Strain producing α-glucosidase, construction method and application thereof

Abstract

The present disclosure belongs to the field of genetic engineering technology, and specifically relates to strains for producing α-glucosidase, and methods for constructing and applying the strains. The present disclosure transfers α-glucosidase derived from Aspergillus niger into Pichia pastoris host cells, constructs a Pichia pastoris engineered strain PSC-α-glu using ethanol as a substrate, and realizes efficient biosynthesis of α-glucosidase from ethanol for the first time. The present disclosure 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 achieves a yield of ten grams per liter. It is easy to ferment on a large scale, is low in cost, and has high safety, making it more convenient for subsequent applications in the fields of food or medicine.

Description

Technical Field

[0001] This disclosure belongs to the field of genetic engineering technology, specifically relating to a strain for producing α-glucosidase, its construction method and application, and a method for efficiently producing α-glucosidase. Background Art

[0002] α-Glucosidase (EC 3.2.1.20), also known as α-D-glucosidase, catalyzes the hydrolysis of oligosaccharides to produce glucose. α-Glucosidases are widely distributed in nature, with numerous species and diverse properties. α-Glucosidases derived from Aspergillus, Saccharomyces cerevisiae, and Bacillus can not only hydrolyze glycosides but also synthesize certain oligosaccharides through transglycosylation reactions. Among them, Aspergillus niger (EC 3.2.1.20) is a notable example. Aspergillus niger α-Glucosidase has great potential for industrial applications due to its excellent transglycosylation ability.

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

[0004] Pichia pastoris ( Shepherd's pie, Also known as Komagataella phaffii Pichia pastoris expression systems are considered more suitable for industrial-scale secretory expression systems due to their stable genetic expression, post-translational processing capabilities, high expression efficiency, secretion of products into culture media, and suitability for high-density fermentation. Currently, methanol is commonly used as an inducer in Pichia pastoris to induce the expression of exogenous genes, but methanol's toxicity limits its application in food. Ethanol, as a raw material, is widely used in food production. Therefore, this disclosure aims to construct a high-yield Pichia pastoris strain that utilizes ethanol to produce α-glucosidase, thereby broadening the routes for ethanol utilization and α-glucosidase production, and exploring the application potential and prospects of Pichia pastoris as a microbial cell factory. Summary of the Invention

[0005] The purpose of this disclosure is to provide vectors and strains for the efficient expression of α-glucosidase, thereby increasing the expression level of α-glucosidase and, more importantly, to utilize ethanol conversion to produce α-glucosidase.

[0006] To achieve the above-mentioned technical objectives, the present disclosure proposes the following technical solutions:

[0007] In one aspect, this disclosure provides 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 artificially synthesized promoter with a nucleotide sequence as shown in SEQ ID NO: 1.

[0008] In another respect, this disclosure provides a host cell comprising the aforementioned α-glucosidase expression vector.

[0009] In another aspect, this disclosure provides a method for constructing a host cell expressing α-glucosidase, comprising:

[0010] (1) The coding sequence of the α-glucosidase is ligated into 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 artificially synthesized promoter with a nucleotide sequence as shown in SEQ ID NO: 1;

[0011] (2) Transform the recombinant expression vector obtained in step (1) into host cells;

[0012] (3) Screening to obtain host cells with high α-glucosidase expression levels.

[0013] In another aspect, this disclosure provides a method for producing α-glucosidase, comprising culturing the aforementioned host cells to obtain a culture comprising the α-glucosidase.

[0014] On the other hand, this disclosure provides the 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 pharmaceuticals.

[0015] This disclosure describes the transformation of α-glucosidase derived from Aspergillus niger into Pichia pastoris host cells, constructing an engineered Pichia pastoris strain PSC-α-glu using ethanol as both a substrate and inducer. This represents the first time that highly efficient biosynthesis of α-glucosidase from ethanol has been achieved. This disclosure also describes the construction and cultivation method of an engineered Pichia pastoris strain that induces α-glucosidase expression using food-grade ethanol, achieving highly efficient heterologous recombinant expression of α-glucosidase with yields reaching tens of grams per liter. This method is suitable for large-scale fermentation, is low-cost, and has high safety, making it more suitable for subsequent applications in the food or pharmaceutical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The image shows the spectrum of the recombinant plasmid vector pPIC9K-PSC-α-glu.

[0017] Figure 2 To screen the SDS-PAGE results of recombinant Pichia pastoris genetically engineered strains, M represents protein markers ranging from 10 to 170 kDa, and lanes 1-11 represent different transformants of the recombinant Pichia pastoris genetically engineered strains.

[0018] Figure 3 The enzyme activity of α-glucosidase at different temperatures.

[0019] Figure 4 The enzyme activity of α-glucosidase under different pH conditions.

[0020] Biological Preservation Instructions

[0021] Recombinant Pichia pastoris ( Komagataella phaffii Also known as Shepherd's pie The genetically engineered bacteria are deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, with accession number CCTCC M20242483, deposited on November 7, 2024. The culture name is... Komagataella phaffii CJ-PP-GU. DETAILED DESCRIPTION

[0022] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the described embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0023] This disclosure may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this disclosure may be combined with technical features of any or more other embodiments to obtain further embodiments. This disclosure includes such further embodiments obtained through combination.

[0024] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. In the event of any conflict between the use or terminology used in any publications and patents incorporated by reference and the use or terminology used in this disclosure, the use and terminology of this disclosure shall prevail.

[0025] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[0026] (I) Definitions or terms

[0027] 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 pertains. For purposes 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.

[0028] Unless otherwise expressly stated, the singular forms “a,” “an,” “the,” “the,” and similar designations used in this specification and the appended claims include plural designations.

[0029] As used in this article, the conjunction term “and / or” between multiple elements means to include both “and” and “or”. For example, the phrase “A, B and / or C” is intended to cover each of the following: 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).

[0030] As used herein, the terms “comprises,” “comprising,” “having,” and “containing,” and any variations thereof, are intended to cover non-exclusive inclusion. The term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially consisting of”.

[0031] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 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.

[0032] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0033] In the description herein, references to “some implementations,” “some implementations,” or “some embodiments” describe a subset of all possible embodiments. However, it is understood that “some implementations” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0034] As used herein, the terms “optional,” “optional,” “any,” “arbitrary,” or “any one” mean that the event or circumstance described below may, but does not have to, occur, including the circumstances in which the event or circumstance may or may not occur.

[0035] As used herein, the terms “peptide” and “protein” are used interchangeably to refer to a string of at least two amino acid residues linked together by covalent bonds (e.g., peptide bonds), and can be recombinant, natural, or synthetic peptides. Peptides can be linear or branched, can contain modified amino acids, and can be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).

[0036] As used herein, the term "amino acid" refers to the 20 naturally occurring amino acids that can be encoded and translated by genes and that make up protein segments, including L-amino acids and D-amino acids, unless otherwise stated. It also refers to chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not normally bound to proteins such as leucine, and chemically synthesized compounds that have amino acid properties known in the art. For example, analogs or mimics of Phe or Pro that can restrict the conformation of peptide compounds to the same level as native phenylalanine or proline are included in the definition of "amino acid" and are known to those skilled in the art. Analogs and mimics as used herein 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).

[0037] As used herein, the term "amino acid substitution" refers to the replacement of an amino acid residue present in the parental sequence with another amino acid residue. The amino acid in the parental sequence can be substituted, for example, via chemical peptide synthesis or by recombination methods known in the art. Therefore, reference to "substitution at position X" means the replacement of the amino acid present at position X with an alternative amino acid residue. In some embodiments, the substitution type may be described according to the pattern AXY, where A is a single-letter code corresponding to the amino acid naturally present at position X, and Y is the substituted amino acid residue. In other aspects, the substitution type may be described according to the pattern XY, where Y is a single-letter code corresponding to the substitution of the amino acid residue naturally present at position X.

[0038] As used herein, the term "nucleic acid molecule" or "nucleic acid" refers to any one or more nucleic acid segments, such as DNA, cDNA, or RNA fragments, present in a polynucleotide. 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; for example, for the purposes of this disclosure, a recombinant polynucleotide encoding an antigen-binding protein contained in a vector is considered isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell 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. Isolated polynucleotides or nucleic acids according to this disclosure further include such molecules synthesized. Furthermore, polynucleotides or nucleic acids may include regulatory elements, such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0039] As used herein, the term “polynucleotide” or “nucleotide” is intended to cover both single and multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs such as messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA).

[0040] As used herein, the term "homology" has a universally accepted meaning in the field and is a central concept in comparative biology. The basic meaning of homology is that two samples being compared (e.g., an amino acid sequence or a nucleotide sequence) share a common ancestor. Generally, two traits (states) in two species can be considered a pair of homologous traits if either of the following two conditions is met: 1. They are identical to a trait found in the ancestral groups of these species; 2. They are distinct traits with an ancestor-descendant relationship. Amino acid sequence homology can be determined using known methods. For example, amino acid sequence homology (%) can be determined using procedures 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 Needleman et al. (1970) (J. Mol. Biol. 48:444-453), Myers and Miller (CABIOS, 1988, 4: 11-17), etc. Needleman et al.'s algorithm has been integrated into the GAP program of the GCG software package (available at www.gcg.com). Homology (%) can be determined, for example, using the BLOSUM 62 matrix or PAM250 matrix, and any of the following: gap weights (16, 14, 12, 10, 8, 6, or 4) and length weights (1, 2, 3, 4, 5, or 6). Additionally, Myers and Miller's algorithm has been integrated into the ALIGN program, which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, for example, a PAM120 weighted residue table, gap length penalty, and gap penalty can be used.

[0041] While this disclosure provides specific amino acid or nucleotide sequences, such as those shown in the sequence listing, it should be understood that a specific amino acid or nucleotide sequence includes variants with conserved modifications, such as sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% homology with it, provided that the biological function or activity of that specific amino acid or nucleotide sequence is not lost.

[0042] The term "conservatively modified variant" is used to refer to amino acid and nucleic acid sequences. With respect to a specific nucleic acid sequence, a conservatively modified variant refers to those nucleic acids that encode the same or a conservatively modified amino acid sequence variant. 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; a degenerate codon is a different three-letter codon used to designate the same amino acid, for example, codons GCA, GCC, GCG, and GCU all encode alanine. Therefore, at any position where alanine is designated by a codon, that codon can be changed to any corresponding codon without changing the encoded polypeptide. This nucleic acid variation is a "silent variant," a variant of conserved modification. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG and UGG, where AUG is generally just a codon for methionine and UGG is just a codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent variant of the nucleic acid encoding the polypeptide of this disclosure is undoubtedly present in each stated polypeptide sequence, which is included within the scope of this disclosure.

[0043] For amino acid sequences, those skilled in the art will recognize that each substitution, deletion, or addition in a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds, or omits a single amino acid or a small number of amino acids is a “conserved variant,” where the change results in the substitution of the amino acid with a chemically similar amino acid. Thus, any integer number of amino acid residues selected from 1 to 15 can be altered. For example, 1, 2, 3, 4, 5, 7, or 10 variations can be produced. Conserved variants typically provide biological activity similar to that of unmodified polypeptide sequences derived therefrom. For example, substrate specificity, enzyme activity, or ligand / receptor binding affinity is typically at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein and its native substrate. Those skilled in the art are well aware that conserved representations of amino acids provide functional similarity. For example, modifications can be introduced into the sequence listings described herein using standard techniques known in the art, such as gene synthesis and PCR-mediated mutagenesis. Conservative modification includes conservative substitution of amino acids. "Conservative substitution" means that one amino acid is replaced by another amino acid with similar properties, such as replacing it with an amino acid with a similar side chain, so that those skilled in peptide chemistry expect that the secondary structure and hydrophilic properties of the peptide remain substantially unchanged. Families of amino acid residues with similar side chains are well-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 nonpolar 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).

[0044] As used herein, the term "vector" refers to a vector that can autonomously replicate in a host cell, preferably a multi-copy vector. The term "vector" as used herein refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is linked. Furthermore, vectors typically possess markers such as antibiotic resistance genes for selecting transformants. Additionally, vectors may have promoters and / or terminators for expressing the introduced gene. Vectors can be, for example, vectors derived from bacterial plasmids, viral vectors, vectors derived from yeast plasmids, vectors derived from bacteriophages, granules, phage particles, etc. The term "recombinant vector" refers to a vector modified as needed for carrying exogenous DNA fragments (DNA fragments of the gene of interest) into host cells for replication and expression, including recombinant cloning vectors and recombinant expression vectors, preferably recombinant expression vectors. In some instances, the recombinant vector may contain various elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The vector may contain the nucleic acids of this disclosure for introduction into cells for expression. The vector may contain expression control elements operatively linked to the nucleic acid, such as promoters, terminators, and / or enhancers.

[0045] 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, etc.

[0046] Suitable yeasts for use in this disclosure include any of the following genera or their derivatives and hybrid varieties: Candida (e.g., Candida albicans) Candida albicans Candida cherubicin (Candida) Etchell's ), and Candida albicans ( Candida guilliermondii ), Candida flatus ( White humble ), Candida albicans ( Candida lipolytica ), Candida parasiticus ( White orthopsilosis ), Candida palmis ( Candida palmioleophila ), Candida tropicalis ( White pseudotropical ), species of the genus Candida, Candida utilis ( Useful Candida ), Candida albicans ( Versatile white )), Cladosporium ( Cladosporium Cryptococcus genus ( Cryptococcus (For example, Cryptococcus terrestrialus) Cryptococcus terricolus Cryptococcus curvilinearis ( ) Cryptococcus curvatus ), Debali yeast ( Debaryomyces (For example, Hansenula polymorpha) Debaryomyces hansenii ), Endosporium ( Endomyces (For example, Lipid-producing Inonotus) Endomyces vernalis ), genus Nematophora ( Endomycopsis (For example, lipophilic nematodes) Endomycopsis vernalis )), Pseudomonas ( Hermitage (For example, *Pseudomonas aeruginosa*) Eremothecium ashbyii ), Hansenula genus ( Little Hansen (For example, species of the genus *Hansenula*, *Hansenula polymorpha*) Hansenula polymorpha Kluyveromyces ( )), Kluyveromyces ( Kluyveromyces (For example, species of the genus Kluyveromyces, Kluyveromyces lactis) Kluyveromyces lactis ), Kluyveromyces lactic acid variant ( Kluyveromyces marxianus var.lactis ), Max Kluyveromycin ( Kluyveromyces Marxian ), heat-resistant Kluyveromycin ( Kluyveromyces thermotolerans ), oleosomalidase ( Lipomyces (For example, *Saccharomyces davidii*) Lipomyces starkeyi ), oil-producing yeast ( Lipomyces fat-bearing ), Ogata yeast ( Ogataea (For example, tiny Osamucrozyme) Ogataea minuta ), Pichia pastoris ( Peach (For example, species of the genus Pichia, Pichia pastoris) Komagataella phaffii Also known as Peach shepherd Pichia pastoris (Finnish Pichia pastoris) Finnish Pichia ), Pichia pastoris (trehalose) Peach trehalophila ), Pichia pastoris ( ), Pichia koclamae ), Pichia pastoris ( ) Peach membrane-forming ), Pichia pastoris ( A few minutes ), Pichia pastoris ( Pichia lindneri Pichia pastoris ( ), cactus yeast Prickly pear ), heat-resistant Pichia pastoris ( Pichia thermotolerans ), Rhizopus oryzae ( Willow tree Pichia pastoris () Pichia guercuum Pichia pastoris (), Peach piper Pichia pastoris (Tree trunk yeast) Pichia stiptis ), Pichia pastoris () Pichia methanolica ), genus *Rhodotorula* ( Rhodosporidium (For example, Rhodotorula buergerianum) Rhodosporidium toruloides ), Rhodotorula genus ( Rhodotorula (For example, species of the genus *Rhodotorula*, *Rhodotorula lepidotorula*) (Rhodotorula gracilis ), sticky red yeast ( Rhodotorula glutinis ), Heben Red Yeast (Rhodotorula graminis )), Yeast () Saccharomyces (For example, species of the genus *Yeast*, *Bacillus* ( Saccharomyces bayanus ), brewer's yeast ( Saccharomyces beticus ), brewer's yeast ( Saccharomyces cerevisiae ), Schafera yeast ( Saccharomyces chevalieri ), saccharified yeast ( Saccharomyces diastaticus ), wine yeast ( Saccharomyces ellipsoideus Oligosaccharomyces ( ), Saccharomyces exiguus ), Flores yeast ( Saccharomyces florentinus ), crispy-wall yeast ( Saccharomyces fragilis Pasteur yeast ( Saccharomyces pastorianus ), millet wine yeast ( Saccharomyces pombe Sake yeast ( Saccharomyces sake ), grape juice yeast ( Saccharomyces uvarum )), genus *Pterocarya* ( Sporobolomyces (For example, *Pseudomonas erythrosporum*) Sporobolomyces roseus ), genus *Leptospora* ( Sporidiobolus (For example, Johnson lock-throw yeast) Sporidiobolus johnsonii ), salmon-colored yeast ( Sporidiobolus salmonicolor ), genus *Mycosporea* ( Trichosporon (For example, *Hydrocotyle spp.*) Trichosporon cacaoliposimilis ), a new species of lipophilic yeast ( Trichosporon oleaginosus sp.nov. ), a new species of cocoa butter mycelium ( Trichosporon cacaoliposimilis sp.nov. ), Fragile Mycospores ( Trichosporon gracile Euonymus spp. ( Trichosporon dulcitum ), *Saccharomyces gibberellii* Trichosporon jirovecii ), Insect filamentous yeast ( Trichosporon insectorum ), Lutein yeast ( Xanthophyllomyces (For example, dendritic lutein yeast) Xanthophyllomyces dendrorhous ), Yersinia ( Yarrowia (For example, Yersinia lipolyticis) Yarrowia lipolytica ), and genus *Zygosaccharomyces* ( Zygosaccharomyces (For example, *Rhodotorula rubescens*) Zygosaccharomyces rouxii ).

[0047] The commonly used yeast expression system is Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Expression systems and methanol-nutritional yeast expression systems:

[0048] ① Saccharomyces cerevisiae expression system: Saccharomyces cerevisiae has been used in the brewing and bread industries for thousands of years and is considered a GRAS (generally recognized as safe) organism, meaning it does not produce toxins and has been recognized as a safe organism by the US FDA. However, Saccharomyces cerevisiae is difficult to cultivate at high densities, has low secretion efficiency, almost never secretes exogenous proteins with a molecular weight greater than 30 kD, and cannot properly glycosylate the expressed exogenous proteins. Moreover, the C-terminus of the expressed protein is often truncated. Therefore, Saccharomyces cerevisiae is generally not used as a host organism for recombinant protein expression.

[0049] ② Methanol-based yeast expression system: The methanol-based yeast expression system is the most widely used yeast expression system. Methanol-based yeasts mainly include those in the genus *Hansenula* (…). Hansenula Pichia pastoris ( ) Pichia ), genus *Gastromycium* Torulopsis Pichia pastoris is the most commonly used genera for expressing exogenous proteins. The expression vectors for *Methanol yeast* are integrative plasmids containing sequences homologous to those in the yeast chromosome, making them relatively easy to integrate into the chromosome. Most *Methanol yeast* expression vectors contain the *Methanol yeast* alcohol oxidase gene-1 (AOX1), and the exogenous gene is expressed under the action of its promoter (PAOX1). *Methanol yeast* is typically grown in a glycerol-containing medium to a high concentration, and then methanol is used as the carbon source to induce the expression of the exogenous protein, which can greatly increase the expression yield. The yield of exogenous proteins expressed using *Methanol yeast* can often reach the gram level. Compared with *Saccharomyces cerevisiae*, its post-translational processing is closer to that of mammalian cells, and hyperglycosylation does not occur.

[0050] The vector is matched to the host cell and, depending on the host, is classified into prokaryotic (bacterial) expression vectors, yeast expression vectors, plant expression vectors, mammalian expression vectors, insect expression vectors, etc. The vector contains a foreign gene fragment. Through vector-mediated expression, the foreign gene can be expressed in the host. In some embodiments, the expression vectors used for Pichia pastoris in this disclosure are preferably the pPIC9K vector and the pGAPZαA vector.

[0051] As used herein, the term "codon optimization" refers to the codon changes in the gene or coding region of a nucleic acid molecule used to transform various hosts, reflecting the typical codon usage of the host organism without altering the DNA-encoded polypeptide. Such optimization involves replacing at least one or more codons with one or more codons that are used more frequently in the organism's gene. By utilizing knowledge of codon usage or codon preferences in each organism, those skilled in the art can apply these frequencies to any given polypeptide sequence and produce nucleic acid fragments encoding that polypeptide, but using codon-optimized coding regions with the optimal codons for the given species. Codon-optimized coding regions can be designed using various methods known to those skilled in the art.

[0052] As used herein, the term "expression" refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. 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 host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0053] As used herein, an "expression frame" refers to a gene expression system containing all the necessary elements required to express a target polypeptide. Typically, it includes the following elements: a promoter, a gene sequence encoding the polypeptide, and a terminator; alternatively, it may include a signal peptide coding sequence. These elements are operatively linked.

[0054] As used in this article, a "promoter" is a nucleic acid sequence that is typically located upstream (5' end) of the coding sequence of a target gene and guides the transcription of the nucleic acid sequence into mRNA. Generally, a promoter or promoter region provides recognition sites for RNA polymerase and other factors necessary for proper initiation of transcription.

[0055] As used in this article, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not naturally occurring, then the promoter is exogenous to the target gene. A particular sequence is "exogenous" to the cell in which it is inserted.

[0056] As used in this article, "operationally linked" refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, such that transcription of the nucleic acid sequence is guided by the promoter region, thus the promoter region is "operationally linked" to the nucleic acid sequence.

[0057] In this disclosure, "ethanol induction" or "ethanol culture" refers to adding an appropriate amount of ethanol or other substances as a carbon source and inducer to the Pichia pastoris culture medium, replacing conventional carbon sources such as glucose and glycerol, and conventional inducers such as methanol. However, for methanol-nutritional yeasts like Pichia pastoris, "ethanol induction" is a crucial difference; therefore, modifying the expression system and optimizing suitable expression elements is challenging but highly significant. For the production of α-glucosidase, ethanol induction is clearly safer.

[0058] (II) Detailed Technical Solution

[0059] This disclosure provides an expression vector for an α-glucosidase gene and constructs an engineered Pichia pastoris strain that produces high levels of α-glucosidase through ethanol fermentation, enabling it to efficiently secrete and express α-glucosidase. This strain can then be used in food and other fields by producing α-glucosidase through high-density fermentation.

[0060] In one aspect, this disclosure provides an α-glucosidase expression vector, comprising a PSC sequence, an α-signal peptide coding sequence, and an α-glucosidase coding sequence; wherein the PSC is a synthetic promoter that can bind to an artificial transcription activator in modified chassis cells, wherein the artificial transcription activator is expressed by an ethanol-inducible promoter (see disclosed patent CN118726120A), thus enabling ethanol-induced recombinant expression of α-glucosidase. The nucleotide sequence of the PSC is shown below:

[0061] TGCTGTATATAAAACCAGTGGTTATATGTACAGTACGTCGAGTGCTGTATATAAAACCAGTGGTTATATGTACAGTACGTCGAGTGCTGTATATAAAACCAGTGGTTATATGTACAGTACGTCGAGCTAACCCCTACTTGACAGCAATATATAA ACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAA (SEQ ID NO: 1).

[0062] In some embodiments, the α-glucosidase is derived from Aspergillus niger, and its amino acid sequence is as follows:

[0063] 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).

[0064] In some embodiments, this disclosure utilizes a method of artificial gene synthesis to modify the codons of the α-glucosidase gene to conform to the codon preferences of Pichia pastoris, laying the foundation for its high-level expression in Pichia pastoris and constructing an engineered Pichia pastoris strain that expresses phytase at a high level. In some embodiments, the Aspergillus niger α-glucosidase coding sequence is artificially synthesized according to the codon preferences of the host cell, and its nucleotide sequence is as follows:

[0065]

[0066] In some embodiments, the α-glucosidase is secreted extracellularly by an α-signal peptide, preferably a modified signal peptide. The original α-mating factor signal peptide of *Saccharomyces cerevisiae* is widely used in *Pichia pastoris*-guided exogenous protein secretion expression. Its terminal amino acid sequence is ultimately cleaved twice by the dipeptidase Ste13 to remove two duplicate EAs. However, due to the influence of various factors on its cleavage efficiency, amino acid sequence residues may remain. The modified α-signal peptide has a C-terminal amino acid of KR, which can be recognized and effectively cleaved by the *Pichia pastoris* Kex2 protease, ensuring that the secreted exogenous protein does not contain redundant amino acids. In some embodiments, the amino acid sequence of the α-signal peptide is as follows:

[0067] MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 14).

[0068] In some embodiments, the α-glucosidase expression vector is pPIC9K-PSC-α-glu, obtained through the following methods:

[0069] Primers F1 (sequence AATGTGATCTCAAAGCGTGCTACGC, SEQ ID NO: 4) and R1 (sequence TCAGAGCTCATACCGGATCGAG, SEQ ID NO: 5) were designed. Using the synthesized α-glucosidase gene as a template, PCR amplification was performed to obtain the α-glucosidase gene sequence, as 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, as shown in SEQ ID NO: 1. Primers F3 (sequence TATTCGAAATGAGATTTCCTTCAATTTTTAC, SEQ ID NO: 8), R3 (sequence CACGCTTTGAGATCACATTTCTTTTCTCGAGAGATACCCC, SEQ ID NO: 9), F4 (sequence TCCGGTATGAGCCTCTGATCAAGAGGATGTCAGAATGCCA, SEQ ID NO: 10), and R4 (sequence CGAATAATAACTGTTATTTTTCAG, SEQ ID NO: 11) containing a 20bp homologous arm at the 5' end were designed to amplify the α signal peptide (sequence shown in SEQ ID NO: 1) on the vector pPIC9K (purchased from Invitrogen). (Shown NO: 14) and the backbone, after amplification of the product by agarose gel electrophoresis, the PSC promoter, α signal peptide, α-glucosidase gene sequence, and plasmid pPIC9K-PSC outer loop were seamlessly assembled. After verification with primers F5 (sequence CAGCTTATCATCGATAAGCTG, SEQ ID NO: 12) and R5 (sequence GTGGGAAATACCAAGAAAAAC, SEQ ID NO: 13), the expression vector pPIC9K-PSC-α-glu was obtained. In some embodiments, the structure of the expression vector is as follows: Figure 1 As shown.

[0070] In another respect, this disclosure provides a host cell comprising the aforementioned α-glucosidase expression vector.

[0071] In some embodiments, the host cell includes 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, a variant of Aspergillus buergerianus ( Aspergillus niger var.awamori Aspergillus oryzae ( ) Aspergillusoryzae ), Candida japonicus ( Candida guilliermondii ), Candida albicans ( Candida lipolytica ), Tropical Candida ( Candida pseudotropicalis ), Candida utilis ( Candida utilis ), chestnut blight ( Endothia parasitica Ashu pseudocystis ( ) Eremothecium ashbyii Fusarium moniliforme () Fusarium moniliforme Kluyveromycin (lactic acid yeast) Kluyveromyces lactis ), Max Kluyveromycin ( Kluyveromyces marxianus ), Morteirellavinaceae var raffinoseutilizer ( Morteirella vinaceae var. raffinoseutilizer ), Mucor ( ) Mucormycosis ), Mucor cultivar Cooney et Emerson ( Mucer miehei var. Cooney and Emerson ), Mucor pusillus Lindt Penicillium loudi ( Penicillium roquefortii Pichia pastoris ( Shepherd's pie ), Rhizopus spp. Rhizopus niveus ), brewing yeast ( Saccharomyces cerevisiae ), crispy-wall yeast ( Saccharomyces fragile Trichoderma reesei ( Trichoderma reesei ), thermophilic hygrophytes ( Myceliophthora thermophile )and Chrysosporium lucknowense .

[0072] In some embodiments, the host cell is Pichia pastoris, preferably Pichia pastoris (Pichia pastoris). Komagataella phaffii Also known as Shepherd's pie (More preferably, Pichia pastoris strains X33, GS115, SMD1168, KM71, or KM71H.)

[0073] 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 It is deposited at the China Center for Type Culture Collection, located at Wuhan University, with accession number CCTCC M 20242483.

[0074] In another aspect, this disclosure provides a method for constructing a host cell expressing α-glucosidase, comprising:

[0075] (1) The coding sequence of the α-glucosidase is ligated into 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 artificially synthesized promoter with a nucleotide sequence as shown in SEQ ID NO: 1;

[0076] (2) Transform the recombinant expression vector obtained in step (1) into host cells;

[0077] (3) Screening to obtain host cells with high α-glucosidase expression levels.

[0078] In some embodiments, the host cell is *Pichia pastoris*. In some preferred embodiments, the host cell is a modified *Pichia pastoris* GS115.

[0079] In some embodiments, the method further includes a codon optimization step for the coding sequence of the α-glucosidase prior to step (1). In some embodiments, the recombinant expression vector obtained in step (1) is localized and transformed into host cells in step (2). In some embodiments, the recombinant expression vector is linearized using a SalI restriction site.

[0080] In another aspect, this disclosure provides a method for producing α-glucosidase, comprising culturing the aforementioned host cells to obtain a culture comprising the α-glucosidase.

[0081] In some embodiments, the host cell is Pichia pastoris (… Shepherd's pie, Also known as Komagataella phaffii The *Pichia pastoris* strain contains the expression vector pPIC9K-PSC-α-glu, whose accession number is CCTCC M 20242483; the supernatant of the culture contains the α-glucosidase.

[0082] In some embodiments, the culture includes culturing the host cells in YPD growth medium for 16-24 hours, then seeding them into BMEY medium and culturing for 96-120 hours, with the addition of 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 added in a flow-through ratio of 0.5-1% by volume during culturing in BMEY medium.

[0083] In some embodiments, the culture further includes the step of activating the host cells and preparing a seed culture. In some embodiments, the host cells are activated in 5 ml of YPD medium and cultured overnight at 200 rpm and 30°C for 14-16 h to obtain a seed culture. In some embodiments, the seed culture is inoculated into shake flasks at an inoculum of 5%-10% for fermentation. In some embodiments, the cells are first cultured in 50 ml of YPD growth medium at 30°C and 250 rpm for 16-24 h, then inoculated into 100 ml of BMEY medium and induced to culture at 30°C and 250 rpm for 120 h, with 0.5% (v / v) ethanol added every 24 h. After centrifugation of the fermentation broth, the cell precipitate is removed, and the fermentation supernatant contains the α-glucosidase.

[0084] In some embodiments, the method for producing α-glucosidase further includes a method for isolating and purifying the α-glucosidase from the culture. In some embodiments, the isolation and purification includes:

[0085] (1) After centrifuging the culture, take the supernatant to obtain a solution, which yields crude α-glucosidase solution;

[0086] (2) Adjust the pH of the crude α-glucosidase solution obtained in step (1) to 7.0-8.0, filter and purify to obtain the α-glucosidase.

[0087] In some embodiments, the filtration is performed using 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.

[0088] In some embodiments, the purified α-glucosidase is desalted again and freeze-dried under vacuum to obtain α-glucosidase lyophilized powder.

[0089] In some embodiments, the culture, i.e., the fermentation broth, is centrifuged to remove the cell precipitate, and the fermentation supernatant contains the α-glucosidase, which is the crude α-glucosidase enzyme solution.

[0090] Take 10 mL of crude α-glucosidase solution, adjust the pH to 7.0 / 8.0 with NaOH, and filter it using a 0.45 μm sterile needle filter.

[0091] For analysis using an AKTA Pure 150 chromatography column, the entire flow path was rinsed with ultrapure water for 5 column volumes at a flow rate of 5 mL / min. Five column volumes were then equilibrated with buffer A at a flow rate of 5 mL / min. Before loading, the loop was washed with buffer A at a flow rate of 3 mL / min. Elution was performed in stages using buffers containing 100, 200, 300, 400, and 500 mM NaCl at a flow rate of 5 mL / min. Elution peaks were collected at each stage, and the molecular weight and purity of the fusion protein were determined by SDS-PAGE. Five column volumes were then washed with ultrapure water, followed by five column volumes of 20% ethanol at a flow rate of 5 mL / min. After optimization, the recombinant protein was purified using a Hitrap Capto Q 5 mL strong anion exchange chromatography column with a loading volume of 2 mL. Elution was performed with buffers containing 100–600 mM NaCl, with 300 mM NaCl eluting yielding α-glucosidase. The purified enzyme was desalted again and freeze-dried under vacuum to obtain α-glucosidase lyophilized powder.

[0092] On the other hand, this disclosure provides the 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 pharmaceuticals.

[0093] This invention constructs an engineered Pichia pastoris strain and cultivation method for expressing α-glucosidase using food-grade ethanol, achieving highly efficient heterologous recombinant expression of α-glucosidase with a yield of up to ten grams per liter. This method is easy for large-scale fermentation, low in cost, and highly safe, facilitating subsequent applications in food and pharmaceutical fields. Industrially, α-glucosidase can be used in the production of isomaltooligosaccharides (IMOs), which are among the most produced and sold functional oligosaccharides globally, widely used in food, feed, and pharmaceutical production. IMOs primarily promote the proliferation of beneficial intestinal flora, inhibit the growth of many pathogenic and putrefactive bacteria, enhance human immunity, reduce serum cholesterol, triglycerides, and free fatty acid levels, and eliminate the causes of cardiovascular diseases. They also have excellent anti-caries properties, are difficult or not easily digested and absorbed by the human body, and provide very little or no energy, making them suitable as a health-promoting sweetener for diabetics, obese patients, and those with hypoglycemia. In the feed industry, isomaltooligosaccharide is considered to improve poultry survival rates without the side effects on humans caused by synthetic feeds. It has been reported that about 50% of feed in Japan contains isomaltooligosaccharide.

[0094] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate this disclosure. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on this disclosure. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in numerous publications.

[0095] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions in the field of genetic engineering or under conditions recommended by the manufacturer.

[0096] Plasmids were constructed using the Seamless Cloning Kit from Novizan Biosciences.

[0097] The enzymes used were purchased from Tiangen Biotech (Beijing) or TaKaRa Biotechnology (Dalian). The specific reaction conditions and methods used were in accordance with the product instructions.

[0098] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0

[0099] YPD medium: glucose 20 g / L, tryptone 20 g / L, yeast extract 10 g / L, pH adjusted to 6.0-7.0.

[0100] YND medium: YNB 6.7g / L, glucose 10.0g / L; solid medium supplemented with agar 20g / L.

[0101] BMEY medium: peptone 20.0 g / L, yeast extract 10.0 g / L, YNB 13.4 g / L, 0.5% ethanol.

[0102] 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.

[0103] All the above culture media were autoclaved at 121°C for 20 minutes. Specifically, the glucose solution was autoclaved at 115°C for 30 minutes, and ethanol was added at room temperature before use. All the solvents for the above culture media were deionized water or tap water.

[0104] Example 1. Construction of an expression vector containing the α-glucosidase gene

[0105] (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 Pichia pastoris host and synthesized by Qingke Biotechnology Co., Ltd. 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, which is shown in SEQ ID NO: 3. The PCR amplification system was 50 μl: 1 μL template, 2 μl each of F / R primers, 25 μl PrimeSTAR Max Premix (2×) enzyme, and 33 μL sterile deionized water. PCR conditions: pre-denaturation 98°C, 5 min; denaturation 98°C, 10 s; annealing 60°C, 20 s; extension 72°C, 1 min, for a total of 30 cycles; final extension 72°C, 10 min.

[0106] (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) with a 20bp homologous arm 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). The amplified product (shown in SEQ ID NO: 14) and its backbone were used to ligate the PSC promoter, α signal peptide, α-glucosidase gene sequence, and pPIC9K vector backbone fragment at 50°C for 20 min using a homologous recombination kit (purchased from Novizan Biosciences Co., Ltd.). The ligation product was then chemically transformed into E. coli DH5α competent cells (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B528413). The transformation solution was plated on LB agar plates containing ampicillin (100 mg / L) and incubated overnight at 37°C. Positive transformants were selected, and colony PCR was performed using primers F5 (sequence shown in SEQ ID NO: 12) and R5 (sequence shown in SEQ ID NO: 13) for verification. After sequencing by Sangon Biotech (Shanghai) Co., Ltd., the cells were incubated overnight at 37°C on a shaker, and the plasmid was extracted and named pPIC9K-PSC-α-glu. The plasmid map is shown in [image missing]. Figure 1 As shown.

[0107] Example 2. Construction of Pichia pastoris engineered strain containing α-glucosidase gene

[0108] (1) Preparation of competent cells

[0109] Select *Pichia pastoris* strain Δku70-I_AM (refer to published patent CN 118726120 A) containing an ethanol-inducible promoter expressing artificial transcription activator, and culture overnight at 30°C with shaking in 10 ml of YPD liquid medium. Then, transfer 100 μl of the culture to 100 mL of YPD liquid medium and culture until OD600 = 1.2. Centrifuge at 5000 rpm for 3 min at 4°C and collect the cells. Mix the cells with pre-chilled 0.1 M LiAc buffer and DTT buffer by pipetting and aspirating. Incubate the centrifuge tubes at 200 rpm for 30–45 min at 30°C on a shaker, centrifuge at 5000 rpm for 3 min at 4°C, then wash the cells twice with pre-chilled 1 M sorbitol buffer. Finally, resuspend the cells in 1 ml of pre-chilled 1 M sorbitol buffer and aliquot into 80 μL tubes.

[0110] (2) Electro-induced conversion and transformant screening

[0111] The recombinant plasmid pPIC9K-PSC-α-glu obtained in Example 1 was linearized by SalI digestion. 10 μg of the linearized recombinant plasmid was mixed thoroughly with 80 μL of competent cells and transferred to a 0.2 cm electroporation cuvette that had been chilled at 4°C. The cuvette containing the mixture was then incubated on ice for 5 min. The electroporator parameters were adjusted to the Pichia pastoris setting: voltage 1.5 kV, capacitance 25 μF, resistance 200 ohms, and time approximately 5 ms. After electroporation, 1 ml of pre-chilled 1 M sorbitol solution was quickly added to the cuvette, and the mixture was gently aspirated and transferred to a centrifuge tube. The mixture was incubated at 30°C and 200 rpm for 1-2 h, followed by centrifugation at 3000 rpm for 5 min. The supernatant was discarded, and the mixture was plated on YND plates and incubated at 30°C for 2-4 days until single colonies appeared.

[0112] Single colonies were selected and yeast colony PCR was performed using primers F5 (sequence shown in SEQ ID NO: 12) and R5 (sequence shown in SEQ ID NO: 13). The correct bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced strain was identified as the recombinant strain PSC-α-glu expressing α-glucosidase, and subsequent fermentation experiments were conducted.

[0113] Example 3. Induced expression of α-glucosidase

[0114] In the recombinant strain PSC-α-glu obtained in Example 2 above, the PSC artificial promoter regulating α-glucosidase expression can bind to an artificial transcription activator, which is expressed by an ethanol-inducible promoter (see published patent CN 118726120 A). Therefore, ethanol can be used as a substrate to induce recombinant α-glucosidase expression indirectly. Eleven single colonies verified correctly in Example 2 were picked and cultured overnight at 200 rpm and 30°C for 14-16 h in 5 mL YPD medium. Then, they were inoculated into shake flasks at an inoculum size of 5%-10% for fermentation. First, the cells were cultured in 50 mL YPD growth medium at 30°C and 250 rpm for 16-24 h. Then, the cells were inoculated into 100 mL BMEY medium and induced to culture at 30°C and 250 rpm for 120 h, with 0.5% (V / V) ethanol added every 24 h.

[0115] The fermentation broth was centrifuged at 10,000 rpm for 5 minutes to remove the bacterial precipitate. The supernatant was collected as the crude α-glucosidase enzyme solution, and 10 μl was analyzed by SDS-PAGE. The detection results of the crude enzyme solution obtained from the fermentation of 11 single colonies (numbered 1-11) are as follows. Figure 2 As shown, colony 1 had the highest α-glucosidase expression level. This recombinant Pichia pastoris (… Komagataella phaffii Also known as Shepherd's pie The genetically engineered bacteria are deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC M 20242483 and deposit date November 7, 2024.

[0116] Example 4. Reactor fermentation of recombinant Pichia pastoris PSC-α-glu

[0117] Example 3 Figure 2 The transformant with the highest expression level (transformer #1) was further fermented in a 5L reactor. The engineered bacteria were inoculated into YPD liquid medium and cultured until OD500 reached. 600 The seed culture was obtained at a concentration of approximately 7. Then, the seed culture was inoculated into a 5L reactor (3L BMEY fermentation medium) at an inoculation rate of 5% (v / v). Fermentation was carried out at a temperature of approximately 30°C, a pH of approximately 6, and a dissolved oxygen concentration greater than 30%. The dissolved oxygen concentration was kept stable. If the dissolved oxygen concentration suddenly increased, the high dissolved oxygen concentration was maintained for about 20 minutes before adding glucose solution. The addition was stopped when the wet weight of the cells increased to 150 g / L.

[0118] When glucose is depleted and dissolved oxygen levels recover, ethanol is added at a volume ratio of 0.5–1% and trace elements at a volume ratio of 0.01% to maintain dissolved oxygen levels above 25%. Fermentation continues for 96 hours, and the supernatant is collected as the crude α-glucosidase enzyme solution. Using a BCA protein concentration assay kit (purchased from Beyotime Biotechnology Co., Ltd.) with bovine serum albumin (BSA) as a standard, the expression level of α-glucosidase can be detected to reach approximately 10 g / L.

[0119] Example 5. Purification method of recombinant Pichia pastoris expressing α-glucosidase

[0120] Take 10 mL of the crude α-glucosidase solution from Example 4, adjust the pH to 7.0 with NaOH, and filter it using a 0.45 μm sterile needle filter.

[0121] For analysis using an AKTA Pure 150 chromatography column, the entire flow path was rinsed with ultrapure water for 5 column volumes at a flow rate of 5 mL / min. Five column volumes were then equilibrated with buffer A at a flow rate of 5 mL / min. Before loading, the loop was washed with buffer A at a flow rate of 3 mL / min. Elution was performed in stages using buffers containing 100, 200, 300, 400, and 500 mM NaCl at a flow rate of 5 mL / min. Elution peaks were collected at each stage, and the molecular weight and purity of the fusion protein were determined by SDS-PAGE. Five column volumes were then washed with ultrapure water, followed by five column volumes of 20% ethanol at a flow rate of 5 mL / min. After optimization, the recombinant protein was purified using a Hitrap Capto Q 5 mL strong anion exchange chromatography column with a loading volume of 2 mL. Elution was performed with buffers containing 100–600 mM NaCl, with 300 mM NaCl eluting yielding α-glucosidase. The purified enzyme was desalted again and freeze-dried under vacuum to obtain 87 mg of α-glucosidase lyophilized powder with a purity of 92%, with a yield of 87%.

[0122] Example 6. Enzyme activity analysis of α-glucosidase

[0123] Determination of α-glucosidase activity:

[0124] (1) Reagents and solutions:

[0125] Acetic acid buffer: Dissolve 4.76 g of sodium acetate trihydrate (NaCH3COO·3H2O, MW=136.08) in approximately 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. Adjust the final volume to 500 ml.

[0126] Stop the reaction (sodium carbonate) solution: Dissolve 15.9g of sodium carbonate (Na2CO3, MW=105.99) in 400 ml of water and bring the volume up to 500 ml.

[0127] Substrate (p-Nitrophenyl-α-D-glucopyranoside, PNPG) solution (0.1% w / v, 10 ml): Dissolve 10.0 mg PNPG in 10 ml acetate buffer. Heat the acetate buffer to 40°C to accelerate the dissolution of the PNPG powder.

[0128] p-Nitrophenol stock solution (PNP): Dissolve 14 mg of p-nitrophenol (PNP, MW=139.11) in 10 ml of acetate buffer to prepare a 10 mM PNP stock solution. After the PNP is completely dissolved, take 1 ml of the PNP stock solution and place it in a sterile 15 ml centrifuge tube. Then add 9 ml of acetate buffer to dilute the PNP 10-fold to a final concentration of 1 mM.

[0129] (2) Preparation of standards and samples

[0130] Standards: PNP standards with concentrations of 6, 20, and 50 µM were prepared from 1 mM stock solution. The absorbance (A400) was measured at 400 nm, and the net A400 value was obtained by subtracting the blank value from the PNP standard value. These values ​​were plotted according to Beer-Lambert's law to calculate PNP (…). The millimolecular extinction coefficient of )

[0131]

[0132] in: λ represents the absorbance at wavelength λ. λ is the molar extinction coefficient at wavelength λ, c is the molar concentration of the solution, and l is the path length (the standard spectrophotometer cuvette is 1 cm).

[0133] (3) Sample solution:

[0134] Dilute the α-glucosidase powder sample obtained in Example 5 in sufficient acetate buffer at least twice, with an activity range of 0.4 to 0.8 GAU / ml. If the sample activity cannot be estimated / is 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).

[0135] (4) Steps:

[0136] Measure the absorbance of three PNP standard solutions and calculate the molar extinction coefficient.

[0137] For the blank solution, mix 200 µl of PNPG substrate with 300 µl of stop solution and 200 µl of acetate buffer.

[0138] For active samples, add 200 µl of PNP substrate to a centrifuge tube and pre-incubate for at least 5 minutes in a water bath at different temperatures (20°C, 30°C, 40°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 80°C, pH 4.5) and different pH values ​​(adjusted to pH 3, 4, 4.5, 5, 5.5, 6, 7, and 8 using hydrochloric acid and sodium hydroxide solutions, at 50°C). After pre-incubation, add 200 µl of each enzyme / sample diluent to each replicate tube at 15-second intervals. Once the sample and substrate have bound in the first tube (incubated for 10 minutes), start a timer set to 10 minutes. After 10 minutes of incubation, immediately add 300 µl of stop solution to each of the three replicate tubes at 15-second intervals. Before measuring absorbance, mix the tubes on a vortex mixer and incubate at room temperature for 10 minutes. The absorbance of the resulting 700 μl mixture was then measured at 400 nm using a spectrophotometer.

[0139] (5) Enzyme activity calculation:

[0140] Enzyme activity definition: One unit of glucosylamylase activity (GAU) is defined as the amount of glucosylamylase that releases 1 μmol of nitrophenol (PNP) in 1 hour under experimental conditions (pH 5.0, 50°C). Enzyme activity can be calculated using the following formula:

[0141]

[0142] A sample The absorbance of the sample was measured at 400 nm.

[0143] A blank The absorbance of the blank solution at 400 nm

[0144] d is the dilution factor (the factor by which the sample is diluted before activity measurement).

[0145] according to Figure 3 、 Figure 4 The results showed that the optimal pH for α-glucosidase activity expressed by recombinant Pichia pastoris PSC-α-glu was 5.0, and the optimal temperature was 60°C. The enzyme activity was 15117.6 U / g at pH 5.0 and 60°C. Furthermore, this α-glucosidase exhibited good thermostability, maintaining a relative activity of 80% or higher within the range of 50-70°C; it also showed some resistance to acids and alkalis, maintaining a relative activity of 80% or higher within the pH range of 4.0-6.5.

[0146] The method disclosed herein can be used to produce α-glucosidase, which can be induced by ethanol to secrete α-glucosidase into the supernatant of fermentation broth. The extracellular expression level can reach 10 g / L in a 5L fermenter, which is high in yield. Purification does not require cell disruption, thus reducing production costs.

[0147] All the embodiments described above are merely illustrative examples to clearly illustrate actual operations and are not intended to limit the implementation. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively list all implementations here, but obvious variations derived therefrom remain within the scope of this disclosure.

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 20242483, and the Pichia pastoris has an artificial synthetic promoter with a nucleotide sequence as shown in SEQ ID NO: 1, an α-signal peptide coding sequence as shown in SEQ ID NO: 14, and an α-glucosidase coding sequence as shown in SEQ ID NO:

3.

2. A method for producing α-glucosidase, comprising culturing the Pichia pastoris according to claim 1 to obtain a culture containing the α-glucosidase.

3. The method according to claim 2, wherein: The culture supernatant contains the α-glucosidase.

4. The method according to claim 3, wherein: The culturing comprises culturing the Pichia pastoris in a YPD growth medium for 16-24 hours, inoculating the Pichia pastoris into a BMEY medium for culturing for 96-120 hours, and adding ethanol with a volume percentage of 0.5-1%.

5. Use of the Pichia pastoris according to claim 1 in the production of food, feed or medicine.

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