Strain for producing alpha-glucosidase as well as construction method and application of strain

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a strain for producing alpha-glucosidase as well as a construction method and application of the strain. Alpha-glucosidase from aspergillus niger is transferred into a pichia pastoris host cell, a pichia pastoris engineering strain PSC-alpha-glu with ethanol as a substrate is constructed, and efficient biosynthesis from ethanol to alpha-glucosidase is realized for the first time. According to the present invention, the pichia pastoris engineering strain for inducing the expression of alpha-glucosidase by using the food-grade ethanol and the culture method are constructed, the efficient heterologous recombinant expression of the alpha-glucosidase is achieved, the yield achieves the 10 g level per liter, the large-scale fermentation is easily performed, the cost is low, the safety is high, and the application in the subsequent fields such as food or medicine is convenient.

Description

Technical Field

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

[0002] α-Glucosidase (EC 3.2.1.20), also known as α-D-glucoside hydrolase, can catalyze the hydrolysis of oligosaccharides to produce glucose. α-Glucosidase is widely distributed in nature, with various types and properties. α-Glucosidase from Aspergillus, Saccharomyces cerevisiae, and Bacillus can not only hydrolyze glycosides, but also synthesize certain oligosaccharides through transglycosidation reactions. Among them, Aspergillus niger ( Aspergillus niger )α-Glucosidase has great application prospects in industry due to its excellent transglycosidation ability.

[0003] Aspergillus niger α-glucosidase can cut the α-1,4 glycosidic bond of maltose and transfer the free glucose residue to other sugar substrates in the form of α-1,6 to form isomaltooligosaccharides (IMOs). IMOs can promote the growth and reproduction of beneficial bacteria in the human body, and have the functions of preventing dental caries, lowering cholesterol, improving intestinal function, and enhancing body immunity. They are widely used in the food and pharmaceutical industries. However, due to the low expression level of natural Aspergillus niger α-glucosidase, it is difficult to achieve a major breakthrough in enzyme production by simply screening bacteria and optimizing fermentation conditions. Traditional mutagenesis breeding has a certain degree of randomness, a long development cycle, and it is difficult to quickly increase the expression level in a short period of time. In order to overcome the low production of α-glucosidase in wild Aspergillus niger strains, the use of genetic engineering methods to achieve efficient expression of α-glucosidase has become a hot topic in current research.

[0004] Pichia pastoris ( Shepherd's pie, Also known as Komagataella phaffii ) The expression is genetically stable, the protein can be post-translationally processed, the expression efficiency is high, the product can be secreted into the culture medium and is suitable for high-density fermentation. Therefore, the Pichia pastoris expression system is considered to be a secretory expression system that is more suitable for industrial production. At present, Pichia pastoris generally uses methanol as an inducer to induce the expression of exogenous genes, but methanol is toxic, which limits its application in food. Ethanol is widely used as a raw material in food production. Therefore, the present disclosure aims to construct a high-yield strain of Pichia pastoris that produces α-glucosidase by converting ethanol. On the one hand, it broadens the route of ethanol utilization and α-glucosidase production, and on the other hand, it explores the application potential and prospects of Pichia pastoris as a microbial cell factory. Summary of the invention

[0005] The purpose of the present disclosure is to provide a vector and a strain for efficiently expressing α-glucosidase, to increase the expression amount of α-glucosidase, and to produce α-glucosidase by ethanol conversion.

[0006] In order to achieve the above technical objectives, the present disclosure proposes the following technical solutions: In one aspect, the present disclosure provides an α-glucosidase expression vector, which includes a PSC sequence, an α signal peptide coding sequence and an α-glucosidase coding sequence; wherein the PSC sequence is an artificial synthetic promoter with a nucleotide sequence as shown in SEQ ID NO: 1.

[0007] In another aspect, the present disclosure provides a host cell comprising the aforementioned α-glucosidase expression vector.

[0008] In another aspect, the present disclosure provides a method for constructing a host cell expressing α-glucosidase, comprising: (1) connecting the coding sequence of α-glucosidase to a vector including a PSC sequence and an α signal peptide coding sequence to obtain a recombinant expression vector, wherein the PSC sequence is an artificial synthetic promoter having a nucleotide sequence as shown in SEQ ID NO: 1; (2) transforming the recombinant expression vector obtained in step (1) into a host cell; (3) Screening and obtaining host cells with high expression levels of the α-glucosidase.

[0009] In another aspect, the present disclosure provides a method for producing α-glucosidase, comprising culturing the aforementioned host cell to obtain a culture comprising the α-glucosidase.

[0010] In another aspect, the present disclosure provides use of the aforementioned expression vector, the aforementioned host cell or the α-glucosidase produced by the aforementioned method in the production of food, feed or medicine.

[0011] The present invention transfers α-glucosidase from Aspergillus niger into Pichia pastoris host cells, constructs a Pichia pastoris engineered strain PSC-α-glu using ethanol as a substrate and an inducer, and realizes efficient biosynthesis of α-glucosidase from ethanol for the first time. The present invention constructs a Pichia pastoris engineered strain and a culture method for inducing the expression of α-glucosidase using food-grade ethanol, realizes efficient heterologous recombinant expression of α-glucosidase, and the yield reaches ten grams per liter. It is easy to ferment on a large scale, low cost, high safety, and more convenient for subsequent applications in the fields of food or medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a map of the recombinant plasmid vector pPIC9K-PSC-α-glu.

[0013] Figure 2 The SDS-PAGE test results for screening the recombinant Pichia pastoris genetically engineered bacteria, M is a 10-170 kDa protein marker, and lanes 1-11 are different transformants of the recombinant Pichia pastoris genetically engineered bacteria.

[0014] Figure 3 is the enzyme activity of α-glucosidase at different temperatures.

[0015] Figure 4 is the enzyme activity of α-glucosidase under different pH conditions.

[0016] Biological Deposit Description 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 M20242483, the deposit date November 7, 2024, and the culture name Komagataella phaffii CJ-PP-GU. DETAILED DESCRIPTION

[0017] 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.

[0018] 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.

[0019] 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.

[0020] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0021] (I) Definitions or terms 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.

[0022] 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.

[0023] 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).

[0024] 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."

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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).

[0030] 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 with 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 mentioned 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).

[0031] 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.

[0032] 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.

[0033] 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).

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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).

[0038] 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.

[0039] 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.

[0040] 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 ) and Rhodotorula ( Rhodotorula ) (e.g., Rhodotorula species, Rhodotorula gracilis (Rhodotorula gracilis )、Rhodotorula glutinosae( Rhodotorula glutinis )、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 ).

[0041] The commonly used yeast expression system is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) expression system and methanol nutritional yeast expression system: 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] In the present disclosure, the "ethanol induction" or "ethanol culture" refers to adding an appropriate amount of ethanol as a carbon source and inducer to the Pichia culture medium to replace conventional carbon sources such as glucose and glycerol and conventional inducers such as methanol. However, for methanol-nutritional yeasts such as Pichia, "ethanol induction" is a key difference, so it is difficult but significant to transform the expression system and optimize the elements suitable for expression. For the production of α-glucosidase, ethanol induction safety is obviously more ideal.

[0051] (II) Detailed technical solution The present invention provides an expression vector of an α-glucosidase gene, and constructs an engineered Pichia pastoris strain that can produce high α-glucosidase by ethanol fermentation, so that the strain can secrete and express α-glucosidase efficiently, and can produce α-glucosidase by high-density fermentation for use in food and other fields.

[0052] In one aspect, the present disclosure provides an α-glucosidase expression vector, which includes a PSC sequence, an α signal peptide coding sequence and an α-glucosidase coding sequence; wherein the PSC is an artificial synthetic promoter, and the artificial synthetic promoter can be combined with an artificial transcription activator in a modified chassis cell, wherein the artificial transcription activator is expressed by an ethanol-inducible promoter (see patent publication CN118726120A), thereby achieving ethanol-induced recombinant expression of α-glucosidase, and the PSC nucleotide sequence is as follows: TGCTGTATATAAAACCAGTGGTTATATGTACAGTACGTCGAGTGCTGTATATAAAACCAGTGGTTATATGTACAGTACGTCGAGTGCTGTATATAAAACCAGTGGTTATATGTACAGTACGTCGAGCTAACCCCTACTTGACAGCAATATATAA ACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAA (SEQ ID NO: 1).

[0053] In some embodiments, the α-glucosidase is derived from Aspergillus niger, and its amino acid sequence is as follows: Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser Asn Glu AlaThr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala Asp Gly Ala Trp Val SerGly Ala Asp Ser Gly Ile Val Val Ala Ser Pro Ser Thr Asp Asn Pro Asp Tyr PheTyr Thr Trp Thr Arg Asp Ser Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe ArgAsn Gly Asp Thr Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala IleVal Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu Gly GluPro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp Gly Arg Pro Gln ArgAsp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile Gly Phe Gly Gln Trp Leu Leu AspAsn Gly Tyr Thr Ser Thr Ala Thr Asp Ile Val Trp Pro Leu Val Arg Asn Asp LeuSer Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val AsnGly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser AlaPhe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln Ala Pro Glu IleLeu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe Ile Leu Ala Asn Phe Asp SerSer Arg Ser Gly Lys Asp Ala AsnThr Leu Leu Gly Ser Ile His Thr Phe Asp ProGlu Ala Ala Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala AsnHis Lys Glu Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu SerAsp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr Asn Gly AsnPro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu Tyr Asp Ala Leu Tyr GlnTrp Asp Lys Gln Gly Ser Leu Glu Val Thr Asp Val Ser Leu Asp Phe Phe Lys AlaLeu Tyr Ser Asp Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser SerIle Val Asp Ala Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr HisAla Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu Gln LeuSer Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr Ala Asn Asn Arg ArgAsn Ser Val Val Pro Ala Ser Trp Gly Glu Thr Ser Ala Ser Ser Val Pro Gly ThrCys Ala Ala Thr Ser Ala Ile Gly Thr Tyr Ser Ser Val Thr Val Thr Ser Trp ProSer Ile Val Ala Thr Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly SerVal Thr Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys ThrSer Thr Thr Thr ArgSer Gly Met Ser Leu (SEQ ID NO: 2).

[0054] In some embodiments, the present disclosure utilizes a method for artificial gene synthesis to transform the codons of the α-glucosidase gene to conform to the codon preference of Pichia pastoris, laying the foundation for achieving high-level expression in Pichia pastoris, and constructing a Pichia pastoris engineered bacterium that expresses phytase at a high level. In some embodiments, the Aspergillus niger α-glucosidase coding sequence is artificially synthesized according to the codon preference of the host cell, and its nucleotide sequence is as follows:

[0055] In some embodiments, the α-glucosidase is secreted outside the host cell under the guidance of the α signal peptide. Preferably, the α signal peptide is an improved 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 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 is: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 14).

[0056] In some embodiments, the α-glucosidase expression vector is pPIC9K-PSC-α-glu, which is obtained by: Primers F1 (sequence: AATGTGATCTCAAAGCGTGCTACGC, SEQ ID NO: 4) and R1 (sequence: TCAGAGGCTCATACCGGATCGAG, SEQ ID NO: 5) were designed, and PCR amplification was performed using the above-synthesized α-glucosidase gene as a template to obtain the α-glucosidase gene sequence, whose sequence is shown in SEQ ID NO: 3. Primers F2 (sequence: ACAGTTATTATTCGATGCTGTATATAAAACCAG, SEQ ID NO: 6) and R2 (sequence: GAAATCTCATTTCGAATAATTAGTTGTTT, SEQ ID NO: 7) were designed to amplify the PSC promoter, and the sequence is shown in SEQ ID NO: 1. Primers F3 (sequence: TATTCGAAATGAGATTTCCTTCAATTTTTAC, SEQ ID NO: 8) and R3 (sequence: CACGCTTTGAGATCACATTTCTTTTCTCGAGAGATACCCC, SEQ ID NO: 9), F4 (sequence: TCCGGTATGAGCCTCTGATCAAGAGGATGTCAGAATGCCA, SEQ ID NO: 10) and R4 (sequence: CGAATAATAACTGTTATTTTTCAG, SEQ ID NO: 11) containing a 20 bp homology arm at the 5' end were designed to amplify the α signal peptide (sequence: SEQ ID NO: 14) and the backbone, after the amplified product was recovered by agarose gel, the PSC promoter, α signal peptide, α-glucosidase gene sequence and plasmid pPIC9K-PSC outer loop were seamlessly assembled, and the expression vector pPIC9K-PSC-α-glu was obtained after verification by primers F5 (sequence CAGCTTATCATCGATAAGCTG, SEQ ID NO: 12) and R5 (sequence GTGGGAAATACCAAGAAAAAC, SEQ ID NO: 13). In some embodiments, the structure of the expression vector is as follows Figure 1 shown.

[0057] In another aspect, the present disclosure provides a host cell comprising the aforementioned α-glucosidase expression vector.

[0058] 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 ( Aspergillusoryzae ), 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( Fusarium moniliforme )、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 .

[0059] 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.

[0060] In some embodiments, the host cell is Pichia pastoris containing the expression vector pPIC9K-PSC-α-glu ( Komagataella phaffii , also known as Shepherd's pie ), which is deposited in the China Type Culture Collection at Wuhan University with the deposit number CCTCC M 20242483.

[0061] In another aspect, the present disclosure provides a method for constructing a host cell expressing α-glucosidase, comprising: (1) connecting the coding sequence of α-glucosidase to a vector including a PSC sequence and an α signal peptide coding sequence to obtain a recombinant expression vector, wherein the PSC sequence is an artificial synthetic promoter having a nucleotide sequence as shown in SEQ ID NO: 1; (2) transforming the recombinant expression vector obtained in step (1) into a host cell; (3) Screening and obtaining host cells with high expression levels of the α-glucosidase.

[0062] In some embodiments, the host cell is Pichia pastoris. In some preferred embodiments, the host cell is a modified Pichia pastoris GS115.

[0063] In some embodiments, the method further comprises a step of codon optimization of the coding sequence of the α-glucosidase before step (1). In some embodiments, in step (2), the recombinant expression vector obtained in step (1) is linearized and then transformed into a host cell. In some embodiments, the recombinant expression vector is linearized using a SalI restriction site.

[0064] In another aspect, the present disclosure provides a method for producing α-glucosidase, comprising culturing the aforementioned host cell to obtain a culture comprising the α-glucosidase.

[0065] In some embodiments, the host cell is Pichia pastoris ( Shepherd's pie, Also known as Komagataella phaffii ), the Pichia pastoris contains the expression vector pPIC9K-PSC-α-glu, whose deposit number is CCTCC M 20242483; the supernatant of the culture contains the α-glucosidase.

[0066] In some embodiments, the culturing comprises culturing in YPD growth medium for 16-24 hours, then inoculating the host cells into BMEY medium for 96-120 hours, and adding 0.5-1% ethanol by volume. In some embodiments, 0.5% ethanol by volume is added every 24 hours during culturing in BMEY medium. In some embodiments, ethanol is fed at a volume ratio of 0.5-1% during culturing in BMEY medium.

[0067] In some embodiments, the culture further includes the step of activating the host cells and preparing a seed solution. In some embodiments, the host cells are activated in 5 ml of YPD medium, cultured overnight at 200 rpm and 30°C for 14-16 hours to obtain a seed solution. In some embodiments, the seed solution is inoculated into a shake flask at an inoculation rate of 5%-10% for fermentation. In some embodiments, first, culture in 50 ml of YPD growth medium at 30°C and 250 rpm for 16-24 hours, inoculate the bacteria into 100 ml of BMEY medium, induce culture at 30°C and 250 rpm for 120 hours, and add 0.5% (V / V) ethanol every 24 hours. After the fermentation broth is centrifuged, the bacterial precipitate is removed, and the fermentation supernatant contains the α-glucosidase.

[0068] In some embodiments, the method for producing α-glucosidase also includes a method for separating and purifying the α-glucosidase from the culture. In some embodiments, the separation and purification includes: (1) After centrifuging the culture, taking the supernatant to obtain a liquid to obtain a crude α-glucosidase enzyme liquid; (2) The crude α-glucosidase enzyme solution obtained in step (1) is adjusted to a pH of 7.0-8.0, filtered and purified to obtain the α-glucosidase.

[0069] In some embodiments, the filtration is filtration with a 0.45 μm sterile syringe filter. In some embodiments, the purification method is chromatography. In some embodiments, the purification method is strong anion exchange chromatography.

[0070] In some embodiments, the purified α-glucosidase is desalted again and vacuum freeze-dried to obtain α-glucosidase freeze-dried powder.

[0071] In some embodiments, the culture, ie, the fermentation broth, is centrifuged to remove the bacterial precipitate, and the fermentation supernatant contains the α-glucosidase, ie, the crude α-glucosidase enzyme solution.

[0072] Take 10 mL of crude α-glucosidase enzyme solution, adjust the pH to 7.0 / 8.0 with NaOH, and filter with a 0.45 μm sterile syringe filter.

[0073] AKTA Pure 150 was used for detection. The entire flow path was washed with ultrapure water for 5 column volumes at a flow rate of 5 mL / min. Buffer A was used to balance 5 column volumes at a flow rate of 5 mL / min. The loop was cleaned with buffer A before loading, and the loading flow rate was 3 mL / min. Buffers containing 100, 200, 300, 400, and 500 mM NaCl were used for stage elution at a flow rate of 5 mL / min. The elution peaks of each stage were collected, and the molecular weight and purity of the fusion protein were detected by SDS-PAGE. Ultrapure water was used to wash 5 column volumes, and then 20% ethanol was used to wash 5 column volumes at a flow rate of 5 mL / min. After optimization, the loading amount of the recombinant protein purified by Hitrap Capto Q 5 mL strong anion exchange chromatography column was 2 mL, and the elution was carried out with buffers containing 100 ~ 600 mM NaCl, among which 300 mM NaCl was used to elute α-glucosidase. The purified enzyme was desalted again and vacuum-freeze-dried to obtain α-glucosidase freeze-dried powder.

[0074] In another aspect, the present disclosure provides use of the aforementioned expression vector, the aforementioned host cell or the α-glucosidase produced by the aforementioned method in the production of food, feed or medicine.

[0075] The invention constructs a Pichia yeast engineering strain and a culture method for inducing the expression of α-glucosidase using food-grade ethanol, realizes the efficient heterologous recombinant expression of α-glucosidase, and the yield reaches ten grams per liter. It is easy to ferment on a large scale, has low cost, high safety, and is more convenient for subsequent application in the fields of food or medicine. α-glucosidase can be industrially applied to the production of oligosaccharides (IMOs), which are products with high production and sales volume among functional oligosaccharides in the world and are widely used in food, feed and medicine production. Oligosaccharides mainly promote the proliferation of beneficial bacteria in the human intestine, inhibit the growth of many pathogens and spoilage bacteria, enhance human immunity, reduce the content of serum cholesterol, triglycerides and free fatty acids, and eliminate the causes of cardiovascular diseases; it has excellent anti-caries properties, is difficult or not digested and absorbed by the human body, provides very low or no energy, and can be used as a health sweetener for diabetics, obese patients and hypoglycemic patients. In the feed industry, isomaltooligosaccharide is believed to improve the survival rate of poultry without the side effects of synthetic feed on the human body. It is reported that about 50% of feed in Japan contains isomaltooligosaccharide.

[0076] 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.

[0077] 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.

[0078] Plasmid construction was performed using the seamless cloning kit from Novazonics.

[0079] The tool enzymes used were purchased from Tiangen Biochemical Technology (Beijing) or TaKaRa Biotechnology (Dalian). The specific reaction conditions and methods used were all referred to the product manual.

[0080] LB medium: tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L, pH 7.0 YPD medium: glucose 20g / L, tryptone 20g / L, yeast powder 10g / L, adjusted pH 6.0-7.0 YND medium: YNB 6.7 g / L, glucose 10.0 g / L; solid culture medium plus agar 20 g / L.

[0081] BMEY medium: peptone 20.0 g / L, yeast extract 10.0 g / L, YNB 13.4 g / L, 0.5% ethanol.

[0082] Trace element mixture: CuSO4·5H2O: 6.0 g / L; FeSO4·7H2O: 0.006 g / L; MnSO4·H2O:0.16 g / L; Na2MoO4·2H2O: 0.2 g / L; H3BO3: 0.024 g / L; ZnCl2: 0.008 g / L; CaSO4·2H2O:0.008 g / L; CoCl2·6H2O: 0.05 g / L; NaI: 0.02 g / L; Na2SeO3·5H2O: 0.02 g / L; EDTA:1.0 g / L.

[0083] 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, and ethanol was added at room temperature when used. The above culture media solvents were all deionized water or tap water.

[0084] Example 1. Construction of an expression vector containing α-glucosidase gene (1) Obtaining the α-glucosidase sequence: The α-glucosidase gene from Aspergillus niger published by NCBI (NCBI accession number: XP_001390530.1) was optimized, and its amino acid sequence is shown in SEQ ID NO: 2. The sequence was optimized according to the codon preference of the Pichia pastoris host and commissioned to Qingke Biotechnology Co., Ltd. for synthesis. The synthesized gene was used as a template and PCR amplification was performed with primers F1 (sequence shown in SEQ ID NO: 4) and F2 (sequence shown in SEQ ID NO: 5) to obtain the α-glucosidase gene sequence, whose sequence is shown in SEQ ID NO: 3. The PCR amplification system was 50 μl: 1 μl template, 2 μl F / R primers each, 25 μl PrimeSTAR Max Premix (2×) enzyme, and 33 μl sterile deionized water. PCR conditions: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s; annealing at 60°C for 20 s; extension at 72°C for 1 min, for a total of 30 cycles; final extension at 72°C for 10 min.

[0085] (2) The PSC artificial promoter was synthesized by Qingke Biotechnology Co., Ltd., and its sequence is shown in SEQ ID NO: 1. Primers F2 (sequence shown in SEQ ID NO: 6) and R2 (sequence shown in SEQ ID NO: 7) were designed to amplify the PSC promoter, and its sequence is shown in SEQ ID NO: 1. Primers F3 (sequence shown in SEQ ID NO: 8) and R3 (sequence shown in SEQ ID NO: 9), F4 (sequence shown in SEQ ID NO: 10) and R4 (sequence shown in SEQ ID NO: 11) containing 20 bp homology arms at the 5' end were designed to amplify the α signal peptide (sequence shown in SEQ ID NO: 11) on the vector pPIC9K (purchased from Invitrogen), respectively. NO: 14) and backbone, the amplified product was recovered by agarose gel, and the PSC promoter, α signal peptide, α-glucosidase gene sequence and pPIC9K vector backbone fragment were connected at 50°C for 20 minutes using a homologous recombination kit (purchased from Novozyme Biotechnology Co., Ltd.), and the ligation product was transformed into Escherichia coli DH5α (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B528413) competent cells using a chemical method. The transformation liquid was spread on an LB plate containing ampicillin (100 mg / L) and cultured overnight at 37°C. Positive transformants were selected and colony PCR verification was performed using primers F5 (sequence shown in SEQ ID NO: 12) and R5 (sequence shown in SEQ ID NO: 13), and Sangon Biotech (Shanghai) Co., Ltd. was commissioned to sequence correctly. After overnight culture at 37°C in a shaking incubator, the plasmid was extracted and named pPIC9K-PSC-α-glu. The plasmid map is shown in Figure 1 shown.

[0086] Example 2. Construction of Pichia pastoris engineered bacteria containing α-glucosidase gene (1) Preparation of competent cells Pick up the Pichia yeast strain Δku70-I_AM (reference patent CN 118726120 A) containing an ethanol-inducible promoter expressing an artificial transcription activator, and culture it in 10ml YPD liquid medium at 30°C with shaking overnight, then draw 100μl from it into 100mL YPD liquid medium, culture it to OD600=1.2, centrifuge it at 5000rpm at 4°C for 3min, and collect the bacteria. Use pre-cooled 0.1M LiAc buffer and DTT buffer, pipette and mix well, place the centrifuge tube in a 30°C shaker at 200rpm for 30-45min, centrifuge it at 4°C for 3min, then wash the bacteria twice with pre-cooled 1M sorbitol buffer, and finally resuspend it in 1ml pre-cooled 1M sorbitol buffer, and dispense 80μL / tube.

[0087] (2) Electroporation and transformant screening The recombinant plasmid pPIC9K-PSC-α-glu obtained in Example 1 was linearized by SalI digestion, 10 μg of the linearized recombinant plasmid after digestion was taken and mixed with 80 μL of competent cells, and transferred to a 0.2 cm electroporation cup with an ice bath at 4 ° C, and the electroporation cup containing the mixed solution was ice bathed for 5 min. Adjust the parameters of the electroporation instrument, set it to the Pichia yeast gear, voltage 1.5 kV, capacitance 25 microfarads, resistance 200 ohms, and time is about 5 milliseconds. After the electric shock, 1 ml of 1M sorbitol solution precooled on ice was quickly added to the transformation cup, gently aspirated and mixed, and the mixed solution was aspirated and transferred to a centrifuge tube. 30 ° C, 200 rpm culture for 1-2h, 3000 rpm centrifugation for 5min. Discard the supernatant, spread on a YND plate, and culture in a 30 ° C incubator for 2-4 days until a single colony grows.

[0088] Single colonies were picked and the above-mentioned primers F5 (sequence as shown in SEQ ID NO: 12) and R5 (sequence as shown in SEQ ID NO: 13) were used for yeast colony PCR verification. The ones with correct bands were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The ones with correct sequencing were the recombinant strains PSC-α-glu expressing α-glucosidase, and fermentation tests were subsequently carried out.

[0089] Example 3. Inducible expression of α-glucosidase In the recombinant strain PSC-α-glu obtained in the above Example 2, the PSC artificial synthetic promoter that regulates the expression of α-glucosidase can be combined with an artificial transcription activator, wherein the artificial transcription activator is expressed by an ethanol-inducible promoter (see patent publication CN 118726120 A), thereby indirectly achieving ethanol as a substrate to induce the recombinant expression of α-glucosidase. Pick the 11 single colonies verified correctly in Example 2 in 5mL YPD medium, culture them overnight at 200rpm and 30°C for 14-16h, and inoculate them into shake flasks at an inoculum size of 5%-10% for fermentation. First, culture them at 30°C and 250rpm for 16-24h in 50ml YPD growth medium, inoculate the bacteria into 100ml BMEY medium, induce the culture at 30°C and 250rpm for 120h, and add 0.5% (V / V) ethanol every 24h.

[0090] The fermentation liquid was centrifuged at 10000rpm for 5min, and then the bacterial precipitate was removed. The supernatant was collected as the crude α-glucosidase enzyme solution. 10μl was taken for SDS-PAGE detection. The detection results of the crude enzyme solution obtained by fermentation of 11 single colonies (numbered 1-11) are as follows Figure 2As shown, the expression level of α-glucosidase in colony 1 was the highest. Komagataella phaffii , also known as Shepherd's pie ) The genetically engineered bacteria are deposited in the China Type Culture Collection at Wuhan University, with the deposit number CCTCC M 20242483 and the deposit date of November 7, 2024.

[0091] Example 4. Reactor fermentation of recombinant Pichia pastoris PSC-α-glu Selected Example 3 Figure 2 The No. 1 transformant with the highest expression level was further fermented in a 5L reactor. The engineered bacteria was inoculated into YPD liquid medium and cultured until OD 600 The pH value is about 7 to obtain the seed solution, and then the seed solution is inoculated into a 5L reactor (3L BMEY fermentation medium) at a 5% (v / v) inoculation amount, and the temperature is maintained at 30°C, pH is about 6, and the dissolved oxygen is greater than 30% for fermentation. The dissolved oxygen is kept stable. If the dissolved oxygen increases suddenly, maintain the high dissolved oxygen for about 20 minutes and then add glucose solution until the wet weight of the bacteria increases to 150g / L, then stop adding.

[0092] When glucose is consumed and dissolved oxygen rises, ethanol is added at a volume ratio of 0.5-1% and trace elements are added at a volume ratio of 0.01%, and dissolved oxygen is controlled to be not less than 25%. When fermentation is completed for 96 hours, the supernatant is collected, which is the crude enzyme solution of α-glucosidase. The expression level of α-glucosidase can reach about 10 g / L by using a BCA protein concentration determination kit (purchased from Biyuntian Biotechnology Co., Ltd.) and bovine serum albumin (BSA) as a standard.

[0093] Example 5. Purification method of α-glucosidase expressed by recombinant Pichia pastoris Take 10 mL of the crude α-glucosidase solution in Example 4, adjust the pH to 7.0 with NaOH, and filter with a 0.45 μm sterile syringe filter.

[0094] AKTA Pure 150 was used for detection. The entire flow path was washed with ultrapure water for 5 column volumes at a flow rate of 5 mL / min. Buffer A was used to balance 5 column volumes at a flow rate of 5 mL / min. The loop was cleaned with buffer A before loading, and the loading flow rate was 3 mL / min. Buffers containing 100, 200, 300, 400, and 500 mM NaCl were used for stage elution at a flow rate of 5 mL / min. The elution peaks of each stage were collected, and the molecular weight and purity of the fusion protein were detected by SDS-PAGE. Ultrapure water was used to wash 5 column volumes, and then 20% ethanol was used to wash 5 column volumes at a flow rate of 5 mL / min. After optimization, the loading amount of the recombinant protein purified using Hitrap Capto Q 5 mL strong anion exchange chromatography column was 2 mL, and eluted with buffers containing 100-600 mM NaCl, respectively. Among them, 300 mM NaCl was used to elute α-glucosidase. The purified enzyme was desalted again and vacuum freeze-dried to obtain 87 mg of α-glucosidase freeze-dried powder with a purity of 92% and a yield of 87%.

[0095] Example 6. Analysis of α-glucosidase activity Determination of α-glucosidase activity: (1) Reagents and solutions: Acetate buffer: Dissolve 4.76 g sodium acetate trihydrate (NaCH3COO·3H2O, MW=136.08) in about 300 ml of water and add 1.35 g (or 1.28 ml) of acetic acid (CH3COOH). Adjust the pH to 5.0±0.05. Bring the final volume to 500 ml.

[0096] Stop reaction (sodium carbonate) solution: Dissolve 15.9 g sodium carbonate (Na2CO3, MW=105.99) in 400 ml water and make up to 500 ml.

[0097] Substrate (p-Nitrophenyl-α-d-glucopyranoside, PNPG) solution (%0.1 w / v, 10ml): Dissolve 10.0mg PNPG in 10ml acetate buffer. Heat the acetate buffer to 40°C to make the PNPG powder dissolve faster. p-Nitrophenol stock solution (PNP): Dissolve 14 mg p-nitrophenol (PNP, MW=139.11) in 10 ml acetate buffer to prepare a 10 mM PNP stock solution. After PNP is completely dissolved, take 1 ml from the PNP stock solution and place it in a sterile 15 ml centrifuge tube. Then add 9 ml acetate buffer to dilute PNP 10 times to a final concentration of 1 mM.

[0098] (2) Preparation of standards and samples Standards: Prepare PNP standards at concentrations of 6, 20, and 50 µM from a 1 mM stock solution. Measure absorbance (A400) at 400 nm and subtract the blank from the value of the PNP standard to obtain the net A400 value. Plot these values ​​to calculate PNP ( ) millimolar extinction coefficient:

[0099] in: is the absorbance at wavelength λ, is the molar extinction coefficient at wavelength λ, c is the molar concentration of the solution, and l is the path length (1 cm for a standard spectrophotometer cuvette).

[0100] (3) Sample solution: Dilute the α-glucosidase enzyme powder sample obtained in Example 5 in sufficient acetate buffer solution, at least twice, with an activity range of 0.4 to 0.8 GAU / ml. If the sample activity cannot be estimated / unknown before analysis, prepare 10x to 2500X dilutions using serial dilutions to ensure that at least two dilutions fall within the given activity range. Each dilution should be prepared in triplicate (n=3).

[0101] (4) Steps: Measure the absorbance of the three PNP standard solutions and calculate the molar extinction coefficient.

[0102] For the blank solution, mix 200µl PNPG substrate with 300µl stop solution and 200µl acetate buffer.

[0103] For active samples, 200 µl of PNP substrate was added to a centrifuge tube and pre-incubated for at least 5 min in a water bath at different temperatures (pH 4.5 at 20°C, 30°C, 40°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 80°C) and pH (pH 3, 4, 4.5, 5, 5.5, 6, 7, and 8 at 50°C using HCl and NaOH solutions). After pre-incubation, 200 µl of each enzyme / sample diluent was added to each replicate tube at 15 sec intervals. Once the sample and substrate were combined in the first tube (10 min incubation), a timer was started for 10 min. After the 10 min incubation, 300 µl of stop solution was immediately added to each of the triplicate tubes (15 sec intervals). The tubes were mixed on a vortex mixer and incubated at room temperature for 10 min before measuring the absorbance. The absorbance of the resulting 700 μl mixture was then measured at 400 nm using a spectrophotometer.

[0104] (5) Calculation of enzyme activity: Enzyme activity definition: 1 unit of glucoamylase activity (GAU) is defined as the amount of glucoamylase that releases 1 μmol of nitrophenol (PNP) within 1 hour under the experimental conditions (pH 5.0, 50°C). Enzyme activity can be calculated using the following formula:

[0105] A sample is the sample absorbance measured at 400 nm, A blank The absorbance of the blank solution at 400 nm, d𝑓 is the dilution factor (the factor by which the sample was diluted before activity measurement).

[0106] according to Figure 3 , Figure 4 The results show that the optimal pH and temperature of α-glucosidase expressed by recombinant Pichia pastoris PSC-α-glu are 5.0 and 60°C, respectively, and the enzyme activity is 15117.6U / g at pH 5.0 and 60°C. In addition, the α-glucosidase has good thermal stability, and the relative enzyme activity is maintained at 80% or above within 50-70°C; it also has a certain resistance to acid and alkali, and the relative enzyme activity is maintained at 80% or above within the pH range of 4.0-6.5.

[0107] The method disclosed herein is used to produce α-glucosidase, which can be secreted into the fermentation broth supernatant by inducing ethanol, and the extracellular expression level can reach 10 g / L in a 5L fermenter, with high yield. Cells do not need to be crushed during purification, thus reducing production costs.

[0108] 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 scope of protection of the present invention.

Claims

1. An α-glucosidase expression vector comprising a PSC sequence, an α signal peptide coding sequence and an α-glucosidase coding sequence; wherein: The PSC sequence is an artificial synthetic promoter with a nucleotide sequence as shown in SEQ ID NO:

1.

2. The expression vector according to claim 1, wherein The α-glucosidase coding sequence has a nucleotide sequence shown in SEQ ID NO:

3.

3. The expression vector according to claim 1 or 2, wherein The α signal peptide has an amino acid sequence shown in SEQ ID NO:

14. A host cell comprising the α-glucosidase expression vector according to any one of claims 1 to 3.

5. The host cell according to claim 4, wherein The host cell is Pichia pastoris ( Komagataella phaffii ), the deposit number of the host cell is CCTCC M 20242483.

6. A method for constructing a host cell expressing α-glucosidase, comprising: (1) connecting the coding sequence of α-glucosidase to a vector including a PSC sequence and an α signal peptide coding sequence to obtain a recombinant expression vector, wherein the PSC sequence is an artificial synthetic promoter having a nucleotide sequence as shown in SEQ ID NO: 1; (2) transforming the recombinant expression vector obtained in step (1) into a host cell; (3) Screening and obtaining host cells with high expression levels of the α-glucosidase.

7. The method according to claim 6, wherein: The host cell is Pichia pastoris.

8. The method according to claim 6 or 7, further comprising the step of codon-optimizing the coding sequence of the α-glucosidase.

9. A method for producing α-glucosidase, comprising culturing the host cell according to claim 4 or 5 to obtain a culture comprising the α-glucosidase.

10. The method according to claim 9, wherein: The deposit number of the host cell is CCTCC M 20242483; the supernatant of the culture contains the α-glucosidase.

11. The method according to claim 10, wherein: The culturing comprises culturing in a YPD growth medium for 16-24 hours, inoculating the host cells into a BMEY medium for culturing for 96-120 hours, and adding 0.5-1% by volume of ethanol.

12. Use of the α-glucosidase produced by the expression vector according to any one of claims 1 to 3, the host cell according to claim 4 or 5, or the method according to any one of claims 9 to 11 in the production of food, feed or medicine.

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