesis and PCR-mediated mutagenesis). Conservative modification includes amino acid conservative substitution, "conservative substitution" is one of the amino acids replaced by another amino acid with similar properties, such as replaced by an amino acid with a similar side chain, so that the secondary structure and hydrophilic properties of the polypeptide are substantially unchanged by the technicians in the field of peptide chemistry. Families of amino acid residues with similar side chains are already defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0043] As used herein, the term "vector" refers to a vector that can replicate autonomously in a host cell, preferably a multi-copy vector. As used herein, the term "vector" is capable of transporting a nucleic acid molecule of another nucleic acid to which it is connected. In addition, the vector usually has markers such as antibiotic resistance genes for selecting transformants. In addition, the vector may have a promoter and / or terminator for expressing the introduced gene. The vector may be, for example, a vector derived from a bacterial plasmid, a viral vector, a vector derived from a yeast plasmid, a vector derived from a phage, a clay, a phagemid, etc. The term "recombinant vector" refers to a vector modified as needed for carrying an exogenous DNA fragment (a DNA fragment of a gene of interest) into a host cell for replication and expression, including a recombinant cloning vector and a recombinant expression vector, preferably a recombinant expression vector. In some examples, the recombinant vector may contain a variety of elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site. The vector may contain a nucleic acid of the present disclosure so as to facilitate introduction into a cell for expression. The vector may comprise expression control elements, such as a promoter, terminator and / or enhancer, operably linked to the nucleic acid.

[0044] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, yeast, plant cells, animal cells, and the like.

[0045] Suitable yeasts for use in the present invention include any of the following yeasts or their derivatives or hybrids: Candida (e.g., Candida albicans ( Candida albicans ), Candida etschulensis (Candida) Etchell's )、Candida guilliermonas( Candida guilliermondii )、Candida albicans( White humble ), Candida lipolytica ( Candida lipolytica )、Candida pseudoglabrata( White orthopsilosis )、Candida palmitoleracea( Candida palmioleophila ), Candida pseudotropicalis ( White pseudotropical ), Candida species, Candida utilis ( Useful Candida )、Candida variabilis( Versatile white ) and Cladosporium ( Cladosporium ), Cryptococcus ( Cryptococcus ) (e.g., Cryptococcus terrestris ( Cryptococcus terricolus )、Cryptococcus flexus( Cryptococcus curvatus )), Debaryomyces spp. Debaryomyces ) (e.g., Debaryomyces hansenii ( Debaryomyces hansenii ) and Endosporium ( Endomyces ) (e.g., Endosporium lipolyticum ( Endomyces vernalis )), Pseudomonas ( Endomycopsis ) (e.g., Pseudomonas lipolytica ( Endomycopsis vernalis ) and Pseudomonas ( Hermitage ) (e.g., Pseudomonas aeruginosa ( Eremothecium ashbyii ) and Hansenula ( Little Hansen ) (e.g., Hansenula species, Hansenula polymorpha ( Hansenula polymorpha )), Kluyveromyces ( Kluyveromyces ) (e.g., Kluyveromyces species, Kluyveromyces lactis ( Kluyveromyces lactis ), Kluyveromyces marxianus var. lactis ( Kluyveromyces marxianus var.lactis )、Kluyveromyces marxianus( Kluyveromyces Marxian )、Thermotolerant Kluyveromyces( Kluyveromyces thermotolerans ) and Lipomyces spp. Lipomyces ) (e.g., Lipomyces staryi ( Lipomyces starkeyi )、Oleaginous yeast( Lipomyces fat-bearing ))、Ogata yeast( Ogataea ) (e.g., Microsaccharomyces cerevisiae ( Ogataea minuta ) and Pichia ( Peach ) (e.g., Pichia species, Pichia pastoris ( Komagataella phaffii , also known as Peach shepherd )、Pichia finnisha( Finnish Pichia ), Pichia trehalose-loving yeast ( Peach trehalophila )、Pichia pastoris( Pichia koclamae )、Pichia pastoris( Peach membrane-forming )、Pichia pastoris( A few minutes )、Pichia pastoris( Pichia lindneri )、Pichia cactus( Prickly pear )、Thermotolerant Pichia pastoris( Pichia thermotolerans )、Pichia willowii( Willow tree )、Pichia pine and oak yeast( Pichia guercuum )、Pichia pastoris( Peach piper )、Pichia stipitis( Pichia stiptis )、Pichia methanolica( Pichia methanolica )), Rhodosporidium ( Rhodosporidium ) (e.g., Rhodosporidium toruloides ( Rhodosporidium toruloides ) , Rhodotorula ( Rhododendron ) (e.g., Rhodotorula species, Rhodotorula gracilis (Rhodotorula gracilis) )、Rhodotorula glutinosae( Rhodotorula glutinosa )、Red yeast rice(Rhodotorula graminis ) and Saccharomyces spp. Saccharomyces ) (e.g., Saccharomyces species, Saccharomyces bayanus ( Saccharomyces bayanus )、Saccharomyces cerevisiae( Saccharomyces beticus )、Saccharomyces cerevisiae( Saccharomyces cerevisiae )、Saccharomyces cerevisiae( Saccharomyces chevalieri )、Saccharifying yeast( Saccharomyces diastaticus )、Wine yeast( Saccharomyces ellipsoideus )、Oligospora yeast( Saccharomyces exiguus )、Floral yeast( Saccharomyces florentinus ), Saccharomyces fragilis ( Saccharomyces fragilis )、Pasteurella Saccharomyces pastorianus )、Saccharomyces pombe( Saccharomyces pombe )、Sake yeast( Saccharomyces sake )、Grape juice yeast( Saccharomyces uvarum ) and Sporobloids ( Sporobolomyces ) (e.g., S. Sporobolomyces roseus ) and the genus Desmosaccharomyces ( Sporidiobolus ) (e.g., Johnson's lock yeast ( Sporidiobolus johnsonii )、Salmon-lock yeast( Sporidiobolus salmonicolor )), Trichosporon spp. Trichosporon ) (e.g., Trichosporon cocoa butter-decomposing yeast ( Trichosporon cacaoliposimilis )、New species of Lipomyces oleifera( Trichosporon oleaginosus sp.nov. )、New species of cocoa butter decomposing yeast ( Trichosporon cacaoliposimilis sp.nov. )、Trichosporon fasciatus( Trichosporon gracile )、Trphombium euonymus( Trichosporon dulcitum ), Trichosporon gibbsii ( Trichosporon jirovecii )、Insect Trichosporon ( Trichosporon insectorum ))、Xanthophyllotoxin yeast( Xanthophyllomyces (For example, dendritic lutein yeast ( Xanthophyllomyces dendrorhous )), Yarrowia ( Yarrowia (For example, Yarrowia lipolytica ( Yarrowia lipolytica )), and Zygosaccharomyces ( Zygosaccharomyces ) (e.g., Zygosaccharomyces rouxii ( Zygosaccharomyces rouxii ).

[0046] The commonly used yeast expression system is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) expression system and methanol nutritional yeast expression system:

[0047] ① Saccharomyces cerevisiae expression system: Saccharomyces cerevisiae has been used in the brewing and bread industries for thousands of years. It is considered a GRAS (generally recognized as safe) organism that does not produce toxins and has been confirmed as a safe organism by the US FDA. However, Saccharomyces cerevisiae is difficult to culture at high density, has low secretion efficiency, and almost does not secrete foreign proteins with a molecular weight greater than 30 kD. It also cannot correctly glycosylate the foreign proteins expressed, and the C-terminus of the expressed proteins is often truncated. Therefore, Saccharomyces cerevisiae is generally not used as a host strain for recombinant protein expression.

[0048] ②Methanol nutritional yeast expression system: Methanol yeast expression system is the most widely used yeast expression system. Methanol yeast mainly includes Hansen yeast ( Hansenula ), Pichia ( Pichia ), Torulopsis spp. Torulopsis ) and most of them are used in Pichia pastoris. The expression vector of methanol yeast is an integrative plasmid, which contains sequences homologous to yeast chromosomes, so it is relatively easy to integrate into yeast chromosomes. Most methanol yeast expression vectors contain methanol yeast alcohol oxidase gene-1 (AOX1). Under the action of the promoter of this gene (PAOX1), foreign genes can be expressed. Methanol yeast generally grows in a glycerol-containing medium to a high concentration, and then uses methanol as a carbon source to induce the expression of foreign proteins, which can greatly increase the expression yield. The yield of exogenous proteins expressed by methanol yeast can often reach grams. Compared with brewer's yeast, its post-translational processing is closer to mammalian cells and does not undergo hyperglycosylation.

[0049] The vector matches the host cell and is divided into prokaryotic (bacterial) expression vectors, yeast expression vectors, plant expression vectors, mammalian expression vectors, insect expression vectors, etc. according to the host. The vector contains foreign gene fragments. Through the mediation of the vector, the foreign gene can be expressed in the host. In some embodiments, the expression vector used for Pichia pastoris in the present disclosure is preferably a pPIC9K vector and a pGAPZαA vector.

[0050] As used herein, the term "codon optimization" refers to genes or coding regions of nucleic acid molecules used to transform various hosts, and refers to codon changes in genes or coding regions of nucleic acid molecules that reflect the typical codon usage of the host organism without changing the polypeptide encoded by the DNA. Such optimization includes replacing at least one or more than one or a large number of codons with one or more codons that are more frequently used in the genes of the organism. By utilizing knowledge of codon usage or codon preference in each organism, a person of ordinary skill in the art can apply these frequencies to any given polypeptide sequence and generate nucleic acid fragments that encode the polypeptide but use the codon-optimized coding region of the optimal codon for a given species. Codon-optimized coding regions can be designed by various methods known to those skilled in the art.

[0051] As used herein, the term "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0052] As used herein, "expression cassette" refers to a gene expression system that contains all necessary elements for expressing a target polypeptide, which generally includes the following elements: a promoter, a gene sequence encoding a polypeptide, a terminator, and optionally a signal peptide coding sequence, etc. These elements are operably linked.

[0053] As used herein, "promoter" refers to a nucleic acid sequence that is usually present upstream (5' end) of the target gene coding sequence and can guide the transcription of the nucleic acid sequence into mRNA. Generally, the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for the correct initiation of transcription.

[0054] As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is exogenous to a gene of interest if the combination of the promoter and the gene of interest sequence does not normally occur in nature. A particular sequence is "exogenous" to the cell into which it is inserted.

[0055] As used herein, "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence, so that the transcription of the nucleic acid sequence is guided by the promoter region, and thus the promoter region is "operably linked" to the nucleic acid sequence.

[0056] (II) Detailed technical solution

[0057] The present invention mainly improves the expression level of β-lactoglobulin by integrating a certain number of copies of β-lactoglobulin gene expression frame into the genome and co-expressing the ATPase ssa4 gene involved in protein folding and stress response.

[0058] In one aspect, the present disclosure provides a β-lactoglobulin expression vector comprising a plurality of β-lactoglobulin expression cassettes, wherein the expression cassettes include a promoter, an α signal peptide coding sequence, a β-lactoglobulin coding sequence and a terminator.

[0059] In some embodiments, the β-lactoglobulin is bovine β-lactoglobulin, and the amino acid sequence is:

[0060] LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI* (SEQ ID NO: 1).

[0061] In some embodiments, the β-lactoglobulin is secreted outside the host cell under the guidance of an exogenous signal peptide, preferably, an improved α secretion signal peptide. The original α mating factor signal peptide of Saccharomyces cerevisiae is widely used in Pichia pastoris to guide the secretion expression of exogenous proteins. Its terminal amino acid sequence is KREAEA, which is finally cut twice by the dipeptidase Ste13 to remove two repeated EAs. However, since its cutting efficiency is affected by many factors, it will cause residual amino acid sequences. The C-terminal amino acid of the modified α signal peptide is KR, which can be recognized and effectively removed by the Kex2 protease of Pichia pastoris, so that the secreted exogenous protein does not contain redundant amino acids. In some embodiments, the amino acid sequence of the α signal peptide disclosed in the present invention is:

[0062] MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 2).

[0063] In some embodiments, such as Figure 1 As shown, the β-lactoglobulin expression frame includes AOX1 promoter, α signal peptide coding sequence, β-lactoglobulin coding sequence, and AOX1 terminator from 5' end to 3' end.

[0064] In some embodiments, the β-lactoglobulin and the modified α secretion signal peptide are "coupled optimized" according to the codon usage frequency of the Pichia pastoris host bacteria, and the nucleotide sequence is:

[0065] (SEQ ID NO: 3).

[0066] In some embodiments, the β-lactoglobulin expression vector comprises 2, 3, 4, 5 or 6 β-lactoglobulin expression cassettes. Preferably, the β-lactoglobulin expression vector comprises 3 β-lactoglobulin expression cassettes.

[0067] In some embodiments, the expression vector is based on pPIC9K as the starting vector, and the synthetic β-lactoglobulin and the modified α secretion signal peptide are used as templates for PCR amplification, and the vector is directionally connected to the downstream of the AOX1 promoter of the plasmid pPIC9K through seamless cloning technology to construct a recombinant plasmid pPIC9K-α1-BLG; a complete expression frame including the AOX1 promoter, secretion signal peptide, lactoglobulin gene and AOX1 terminator is amplified, and multiple expression frames are sequentially connected in series to the plasmid pPIC9K-α1-BLG to obtain a gene recombination plasmid pPIC9K-α1-3BLG containing multiple copies, preferably 3 copies.

[0068] In another aspect, the present disclosure provides a vector combination, which includes the aforementioned β-lactoglobulin expression vector and an Ssa4 expression vector.

[0069] Ssa4 is an ATPase involved in protein folding and response to stress. In some embodiments, the Ssa4 expression vector includes the Pichia pastoris SSA4 gene, GenBank number CAY70247.1, and its nucleotide sequence is as follows:

[0070]

[0071] In some embodiments, the Ssa4 expression vector is based on pGAPZαA and is expressed by a GAP constitutive promoter.

[0072] In another aspect, the present disclosure provides a host cell, comprising the aforementioned β-lactoglobulin expression vector or the aforementioned vector combination.

[0073] In some embodiments, the host cell comprises a eukaryotic cell or a prokaryotic cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the fungal cell is selected from Aspergillus niger ( Aspergillus niger ), Aspergillus niger var. awamori ( Aspergillus niger var.awamori ), Aspergillus oryzae ( Aspergillus oryzae ), Candida guillimonensis ( Candida guilliermondii ), Candida lipolytica ( Candida lipolytica ), Candida tropicalis ( Candida pseudotropicalis ), Candida utilis ( Candida utilis )、Crysalis Endothia parasitica )、Pseudomonas aeruginosa( Eremothecium ashbyii )、Fusarium moniliforme( Fusariummoniliforme )、Kluyveromyces lactis( Kluyveromyces lactis )、Kluyveromyces marxianus( Kluyveromyces marxianus ), Morteirellavinaceae var raffinoseutilizer ( Morteirella vinaceae var. raffinoseutilizer )、Mucor miehei( Mucormycosis ), Mucor miehei var. Cooney et Emerson ( Mucer miehei var. Cooney and Emerson )、 Mucor pusillus Lindt Penicillium roqueforti Penicillium roquefortii )、Pichia pastoris( Shepherd's pie )、Rhizopus niveus( Rhizopus niveus )、Saccharomyces cerevisiae( Saccharomyces cerevisiae ), Saccharomyces fragilis ( Saccharomyces fragile )、Trichoderma reesei( Trichoderma reesei )、Thermomycete Myceliophthora thermophile )and Chrysosporium lucknowense .

[0074] In some embodiments, the host cell is Pichia pastoris, preferably Pichia pastoris ( Komagataella phaffii , also known as Shepherd's pie ), more preferably Pichia pastoris X33, GS115, SMD1168, KM71 or KM71H strains.

[0075] In some preferred embodiments, the host cell comprises the aforementioned vector combination, i.e., the aforementioned β-lactoglobulin expression vector and Ssa4 expression vector. In some preferred embodiments, the host cell comprises the β-lactoglobulin expression vector pPIC9K-α1-3BLG and the Ssa4 expression vector pGAPZαA-Ssa4. In some preferred embodiments, the host cell is Pichia pastoris ( Komagataella phaffii , also known as Shepherd's pie ), which is deposited in the China Type Culture Collection at Wuhan University, with the deposit number being CCTCC M 20242480 and the deposit date being November 7, 2024.

[0076] In another aspect, the present disclosure provides a method for constructing a host cell expressing β-lactoglobulin, comprising:

[0077] Transform the host cell with the aforementioned β-lactoglobulin expression vector and Ssa4 expression vector simultaneously; or

[0078] The host cells were transformed with the aforementioned β-lactoglobulin expression vector and Ssa4 expression vector in sequence.

[0079] In some embodiments, the method comprises:

[0080] (1) transforming host cells with the aforementioned β-lactoglobulin expression vector and screening transformants;

[0081] (2) Transforming the transformant obtained in step (1) with the Ssa4 expression vector to obtain a host cell that co-expresses β-lactoglobulin and Ssa4.

[0082] In some embodiments, the host cell is Pichia pastoris, preferably Pichia pastoris, more preferably Pichia pastoris X33, GS115, SMD1168, KM71 or KM71H strain.

[0083] In some embodiments, the multi-copy recombinant expression vector pPIC9K-α1-3BLG constructed in vitro is linearized by enzyme digestion and then electroporated into the competent cell of Pichia pastoris GS115, and the recombinant Pichia pastoris strain with a high copy number of the β-lactoglobulin gene is screened and transformed. On this basis, the gene encoding the overexpressed molecular chaperone Ssa4 is integrated, and then fermentation culture verification is carried out to finally obtain an engineered Pichia pastoris strain that efficiently expresses β-lactoglobulin.

[0084] In another aspect, the present disclosure provides a method for producing β-lactoglobulin, comprising: culturing the aforementioned host cell to obtain a culture comprising the β-lactoglobulin. In some embodiments, the host cell is Pichia pastoris ( Peach), the Pichia yeast has a deposit number of CCTCC M 20242480; the culture supernatant contains the β-lactoglobulin. In some embodiments, the method further comprises the step of separating and purifying the β-lactoglobulin from the culture supernatant.

[0085] In some embodiments, the culture temperature is 30°C. In some embodiments, the culture is cultured at 170-260 rpm, preferably at 200-220 rpm. In some embodiments, the culture is cultured in a growth medium for 16-24 hours, the cells are collected by centrifugation, inoculated into an induction medium and cultured for 120 hours, and 1% (V / V) methanol is added every 24 hours.

[0086] In some embodiments, the culture process is: activating any obtained Pichia yeast strain to prepare a seed liquid, inoculating it into a BMGY growth medium, culturing it at 30°C and 170-260rpm for 16-24h, collecting the bacteria by centrifugation, inoculating it into a BMMY induction medium, inducing and culturing it at 30°C and 170-260rpm for 120h, and adding 1% (V / V) methanol every 24h, centrifuging the fermentation broth, removing the bacterial precipitate, and the fermentation supernatant contains the β-lactoglobulin.

[0087] In some embodiments, the growth medium, BMGY components are: 1% yeast powder, 2% peptone, 1.34% YNB, 1% (V / V) glycerol, and 10% PBS pH 6.0 buffer solution.

[0088] In some embodiments, the induction medium is BMMY, the components of which are: 1% yeast powder, 2% peptone, 1.34% YNB, 1% methanol, and 10% PBS pH 6.0 buffer solution.

[0089] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present disclosure, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in the publication of many publications.

[0090] The experimental methods in the following examples without specifying specific conditions are generally carried out according to conventional conditions in the field of genetic engineering or according to conditions recommended by the manufacturers.

[0091] Plasmid construction was performed using the seamless cloning kit from Novazonics.

[0092] The tool enzymes used were purchased from Tiangen Biochemical Technology (Beijing) or TaKaRa Biotechnology (Dalian). The specific reaction conditions and methods used were referred to the product manual.

[0093] YPD medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L;

[0094] YND medium: glucose 20 g / L, YNB 6.7 g / L, agar 15 g / L;

[0095] DTT buffer: Dissolve 3.09 g DTT in 20 mL 0.01 mol / L sodium acetate solution (pH 5.2);

[0096] BMGY growth medium includes: 1% yeast powder, 2% peptone, 1.34% YNB, 1% (V / V) glycerol, 10% PBS pH 6.0 buffer solution;

[0097] The BMMY induction medium includes: 1% yeast powder, 2% peptone, 1.34% YNB, 1% methanol, and 10% PBS pH 6.0 buffer solution.

[0098] The above culture media were all sterilized by autoclave at 121°C for 20 min. In particular, the glucose solution was sterilized by autoclave at 115°C for 30 min. The above culture media solvents were all deionized water or tap water.

[0099] Example 1. Cloning of β-lactoglobulin gene

[0100] According to the bovine β-lactoglobulin gene (Gene ID: 280838) published by NCBI, its amino acid sequence was analyzed and its own signal peptide was removed. Its amino acid sequence is shown in SEQ ID NO: 1.

[0101] The terminal amino acid of the improved α1 signal peptide is KR, which is obtained by removing the terminal EAEA from the original α signal peptide of Saccharomyces cerevisiae. Its amino acid sequence is shown in SEQ ID NO:2.

[0102] According to the codon preference of the Pichia pastoris host, the β-lactoglobulin gene and the improved α1 signal peptide were subjected to codon "coupling optimization" and fully synthesized by BGI, named α1-BLG, and its nucleotide sequence is shown in SEQ ID NO: 3.

[0103] According to the gene sequence, the upstream and downstream primers α1-F (SEQ ID NO: 7) and BLG-R (SEQ ID NO: 6) were designed with the sequences shown in Table 1. The gene fragment of α1-BLG was cloned by PCR reaction. The PCR conditions were: 98°C for 3min, 98°C for 30s, 55°C for 90s, 72°C for 90s, and 32 cycles. The PCR amplification system was 50μl: 1μl template, 2μl upstream and downstream primers, 25μl high-fidelity premixed enzyme, and 20μl sterilized double distilled water.

[0104] Table 1. Primers used to construct β-lactoglobulin expression strains

[0105]

[0106] Example 2. Construction of recombinant expression vector

[0107] (1) Primers 9K-F (SEQ ID NO: 15) and 9K-R (SEQ ID NO: 16) with sequences as shown in Table 1 were designed to amplify the commercial vector pPIC9K (purchased from Invitrogen). The cloned gene fragment and vector fragment were recovered by agarose gel, connected to the downstream of the AOX1 promoter of pPIC9K using a seamless cloning kit, and transformed into DH5α competent cells (purchased from Sangon Biotech (Shanghai) Co., Ltd., Catalog No. B528413) to obtain the single-copy recombinant plasmid pPIC9K-α1-BLG.

[0108] (2) Using the correctly sequenced plasmid in (1) as a template, amplify the expression cassette containing the promoter, signal peptide, target gene and terminator, and connect two identical expression cassettes in series to pPIC9K-α1-BLG to obtain a three-copy recombinant expression plasmid pPIC9K-α1-3BLG, the map of which is shown in the figure. Figure 1 shown.

[0109] Example 3. Construction and screening of yeast strains expressing recombinant β-lactoglobulin

[0110] (1) Linearization of recombinant plasmid pPIC9K-α1-3BLG: 10 μg of recombinant plasmid pPIC9K-α1-3BLG was linearized by Sal I digestion at 37°C for 4 h. The digestion product was verified by agarose gel electrophoresis. The product was recovered and the DNA concentration was determined. The plasmid fragment was diluted to a concentration of 1 μg / μL and stored for later use.

[0111] (2) Preparation of competent cells of Pichia pastoris GS115: Pick a single colony of GS115 (purchased from Invitrogen) and culture it in 10 ml YPD liquid medium at 30°C overnight with shaking. Then, pipette 100 μl of the colony into 100 mL YPD liquid medium and culture until OD600 = 1.5. Centrifuge at 5000 rpm at 4°C for 3 min to collect the cells. Use pre-cooled 0.1 M LiAc buffer and 100 μL DTT buffer, pipette gently to mix, place the centrifuge tube in a 30°C shaker at 200 rpm for 30-45 min, centrifuge at 4°C for 3 min, then wash the cells twice with pre-cooled 1 M sorbitol buffer, and finally resuspend in 1 ml pre-cooled 1 M sorbitol buffer and dispense 80 μL / tube.

[0112] (3) Electroporation: Take 80 μL of competent cells and add 10 μL of linearized recombinant plasmid pPIC9K-α1-3BLG to mix evenly, and transfer to a 2 mm electroporation cup that has been pre-bathed on ice, and place the electroporation cup containing the mixture on ice for 5 minutes. Adjust the parameters of the electroporation instrument and set it to the "PIC" position. The electroporation conditions are 1500V, 25 microfarads of capacitance, 200 ohms of resistance, and the time is about 5 milliseconds. After electroporation, quickly add 1 ml of 1M sorbitol solution pre-cooled on ice to the electroporation cup, gently blow and mix, and transfer the mixture to a centrifuge tube containing YPD liquid culture medium. After culturing at 30°C for 1-2 hours, centrifuge at 3000 rpm for 5 minutes. Discard the supernatant, resuspend the mixed bacteria with YPD liquid culture medium, and spread on the YND plate. The cells were cultured in a 30°C incubator for 2-4 days. When a single colony was grown, colony PCR was performed using primers LG-yz-F (SEQ ID NO: 21, shown in Table 1) and LG-yz-R (SEQ ID NO: 22, shown in Table 1) to verify whether the transformant was a correct transformant. The correct transformant was the recombinant expression strain containing the pPIC9K-α1-3BLG plasmid.

[0113] Pick a single colony from the above YND plate, spot-spread it on a YPD plate containing 1, 2, 3, and 4 mg / mL G418, and incubate it upside down at 30°C for 3-5 days until a single colony grows out. The recombinant strain may obtain integrated plasmids with different copy numbers during homologous recombination integration. R The gene confers resistance to G418 on yeast cells, and the number of integrated plasmids is positively correlated with the resistance level of G418. Transformants with different copy numbers are obtained by growing them on YPD plates containing different concentrations of G418, thereby obtaining multi-copy, highly expressed genetically engineered strains.

[0114] Example 4. Analysis of β-lactoglobulin copy number in recombinant engineered strains

[0115] In the strains obtained by screening with different concentrations of G418 in Example 3 above, the copy numbers of β-lactoglobulin were different. qPCR quantitative analysis using primers BLG-F (SEQ ID NO: 5) and BLG-R (SEQ ID NO: 6) shown in Table 1 showed that the copy numbers varied from 1 to 8. The results are shown in Table 2.

[0116] Table 2. Effects of different antibiotic concentrations on β-lactoglobulin copy number

[0117]

[0118] Example 5. Amplification of the molecular chaperone ssa4 gene fragment and construction of the expression plasmid

[0119] Using the Pichia pastoris genome as a template, the molecular chaperone Ssa4 encoding gene was amplified using primers Ssa4-F (SEQ ID NO: 17) and Ssa4-R (SEQ ID NO: 18) whose sequences are shown in Table 1. The nucleotide sequence of the gene is shown in SEQ ID NO: 4.

[0120] The fragment was recovered by gel and then connected to the downstream of the GAP promoter of the expression vector pGAPZαA (purchased from Invitrogen). The plasmid was digested with the restriction endonuclease BamHI and verified by PCR and sequencing, and the linearized fragment was purified using a product recovery kit. The obtained pGAPZαA-Ssa4 linear plasmid vector was electroporated into the competent state of the recombinant expression strain obtained in the above Example 3 using the same electroporation transformation method as in Example 3, and was spread on a plate containing bleomycin. After a single colony grew, the primers Ssa4-yz-F (SEQ ID NO: 23, shown in Table 1) and Ssa4-yz-R (SEQ ID NO: 24, shown in Table 1) were used to perform colony PCR to verify whether it was a correct transformant. The correct transformant was the recombinant expression strain pPIC9K-3BLG-Ssa4 that co-expressed the molecular chaperone Ssa4.

[0121] Example 6. Inducible expression of β-lactoglobulin

[0122] Pick the single colony verified correctly in Implementation 3 and Example 5 and place it in 20 mL YPD medium, and culture it overnight at 30°C, 200 rpm. The next day, transfer the bacterial solution to 50 mL BMGY medium and culture it at 30°C, 200 rpm for 24 h. Collect the bacteria by centrifugation at 3000 rpm for 5 min, wash them 3 times with sterile water to remove residual glycerol, then resuspend them in 25 mL BMMY medium, transfer them to a 250 mL conical flask, and culture them at 30°C, 220 rpm for 5 days, and add 1% methanol every 24 hours to allow induction to continue.

[0123] After the fermentation, the supernatant was collected by centrifugation at 6000 rpm for 5 min to obtain the fermentation supernatant containing bovine β-lactoglobulin, and the expression of β-lactoglobulin was detected by SDS-PAGE. Using β-LG as the standard (Sigma, L3908), the detection standard curve was established by high performance liquid chromatography, and the production of β-lactoglobulin secreted by the recombinant strain was detected.

[0124] The results showed that the transformants screened on YPD plates with different G418 concentrations had different β-lactoglobulin copy numbers, among which the β-lactoglobulin production in the fermentation supernatant containing 8 copies was the highest ( Figure 2 On this basis, co-expression ssa4 After the gene was added, the expression level of β-lactoglobulin increased to 1.31 g / L, which was 2.47 times higher than the initial single-copy strain's yield of 0.53 g / L ( Figure 3B ), the strain was deposited in the China Type Culture Collection at Wuhan University, with the deposit number CCTCC M 20242480 and the deposit date being November 7, 2024.

[0125] All the above embodiments are only examples for clearly explaining the actual operation, and are not intended to limit the implementation scheme. Those skilled in the art can make other different forms of changes or modifications based on the above description. It is not necessary and impossible to list all the implementation schemes here, but the obvious changes derived from them are still within the protection scope of the invention.

Claims

1. A Pichia pastoris yeast ( Komagataella phaffii ), which is deposited in the China Type Culture Collection at Wuhan University with the deposit number CCTCC M 20242480.

2. A method for producing β-lactoglobulin, comprising: Cultivate the Pichia pastoris according to claim 1 to obtain a culture containing the β-lactoglobulin. 3 . The method according to claim 2 , further comprising the step of separating and purifying the β-lactoglobulin from the culture supernatant.

4. The method according to claim 2, wherein: The culture temperature is 30°C.

5. The method according to claim 2, wherein: The culture is carried out at 170-260 rpm.

6. The method according to claim 2, wherein: The culture is carried out at 200-220 rpm.

7. The method according to any one of claims 2 to 6, wherein: The culture is carried out in a growth medium for 16-24 hours, the bacteria are collected by centrifugation, inoculated into an induction medium and cultured for 120 hours, and 1% (V / V) methanol is added every 24 hours.

8. The method according to claim 7, wherein: The growth medium is BMGY medium, and the components of the BMGY medium are: 1% yeast powder, 2% peptone, 1.34% YNB, 1% (V / V) glycerol, and 10% PBS pH 6.0 buffer solution.

9. The method according to claim 7 or 8, wherein: The induction culture medium is BMMY culture medium, and the components of the BMMY culture medium are: 1% yeast powder, 2% peptone, 1.34% YNB, 1% methanol, and 10% PBS pH 6.0 buffer solution.

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