Recombinant pichia pastoris expressing recombinant glucoside hydrolase and preparation method, preparation and application thereof

Abstract

The application relates to the field of biotechnology, in particular to recombinant Pichia pastoris expressing recombinant glucoside hydrolase, a preparation method, a preparation and application thereof, further relates to the recombinant glucoside hydrolase, a preparation method of the recombinant glucoside hydrolase, a preparation of the recombinant glucoside hydrolase, a preparation method of the preparation of the recombinant glucoside hydrolase, a product for constructing the recombinant Pichia pastoris and a method for synthesizing ginsenoside CK. The recombinant Pichia pastoris expresses the recombinant glucoside hydrolase, and MicC sRNA and Hfq protein of a target gene. The recombinant Pichia pastoris has the advantages of high expression of the recombinant glucoside hydrolase, high safety, high heat resistance, strong specificity and high transformation efficiency.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to recombinant Pichia pastoris expressing recombinant glucosyl hydrolase, its preparation method, formulation and application, and further to recombinant glucosyl hydrolase, recombinant glucosyl hydrolase preparation method, recombinant glucosyl hydrolase formulation, preparation method of recombinant glucosyl hydrolase formulation, product for constructing recombinant Pichia pastoris and method for synthesizing ginsenoside CK. Background Technology

[0002] Ginsenosides are important active ingredients in ginseng and other ginseng species. Rare ginsenosides are present in very low amounts or not naturally occurring, but they typically possess rich biological activities. Among them, rare ginsenoside CK is a ginsenoside metabolite first discovered in the human gut microbiota. Numerous studies have shown that rare ginsenoside CK, due to its small molecular weight, has high bioavailability and abundant biological activities, including anti-aging, anti-inflammatory, anti-tumor, hypoglycemic, lipid-lowering, hepatoprotective, and neuroprotective effects, as well as skincare effects such as anti-inflammatory, whitening, anti-aging, moisturizing, and anti-allergic properties. In conclusion, rare ginsenoside CK has extremely high application value, and researching and developing methods for its production is of great significance.

[0003] Currently, the mainstream preparation processes for ginsenoside CK include microbial transformation and enzymatic transformation. Both methods synthesize the target product by deglycosylation of the main prototype ginsenosides such as Rb1, Rb2, and Rd. While microbial transformation has the advantages of mild reaction conditions and low production costs, it also has inherent risks compared to enzymatic transformation, such as poor genetic stability of microbial strains, complex intracellular enzyme composition, and difficulty in assessing product safety. Furthermore, it faces technical shortcomings such as complex reaction product composition, longer transformation cycle, and insufficient controllability of a single target product.

[0004] In contrast, the bioenzymatic conversion method, which specifically degrades the glycosidic bonds of proto-ginsenosides to generate rare ginsenosides, has significant advantages such as mild reaction conditions, environmental friendliness, high product purity, and excellent conversion efficiency. It also boasts high efficiency, specificity, and low pollution, making it the most widely used technical route in this field. However, this method still faces two major industrialization bottlenecks: first, the large-scale cultivation of special microbial strains capable of producing high-purity ginsenoside glycosidases is difficult, hindering industrial production; second, commercially available glycosidases generally suffer from weak substrate specificity, insufficient glycosylation activity, large enzyme dosage, and high overall cost.

[0005] Bioenzymes can be obtained from natural microorganisms and recombinant genetically engineered strains. Natural microorganisms, however, require natural selection. This selection process is lengthy and the results are often random, making it difficult to obtain suitable strains.

[0006] Synthetic biology, as a cutting-edge interdisciplinary field in the 21st century, provides powerful tools to support basic research in life sciences and breakthroughs in biotechnology innovation. With the rapid development of synthetic biology techniques, researchers can screen and optimize key functional genes encoding ginsenoside glycoside hydrolases and introduce them into host bacteria for heterologous and efficient expression. The resulting recombinant glycoside hydrolases possess excellent characteristics of high catalytic efficiency and strong substrate specificity, significantly improving the substrate conversion rate of the original ginsenosides and greatly shortening the enzymatic reaction cycle, thereby achieving a comprehensive improvement in production efficiency and an effective reduction in production costs. In recent years, the method of using genetic engineering technology to construct recombinant genetically engineered strains to produce glucosinolate hydrolases and using enzymatic conversion to generate rare ginsenoside CK has attracted much attention. However, the current method of producing glucosinolate hydrolases using recombinant strains still suffers from low efficiency; furthermore, when using commonly used *E. coli* for recombinant expression of glucosinolate hydrolases, the potential food safety risks associated with *E. coli* itself and endotoxin residues limit its application in the food industry.

[0007] In summary, current enzymatic methods for preparing rare ginsenosides (CK) still face numerous technical limitations, primarily manifested in difficulties in obtaining high-yielding bacterial strains, low enzyme expression levels, insufficient substrate conversion efficiency, and the unsuitability of the widely used *E. coli* system for food-grade applications. Therefore, achieving efficient and specific conversion of rare ginsenosides via bioenzymes, promoting industrial-scale production with process adaptation, and ultimately realizing the large-scale preparation of this high-value active ingredient has become a key technical challenge that urgently needs to be overcome by those skilled in the art. Summary of the Invention

[0008] Therefore, it is necessary to provide a recombinant Pichia pastoris expressing recombinant glucosyl hydrolase, as well as its preparation method, formulation, and application.

[0009] In a first aspect, some embodiments provide a recombinant Pichia pastoris expressing a recombinant glucosyl hydrolase; and...

[0010] Expression Target The MicC sRNA and Hfq protein of the gene.

[0011] In an optional embodiment, the nucleic acid molecule encoding the recombinant glucosidase, and the nucleic acid molecule encoding the target... At least one of the nucleic acid molecules of the MicC sRNA of the gene and the nucleic acid molecule encoding the Hfq protein is integrated into the genome of the recombinant Pichia pastoris;

[0012] In an optional embodiment, the nucleic acid molecule encoding the recombinant glucosinolate hydrolase is integrated into the HIS4 site of yeast;

[0013] In an optional implementation, the target is encoded. The nucleic acid molecules of the MicC sRNA of the gene and the nucleic acid molecules encoding the Hfq protein are integrated into the AOX1 promoter region of yeast;

[0014] In an optional embodiment, the genome of the recombinant Pichia pastoris integrates a first nucleic acid molecule, the first nucleic acid molecule containing a coding sequence encoding the recombinant glucosinolate hydrolase;

[0015] In an optional embodiment, a second nucleic acid molecule is integrated into the genome of the recombinant Pichia pastoris, and the second nucleic acid molecule contains the target The coding sequence of the MicC sRNA of the gene and the coding sequence of the Hfq protein;

[0016] In an optional embodiment, the second nucleic acid molecule contains, in tandem, the following coding sequence: Gene-binding sequence, MicC backbone coding sequence, yeast terminator sequence, yeast promoter sequence, and Hfq coding sequence;

[0017] In an optional embodiment, the first nucleic acid molecule is a coding sequence encoding the recombinant glucosinolate hydrolase, the amino acid sequence of which is as shown in SEQ ID NO.1, or has at least 90% identity with the sequence shown in SEQ ID NO.1, and the 233rd amino acid residue is K, the 234th amino acid residue is K, and the 288th amino acid residue is Y;

[0018] In an optional implementation, the The gene-binding sequence is as shown in SEQ ID NO.4, or has at least 90% identity with the sequence shown in SEQ ID NO.4;

[0019] In an optional implementation, the MicC skeleton coding sequence is as shown in SEQ ID NO.5, or has at least 90% identity with the sequence shown in SEQ ID NO.5;

[0020] In an optional embodiment, the yeast terminator sequence includes the AOX1 terminator sequence. Further optionally, the AOX1 terminator sequence is as shown in SEQ ID NO.6, or has at least 90% identity with the sequence shown in SEQ ID NO.6.

[0021] In an optional embodiment, the yeast promoter sequence includes the TEF1 promoter sequence. Further optionally, the TEF1 promoter sequence is as shown in SEQ ID NO.7, or has at least 90% identity with the sequence shown in SEQ ID NO.7.

[0022] In an optional embodiment, the amino acid sequence encoded by the Hfq coding sequence is as shown in SEQ ID NO.8, or has at least 90% identity with the sequence shown in SEQ ID NO.8;

[0023] In an optional implementation, the Hfq encoding sequence is as shown in SEQ ID NO.3, or has at least 90% identity with the sequence shown in SEQ ID NO.3;

[0024] In an optional embodiment, the amino acid sequence encoded by the second nucleic acid molecule is as shown in SEQ ID NO.2, or has at least 90% identity with the sequence shown in SEQ ID NO.2;

[0025] In an optional embodiment, the starting strain of the recombinant Pichia pastoris is Pichia pastoris strain GS115.

[0026] Secondly, some embodiments provide a method for preparing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in the first aspect, the method comprising: mixing a nucleic acid molecule encoding the recombinant glucosyl hydrolase with a nucleic acid molecule encoding the target... The nucleic acid molecules of the MicC sRNA gene and the nucleic acid molecules encoding the Hfq protein were transformed into the starting strain to construct the recombinant Pichia pastoris.

[0027] In an optional embodiment, the preparation method includes preparing a nucleic acid molecule encoding the recombinant glucosidase and a nucleic acid molecule encoding the target... At least one of the nucleic acid molecules of the MicC sRNA of the gene and the nucleic acid molecule encoding the Hfq protein is integrated into the genome of the originating strain;

[0028] In an optional embodiment, the preparation method includes integrating a nucleic acid molecule encoding the recombinant glucosinolate hydrolase into the HIS4 site of yeast;

[0029] In an optional embodiment, the preparation method includes encoding the target The nucleic acid molecules of the MicC sRNA of the gene and the nucleic acid molecules encoding the Hfq protein are integrated into the AOX1 promoter region of yeast;

[0030] In an optional embodiment, the preparation method includes first transforming the nucleic acid molecule encoding the recombinant glucosyl hydrolase into a starting strain, and then screening for positive strains expressing the recombinant glucosyl hydrolase; then transforming the nucleic acid molecule encoding the target... The nucleic acid molecules of the MicC sRNA gene and the nucleic acid molecules encoding the Hfq protein were transformed into the positive strain to construct the recombinant Pichia pastoris.

[0031] In an optional embodiment, the preparation method includes transforming the first nucleic acid molecule and the second nucleic acid molecule described in the first aspect into the starting strain to construct the recombinant Pichia pastoris;

[0032] In an optional implementation, the starting strain includes Pichia pastoris.

[0033] Thirdly, some embodiments provide a recombinant glucosyl hydrolase expressed by recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in the first aspect, or by recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the preparation method described in the second aspect.

[0034] Fourthly, some embodiments provide a method for preparing recombinant glucosyl hydrolase, the method comprising culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in the first aspect or recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the method described in the second aspect; and then isolating the recombinant glucosyl hydrolase expressed by the recombinant Pichia pastoris.

[0035] Fifthly, some embodiments provide a recombinant glucosyl hydrolase preparation comprising the recombinant glucosyl hydrolase described in the third aspect; or comprising a culture of recombinant Pichia pastoris expressing the recombinant glucosyl hydrolase described in the first aspect; or comprising a culture of recombinant Pichia pastoris expressing the recombinant glucosyl hydrolase prepared by the preparation method described in the second aspect.

[0036] In an optional embodiment, the culture includes fermentation broth, fermentation supernatant, bacterial cells, or lysate;

[0037] In an optional embodiment, the recombinant glucosidase preparation further includes a crude extract or solid preparation of the culture after treatment;

[0038] In an optional implementation, the process includes drying;

[0039] In an optional embodiment, the recombinant glucosinolate hydrolase preparation is a solid preparation of the fermentation broth supernatant that has been freeze-dried.

[0040] In a sixth aspect, some embodiments provide a method for preparing a recombinant glucosyl hydrolase preparation, the method comprising culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in the first aspect, or culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the method described in the second aspect; and then collecting the culture.

[0041] In an optional embodiment, the culture supernatant of the culture is separated and then freeze-dried.

[0042] In a seventh aspect, some embodiments provide a product for constructing the recombinant Pichia pastoris described in the first aspect, the product comprising at least one of (i) to (ii):

[0043] (i) A nucleic acid composition comprising the first nucleic acid molecule described in the first aspect and the second nucleic acid molecule described in the first aspect.

[0044] (ii) includes a first carrier and a second carrier, wherein the first carrier carries the first nucleic acid molecule described in the first aspect, and the second carrier carries the second nucleic acid molecule described in the first aspect;

[0045] In an optional embodiment, the first vector is a pPIC9K vector that integrates the first nucleic acid molecule;

[0046] In an optional embodiment, the second vector is a pPICZA vector that integrates the second nucleic acid molecule.

[0047] Eighthly, some embodiments provide that the recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in the first aspect, the recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the preparation method described in the second aspect, the recombinant glucosyl hydrolase as described in the third aspect, the recombinant glucosyl hydrolase preparation as described in the fifth aspect, or the product as described in the fifth aspect are used in at least one of (a) to (b):

[0048] (a) Synthetic ginsenoside CK; and,

[0049] (b) Preparation of products for the synthesis of ginsenoside CK.

[0050] Ninth aspect, some embodiments provide a method for synthesizing ginsenoside CK, the method comprising mixing (I) and (II) below, and preparing ginsenoside CK after reaction;

[0051] (I) The reaction substrate, wherein the reaction substrate comprises at least one of ginsenoside Rb1, ginsenoside Rb2 and ginsenoside Rd;

[0052] (II) The recombinant glucosidase described in the third aspect, or the recombinant glucosidase preparation described in the fifth aspect;

[0053] In an optional embodiment, the reaction conditions include at least one of the following: the pH of the reaction environment is 5 to 5.5, the reaction temperature is 80°C to 85°C, and the reaction time is 3 to 5 hours.

[0054] In an optional embodiment, the scheme includes using ginsenoside Rb1 as a reaction substrate, and mixing the ginsenoside Rb1 with the freeze-dried solid preparation of the fermentation broth supernatant described in the fifth aspect at a weight ratio of (1~2):(1~2) and then reacting.

[0055] Some embodiments of this application provide a recombinant Pichia pastoris that expresses recombinant glucosinolate hydrolase and inhibits... The MicC sRNA of the gene and its molecular chaperone Hfq protein can effectively increase the expression level of recombinant glucosinolate hydrolase.

[0056] The recombinant Pichia pastoris has the following advantages: compared with the Escherichia coli expression system, using Pichia pastoris as the host strain significantly improves the expression level of recombinant glucosinolate hydrolase, and the host strain of Pichia pastoris is safer than the Escherichia coli system. The rare ginsenoside CK obtained can be better adapted for food applications. Attached Figure Description

[0057] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0058] Figure 1 This is a schematic diagram of plasmid design provided in Example 1;

[0059] Figure 2 The results of SDS-PAGE gel electrophoresis of L213A-BgaS(R233K / R234K / W288Y) expressed by GS115-L213A-BgaS(R233K / R234K / W288Y) constructed in Example 1 are shown.

[0060] Figure 3 The yield of L213A-BgaS (R233K / R234K / W288Y) expressed by GS115-L213A-BgaS (R233K / R234K / W288Y)-sRNA-PAS_chr3_1087 constructed in Example 1 and GS115-L213A-BgaS (R233K / R234K / W288Y)-sRNA-PAS_chr3_1087 constructed in Example 2;

[0061] Figure 4 This is a map of sRNA tool plasmids targeting PAS_chr3_1087 provided in Example 2;

[0062] Figure 5 The curves showing the changes in cell weight loss and L213A-BgaS (R233K / R234K / W288Y) yield of recombinant yeast GS115-L213A-BgaS (R233K / R234K / W288Y) over fermentation time;

[0063] Figure 6 The results of HPLC detection in Example 4 of ginsenoside Rb1 and rare ginsenoside CK in the product of the enzyme conversion reaction mediated by the enzyme preparation prepared in Example 3. Detailed Implementation

[0064] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0066] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0067] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0068] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0069] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0070] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0071] In this application, the term "recombinant Pichia pastoris" refers to Pichia pastoris that can or has been introduced with exogenous polynucleotides and / or vectors. The exogenous polynucleotides may or may not be integrated into the genome of the "recombinant Pichia pastoris". Vectors can be introduced into cells to construct recombinant Pichia pastoris, which can then be used to express the target protein. The corresponding target protein can be obtained by culturing the recombinant Pichia pastoris.

[0072] In this application, the term "recombinant glucosinolate hydrolase" refers to an enzyme protein obtained by introducing a nucleic acid molecule encoding a glucosinolate hydrolase into a host cell and expressing it using recombinant DNA technology. This enzyme protein can specifically hydrolyze glycosidic bonds (such as β-glucosinolate bonds at C-3 or C-20) in ginsenoside molecules, converting diol-type ginsenosides (such as Rb1, Rd, etc.) into rare ginsenoside CK. Recombinant glucosinolate hydrolases include both wild-type enzymes with natural amino acid sequences and mutant enzymes obtained by site-directed mutagenesis, truncation, fusion, glycosylation site modification, or sequence optimization based on wild-type enzymes, provided that the mutant enzyme still possesses the catalytic activity to convert diol-type ginsenosides into CK.

[0073] In this application, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a polynucleotide encodes a protein or polypeptide, it optionally encodes either the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0074] In this application, the term "vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. For example, vectors include: plasmids, episome plasmids, microcircular DNA, phage particles, cosmid plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vector (e.g., expression vector) provided in this application contains a nucleic acid sequence encoding a target fragment, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one selection marker.

[0075] In this application, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0076] In this application, the term "identity percentage" refers to the degree to which two nucleotide or amino acid sequences are identical at equivalent positions when optimally aligned. Sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine appropriate parameters for the aligned sequences, including any algorithms required to achieve maximum alignment of the full length of the compared sequences.

[0077] In an optional embodiment, the sequence in this application that is identical to the target sequence differs from the target sequence from conserved amino acid substitutions. The term "conserved amino acid substitution" refers to replacing one amino acid residue with another amino acid residue that is physicochemically similar, such that the substitution does not alter or minimally alters the properties and function of the entire polypeptide or protein. Conserved amino acid substitution is well known to those skilled in the art. Families of amino acid residues with similar side chains are known in the art, including 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 uncharged 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), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Specific forms of conserved amino acid substitutions include those that replace amino acids not in the normal 20 amino acids encoded by the genetic code. In alternative embodiments, the number of "conserved amino acid substitutions" can be, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0078] In this application, "starting strain" refers to a microbial strain that has not undergone this specific genetic modification (such as gene knockout, gene integration, plasmid transformation, or mutagenesis). This strain can be a wild-type strain or an existing modified strain obtained through previous modifications (such as classical mutagenesis or genome simplification), but in the modification steps of this specific implementation, it is further modified as starting material to endow it with new target traits.

[0079] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0080] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0081] In a first aspect, some embodiments provide a recombinant Pichia pastoris expressing a recombinant glucosidase; and, expressing a targeted The MicC sRNA and Hfq protein of the gene.

[0082] MicC is a type of trans-coding small regulatory RNA (sRNA) that was first discovered in E. coli and can interfere with the translation and stability of target mRNA. The locus number of the gene in the Pichia pastoris genome annotation is: It encodes vacuolar aspartic protease A. Hfq protein (Host Factor for RNA phage Qβ) is an RNA molecular chaperone belonging to the Sm-like (LSm) protein family. This application constructs a device carrying... The recombinant sRNA with the MicC backbone targeting the target sequence, together with the Hfq protein, forms a post-transcriptional regulatory system. This recombinant sRNA specifically binds to the target sequence it carries. mRNA is recruited and Hfq protein is introduced; Hfq acts as a molecular chaperone, promoting hybridization between sRNA and target mRNA, and mediating the degradation of target mRNA, thereby inhibiting mRNA degradation. Gene expression is reduced, decreasing the risk of exogenous expression products in recombinant Pichia pastoris being degraded by intracellular proteases. In summary, this recombinant Pichia pastoris, while expressing recombinant glucosyl hydrolase, inhibits... Gene expression increased the yield of recombinant glucosinolate hydrolase.

[0083] In an optional embodiment, the nucleic acid molecule encoding the recombinant glucosyl hydrolase, and the nucleic acid molecule encoding the target At least one of the nucleic acid molecules of the MicC sRNA gene and the nucleic acid molecule encoding the Hfq protein is integrated into the genome of recombinant Pichia pastoris.

[0084] In an alternative embodiment, the nucleic acid molecule encoding recombinant glucosinolate hydrolase is integrated into the HIS4 site of yeast.

[0085] In an optional implementation, coding target The nucleic acid molecules of the MicC sRNA and the nucleic acid molecules encoding the Hfq protein are integrated into the AOX1 promoter region of yeast.

[0086] In an optional embodiment, the genome of the recombinant Pichia pastoris integrates a first nucleic acid molecule containing a coding sequence for a recombinant glucosinolate hydrolase.

[0087] In an optional embodiment, a second nucleic acid molecule is integrated into the genome of the recombinant Pichia pastoris, and the second nucleic acid molecule contains a target The coding sequence of the MicC sRNA and the coding sequence of the Hfq protein.

[0088] In an optional implementation, the second nucleic acid molecule contains, in tandem, the following coding sequence: Gene-binding sequence, MicC backbone coding sequence, yeast terminator sequence, yeast promoter sequence, and Hfq coding sequence. The gene-binding sequence and the MicC backbone coding sequence together form the complete MicC sRNA, and the yeast terminator sequence is used to terminate the process. Transcription of gene-binding sequences and MicC backbone coding sequences, with yeast promoter sequences used to initiate transcription of Hfq coding sequences.

[0089] In an optional embodiment, the first nucleic acid molecule is a coding sequence for a recombinant glucosyl hydrolase, the amino acid sequence of which is shown in SEQ ID NO.1. This recombinant glucosyl hydrolase has advantages such as high heat resistance, high specificity, and high conversion efficiency.

[0090] SEQ ID NO.1:

[0091] MYSFPNSFMFGYSWSGFQFEMGTPGSEDPNTDWYKWVHDPENMAAGLVSGDLPENGPGYWGNYKTFHDNAQKMGLKIARLNVEWSRIFPNPLPRPEDFDESKQDVTEVEINENELKRLDEYANKDA LNHYREIFKDLKSRGLYFILNMYHWPLPLWLHDPIRVRRGDFTGPSGWLSTRTVYEFARFSAYIAWKFDDLVDEYSTMNEPNVVGGLGYVGVKSGFPPGYLSFELSKKAMYNIIQAHARAYDGIKSV SKKPVGIIYANSSFQPLTDKDMEAVEMAENDNRWYFFDAIIRGEITRGDEKIVRDDLKGRLDWIGVNYYTRTVVKRTEKGYVSLGGYGHGCERNSVSLAGLPTSDFGWEFFPEGLYDVLTKYWNRY HLYMYVTENGIADDADYQRPYYLVSHVYQVHRAINSGADVRGYLHWSLADNYEWASGFIWARIFPKPTFDVKVDVEKFGLVYVDFETKKRLYWRPSALVYREIATNGAITDEIEHLNSVPPVKPLRH

[0092] The amino acid sequence of the glucosidase is at least 90% identical to the sequence shown in SEQ ID NO.1 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and the amino acid residue at position 233 is K, the amino acid residue at position 234 is K, and the amino acid residue at position 288 is Y.

[0093] In an optional implementation, The gene-binding sequence is as shown in SEQ ID NO.4, or has at least 90% identity with the sequence shown in SEQ ID NO.4 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0094] SEQ ID NO.4:CATCGTAGTACCGTCAAATATCAT

[0095] In an optional implementation, the MicC skeleton coding sequence is as shown in SEQ ID NO.5, or has at least 90% identity with the sequence shown in SEQ ID NO.5 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0096] SEQ ID NO.5:

[0097] GTTATATGCCTTTATTGTCACAGATTTTATTTTCTGTTGGGCCATTGCATTGCCACTGATTTTCCAACATATAAAAAGACAAGCCCGAACAGTCGTCCGGGCTTTTTTTTT

[0098] In an optional embodiment, the yeast terminator sequence includes the AOX1 terminator sequence. In a further optional embodiment, the AOX1 terminator sequence is as shown in SEQ ID NO.6, or has at least 90% identity with the sequence shown in SEQ ID NO.6 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0099] SEQ ID NO.6:

[0100] TCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGA

[0101] In an optional embodiment, the yeast promoter sequence includes the TEF1 promoter sequence. In a further optional embodiment, the TEF1 promoter sequence is as shown in SEQ ID NO.7, or has at least 90% identity with the sequence shown in SEQ ID NO.7 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0102] SEQ ID NO.7:

[0103] GATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTTTCTTCCTCTAGGGTGCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTC GTCGAAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTCTTGAAAATTTTTTTTTTGTTTTTTTTCTTTCAGTGACCTCCATTGATATTTAAGTTAATAAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAG

[0104] In an optional embodiment, the amino acid sequence encoded by the Hfq coding sequence is as shown in SEQ ID NO.8, or has at least 90% identity with the sequence shown in SEQ ID NO.8 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0105] SEQ ID NO.8

[0106] MAKGQSLQDPFLNALRRERVPVSIYLVNGIKLQGQIESFDQFVILLKNTVSQMVYKHAISTVVPSRPVSHHSNNAGGGTSSNYHHGSSAQNTSAQQDSEETE

[0107] In an optional implementation, the Hfq encoded sequence is as shown in SEQ ID NO.3, or has at least 90% identity with the sequence shown in SEQ ID NO.3 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0108] SEQ ID NO.3:

[0109] ATGGCTAAGGGGCAATCTTTACAAGATCCGTTCCTGAACGCACTGCGTCGGGAACGTGTTCCAGTTTCTATTTATTTGGTGAATGGTATTAAGCTGCAAGGGCAAATCGAGTCTTTTGATCAGTTCGTGATCCTGTTGAAAAACACGGTCAGCC AGATGGTTTACAAGCACGCGATTTCTACTGTTGTCCCGTCTCGCCCGGTTTCTCATCACAGTAACAACGCCGGTGGCGGTACCAGCAGTAACTACCATCATGGTAGCAGCGCGCAGAATACTTCCGCGCAACAGGACAGCGAAGAAACCGAATAA

[0110] In an optional embodiment, the amino acid sequence encoded by the second nucleic acid molecule is as shown in SEQ ID NO.2, or has at least 90% identity with the sequence shown in SEQ ID NO.2 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0111] SEQ ID NO.2:

[0112]

[0113] In an optional implementation, the starting strain of recombinant Pichia pastoris is Pichia pastoris strain GS115.

[0114] Secondly, some embodiments provide a method for preparing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase, comprising: expressing a nucleic acid molecule encoding the recombinant glucosyl hydrolase, and expressing a target... The nucleic acid molecules of the MicC sRNA gene and the nucleic acid molecules encoding the Hfq protein were transformed into the starting strain to construct recombinant Pichia pastoris.

[0115] In an optional embodiment, the preparation method includes preparing a nucleic acid molecule encoding a recombinant glucosidase and a target... At least one of the nucleic acid molecules of the MicC sRNA gene and the nucleic acid molecule encoding the Hfq protein is integrated into the genome of the originating strain.

[0116] In an optional embodiment, the preparation method includes integrating a nucleic acid molecule encoding a recombinant glucosinolate hydrolase into the HIS4 site of yeast.

[0117] In an optional embodiment, the preparation method includes encoding a target The nucleic acid molecules of the MicC sRNA and the nucleic acid molecules encoding the Hfq protein are integrated into the AOX1 promoter region of yeast.

[0118] In an optional embodiment, the preparation method includes first transforming the nucleic acid molecule encoding recombinant glucosyl hydrolase into the starting strain, and then screening for positive strains expressing recombinant glucosyl hydrolase; then encoding the target The nucleic acid molecules of the MicCsRNA gene and the nucleic acid molecule encoding the Hfq protein were transformed into positive strains to construct recombinant Pichia pastoris.

[0119] In an optional embodiment, the preparation method includes transforming the first nucleic acid molecule and the second nucleic acid molecule in the first aspect into the starting strain to construct recombinant Pichia pastoris.

[0120] Thirdly, some embodiments provide a recombinant glucosyl hydrolase expressed by recombinant Pichia pastoris expressing recombinant glucosyl hydrolase according to the first aspect, or by recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the preparation method of the second aspect.

[0121] Fourthly, some embodiments provide a method for preparing recombinant glucosyl hydrolase, the method comprising culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as in the first aspect or recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the method of the second aspect; and then isolating the recombinant glucosyl hydrolase expressed by the recombinant Pichia pastoris.

[0122] Fifthly, some embodiments provide a recombinant glucosyl hydrolase preparation comprising the recombinant glucosyl hydrolase of the third aspect; or comprising a culture of recombinant Pichia pastoris expressing the recombinant glucosyl hydrolase of the first aspect; or comprising a culture of recombinant Pichia pastoris expressing the recombinant glucosyl hydrolase prepared by the preparation method of the second aspect.

[0123] In an optional embodiment, the culture includes fermentation broth, fermentation supernatant, bacterial cells, or lysate;

[0124] In optional embodiments, the recombinant glucosidase preparation may also include crude extracts or solid preparations obtained by treating the culture.

[0125] In an optional implementation, the process includes drying.

[0126] In an optional embodiment, the recombinant glucosinolate hydrolase preparation is a solid preparation of fermentation broth supernatant that has been freeze-dried.

[0127] In an optional embodiment, the recombinant glucosidase preparation further comprises excipients, including but not limited to at least one of stabilizers, protectants, buffer systems, lyophilization protectants, excipients, fillers, preservatives, carriers, and encapsulation materials.

[0128] Sixthly, some embodiments provide a method for preparing a recombinant glucosyl hydrolase preparation, the method comprising culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as in the first aspect, or culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the method of the second aspect; and then collecting the culture.

[0129] In an optional embodiment, the preparation method further includes separating the culture supernatant of the culture and then freeze-drying it.

[0130] In a seventh aspect, some embodiments provide a product for constructing the recombinant Pichia pastoris of the first aspect, the product comprising at least one of (i) to (ii):

[0131] (i) A nucleic acid composition comprising the first nucleic acid molecule of the first aspect and the second nucleic acid molecule of the first aspect.

[0132] (ii) includes a first carrier and a second carrier, wherein the first carrier carries the first nucleic acid molecule in the first aspect, and the second carrier carries the second nucleic acid molecule in the first aspect.

[0133] In an optional implementation, the first vector is a pPIC9K vector that integrates a first nucleic acid molecule.

[0134] In an optional implementation, the second vector is a pPICZA vector that integrates a second nucleic acid molecule.

[0135] Eighthly, some embodiments provide that the recombinant Pichia pastoris of the first aspect, the recombinant glucosidase of the third aspect, the recombinant glucosidase preparation of the fifth aspect, or the product of the seventh aspect are used in at least one of (a) to (b):

[0136] (a) Synthetic ginsenoside CK; and,

[0137] (b) Preparation of products for the synthesis of ginsenoside CK.

[0138] In an optional embodiment, recombinant Pichia pastoris prepared by the first method or the second method can be used directly for whole-cell catalysis. Under conditions suitable for recombinant yeast growth and protein expression, ginsenoside substrates are added to the culture system, allowing the yeast to continuously express recombinant glucosinolate hydrolase and catalyze substrate conversion in situ during growth. After the reaction, the culture is collected and rare ginsenoside CK is extracted.

[0139] In an optional embodiment, the recombinant Pichia pastoris prepared by the first aspect, the recombinant Pichia pastoris prepared by the second aspect, or the product of the seventh aspect is used as the production raw material for the recombinant glucosinolate hydrolase used in the synthesis of ginsenoside CK or the preparation of the product for the synthesis of ginsenoside CK.

[0140] In an optional embodiment, the recombinant glucosinolate hydrolase of the third aspect and the recombinant glucosinolate hydrolase preparation of the fifth aspect are used as the bioenzyme for synthesizing ginsenoside CK by bioenzyme conversion method.

[0141] In an optional embodiment, the recombinant glucosinolate hydrolase of the third aspect and the recombinant glucosinolate hydrolase preparation of the fifth aspect are used as the bioenzyme in the bioenzyme conversion method of the product synthesizing ginsenoside CK.

[0142] Ninth aspect, some embodiments provide a method for synthesizing ginsenoside CK, the method comprising mixing (I) and (II) below, and preparing ginsenoside CK after reaction;

[0143] (I) The reaction substrate includes at least one of ginsenoside Rb1, ginsenoside Rb2 and ginsenoside Rd;

[0144] (II) The third aspect of recombinant glucosinolate hydrolase, or the fifth aspect of recombinant glucosinolate hydrolase preparation;

[0145] In an optional embodiment, the reaction conditions include at least one of the following: the pH of the reaction environment is 5 to 5.5 (e.g., but not limited to 5, 5.2 or 5.5), the reaction temperature is 80°C to 85°C (e.g., but not limited to 80°C, 82°C or 85°C), and the reaction time is 3h to 5h (e.g., but not limited to 3h, 3.5h, 4h, 4.5h or 5h).

[0146] In an optional embodiment, the scheme includes using ginsenoside Rb1 as a reaction substrate, mixing ginsenoside Rb1 with the freeze-dried solid preparation of the fermentation broth supernatant in the fifth aspect at a weight ratio of (1~2):(1~2) and reacting them. The weight ratio of ginsenoside Rb1 and the freeze-dried solid preparation of the fermentation broth supernatant in the fifth aspect is, for example, but not limited to, 1:1, 1:2, 2:3, 3:2 or 2:1.

[0147] The following are some examples.

[0148] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0149] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0150] Example 1: Construction of genetically engineered Pichia pastoris strain

[0151] 1.1 Construction and Validation of Recombinant Plasmids

[0152] (1) The amino acid sequence of the recombinant glycoside hydrolase L213A-BgaS (R233K / R234K / W288Y) is shown in SEQ ID NO:1:

[0153] MYSFPNSFMFGYSWSGFQFEMGTPGSEDPNTDWYKWVHDPENMAAGLVSGDLPENGPGYWGNYKTFHDNAQKMGLKIARLNVEWSRIFPNPLPRPEDFDESKQDVTEVEINENELKRLDEYANKDA LNHYREIFKDLKSRGLYFILNMYHWPLPLWLHDPIRVRRGDFTGPSGWLSTRTVYEFARFSAYIAWKFDDLVDEYSTMNEPNVVGGLGYVGVKSGFPPGYLSFELSKKAMYNIIQAHARAYDGIKSV SKKPVGIIYANSSFQPLTDKDMEAVEMAENDNRWYFFDAIIRGEITRGDEKIVRDDLKGRLDWIGVNYYTRTVVKRTEKGYVSLGGYGHGCERNSVSLAGLPTSDFGWEFFPEGLYDVLTKYWNRY HLYMYVTENGIADDADYQRPYYLVSHVYQVHRAINSGADVRGYLHWSLADNYEWASGFIWARIFPKPTFDVKVDVEKFGLVYVDFETKKRLYWRPSALVYREIATNGAITDEIEHLNSVPPVKPLRH

[0154] (2) Vector construction and sequence optimization

[0155] Using the commercial carrier pPIC9K (carrier map as shown) Figure 1 As shown, the location of the functional region of pPIC9K (which contains the homologous arm of the yeast HIS4 site) is included. Enzyme cleavage sites are designed based on the relevant sequence location of pPIC9K; the target fragment sequence is carried, which encodes the glucosidase L213A-BgaS (R233K / R234K / W288Y).

[0156] This embodiment uses the commercially available plasmid vector pPIC9K, purchased from Beijing Liuhe BGI Genomics Co., Ltd., according to... Figure 1The relevant sequence positions were designed using the EcoRI and NotI restriction enzyme sites. The gene sequence encoding the recombinant glucosyl hydrolase L213A-BgaS (R233K / R234K / W288Y) was obtained through artificial synthesis using artificially optimized E. coli preferred codons. The synthesized full-length DNA fragment of the glycoside hydrolase has one restriction endonuclease site at both the 5' and 3' ends, corresponding to EcoRI and NotI, respectively. The target fragment encoding the glycoside hydrolase was inserted between these two restriction enzyme sites to obtain the expression plasmid encoding the recombinant glycoside hydrolase. The recombinant DNA fragment was synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and the recombinant expression plasmid was constructed. Sequencing results showed 100% sequence identity with the theoretical sequence.

[0157] 1.2 Construction, screening and identification of recombinant strains

[0158] (1) Preparation and transformation of Pichia pastoris competent cells

[0159] a) Using the Pichia pastoris competent cell preparation and transformation kit (Beyotime), the salI-linearized pPIC9K-L213A-BgaS (R233K / R234K / W288Y) plasmids were transformed into Pichia pastoris GS115 strain. See the kit instructions for transformation procedures.

[0160] b) Spread all the resuspended bacterial solution obtained in a) onto MD plates and incubate at 30°C with the plates inverted for 3-4 days.

[0161] (2) Screening of recombinant strains

[0162] a) Observe the transformation plate; several single-clone colonies should be distributed on it.

[0163] b) Pick single colonies from the MD plate in a) and sequentially inoculate them onto YPD plates containing 1 mg / mL, 2 mg / mL and 3 mg / mL G418 for screening. Select strains with good growth on the YPD plate containing 3 mg / mL G418 for the next step of verification.

[0164] (3) Identification of protein expression in recombinant strains

[0165] a) Pick single colonies of GS115-L213A-BgaS (R233K / R234K / W288Y) and inoculate them into 100 mL Erlenmeyer flasks (containing 20 mL YPD medium) and incubate at 30℃ and 220 rpm for 18 h.

[0166] b) Inoculate the seed culture from a) at 2% into a 250 mL Erlenmeyer flask (containing 25 mL of BMGY medium), and incubate at 30°C and 220 rpm for 24 h. Collect the cells and centrifuge at 4°C and 5,000 rpm for 10 min. Discard the supernatant, wash the cells twice with pre-cooled ddH2O, and resuspend them in a 250 mL Erlenmeyer flask (containing 25 mL of fresh BMGY medium). Ferment at 30°C and 220 rpm, and supplement the medium with 0.5% methanol every 12 h.

[0167] c) After fermentation for 72 h, collect the bacterial culture, centrifuge at 10,000 × g for 10 min, and collect the supernatant.

[0168] d) SDS-PAGE gel electrophoresis analysis of the supernatant sample in c) showed that the supernatant sample of GS115-L213A-BgaS (R233K / R234K / W288Y) exhibited a specific band at 60 kDa, consistent with the theoretical molecular weight. Figure 2 ); Determination of L213A-BgaS (R233K / R234K / W288Y) yield in fermentation broth supernatant (BCA protein concentration assay kit; Beyotime) Figure 3 ).

[0169] 1.3 Preservation of microbial strains

[0170] a) Pick the single colony with the thickest target protein band in 1.2 and put it into a shake flask containing YPD medium and incubate overnight at 30°C and 220 rpm.

[0171] b) Take 300 μL of bacterial culture and 300 μL of 50% glycerol, and mix them thoroughly.

[0172] c) Store the bacterial culture in a -80°C refrigerator.

[0173] Example 2: sRNA strategy targeting PAS_chr3_1087 to increase recombinant glycosidase production

[0174] 1.1 Construction and validation of sRNA tool plasmid targeting PAS_chr3_1087

[0175] (1) DNA sequence (SEQ ID NO:2) used to construct the sRNA regulatory strategy, consisting of a PAS_chr3_1087 targeting binding sequence ( The sequence is composed of a gene-binding sequence, a MicC backbone coding sequence, a yeast terminator sequence, a yeast promoter sequence, and an Hfq coding sequence, cascaded together. SEQ ID NO:2 is as follows:

[0176]

[0177] (2) Vector construction and sequence optimization

[0178] Using the commercial vector pPICZA (plasmid map as shown) Figure 4 As shown, including the location of the functional region of pPICZA, the restriction enzyme sites are designed based on the relevant sequence locations of pPICZA; carrying the sequence shown in SEQ ID NO:2.

[0179] This embodiment uses the commercial plasmid vector pPICZA, purchased from Shanghai Kelei Biotechnology Co., Ltd., according to... Figure 4 The relevant sequence positions were designed using EcoRI and ApaI restriction enzyme sites, tandemly targeting the PAS_chr3_1087 binding sequence, the MicC backbone coding sequence, the yeast terminator sequence, the yeast promoter sequence, and the Hfq coding sequence, all of which were artificially synthesized. The synthesized full-length DNA fragment has one restriction endonuclease site at both the 5' and 3' ends, corresponding to EcoRI and ApaI, respectively. The tandem fragment was inserted between these two restriction enzyme sites to obtain the sRNA regulatory strategy tool plasmid targeting PAS_chr3_1087. The recombinant DNA fragment was synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and the recombinant tool plasmid was constructed. Sequencing results showed 100% sequence identity with the theoretical sequence.

[0180] 1.2 Construction, screening and identification of recombinant strains

[0181] (1) Preparation and transformation of Pichia pastoris competent cells

[0182] a) Using the Pichia pastoris competent cell preparation and transformation kit (Beyotime), the SacI-linearized pPICZA-sRNA-PAS_chr3_1087 plasmid was transformed into the GS115-L213A-BgaS (R233K / R234K / W288Y) strain in Case 1. See the kit instructions for transformation procedures.

[0183] b) Spread all the resuspended bacterial solution obtained in a) onto YPD plates containing 100 ng / μL Zeocin and incubate at 30°C inverted plates for 3-4 days.

[0184] (2) Screening of recombinant strains

[0185] a) Observe the transformation plate; several single-clone colonies should be distributed on it.

[0186] b) Pick single colonies from the plates in a) and sequentially inoculate them onto YPD plates containing 0.5 mg / mL, 1 mg / mL, and 2 mg / mL Zeocin for screening. Select strains with good growth on YPD plates containing 2 mg / mL Zeocin for further validation.

[0187] (3) Identification of protein expression in recombinant strains

[0188] a) Pick single colonies of GS115-L213A-BgaS(R233K / R234K / W288Y)-sRNA-PAS_chr3_1087 and inoculate them into 100 mL Erlenmeyer flasks (containing 20 mL YPD medium) and incubate at 30℃ and 220 rpm for 18 h.

[0189] b) Inoculate the seed culture from a) at 2% into a 250 mL Erlenmeyer flask (containing 25 mL of BMGY medium), and incubate at 30°C and 220 rpm for 24 h. Collect the cells and centrifuge at 4°C and 5,000 rpm for 10 min. Discard the supernatant, wash the cells twice with pre-cooled ddH2O, and resuspend them in a 250 mL Erlenmeyer flask (containing 25 mL of fresh BMGY medium). Ferment at 30°C and 220 rpm, and supplement the medium with 0.5% methanol every 12 h.

[0190] c) After fermentation for 72 h, collect the bacterial culture, centrifuge at 10,000 × g for 10 min, and collect the supernatant.

[0191] d) SDS-PAGE gel electrophoresis was used to detect the supernatant sample in c), and the yield of L213A-BgaS (R233K / R234K / W288Y) in the fermentation broth supernatant was determined (BCA protein concentration assay kit; Beyotime). The results are as follows: Figure 3 As shown.

[0192] 1.3 Preservation of microbial strains

[0193] Same as Example 1.

[0194] Example 3: High-density fermentation for the preparation of bio-enzymes

[0195] 1.1 Seed culture

[0196] A small amount of GS115-L213A-BgaS(R233K / R234K / W288Y)-sRNA-PAS_chr3_1087 bacterial culture was streaked onto a YPD plate for isolation. The culture was incubated at 30°C for 72 h. A single colony was picked and inoculated into a 50 mL Erlenmeyer flask (containing 10 mL of BMGY medium). The colony was incubated at 30°C and 220 rpm for 18 h as the primary seed culture. This seed culture was then inoculated at a rate of 2% into a 500 mL Erlenmeyer flask (containing 50 mL of BMGY medium) and incubated until OD (Organic Difference). 600 5% of the secondary seed culture was inoculated into a 5 L fermenter (2 L of liquid).

[0197] 1.2 5-L tank high-density fermentation

[0198] After cleaning and sterilization, the fermenter was filled with BSM (Bio-Salt Medium) culture medium. After sterilization, it was connected to the fermentation control system. The initial pH was adjusted to 6 using 50% ammonia and 50% phosphoric acid. The temperature was controlled at 30°C, the aeration rate was 1 vvm, and the initial rotation speed was 600 rpm. As the cells grew and the cell density increased, the aeration rate was gradually increased to 2-3 vvm, and the rotation speed was increased to 1000 rpm to maintain a dissolved oxygen (DO) value above 15% throughout the fermentation process. After inoculation, the DO value initially dropped to its lowest point and then began to rise. At this point, the glycerol in the initial medium had been depleted. Glycerol was then added to the culture medium at a rate of 18 mL / h·L, while controlling the DO value, until the cell wet weight reached 200 g / L. Once the DO value returned to 100%, the culture was starved for 1 h, the pH was adjusted to 5, and methanol induction was initiated. During the initial acclimatization phase, the methanol flow rate was extremely low. After acclimatization, the methanol flow rate was pulsed and controlled according to the DO-star feeding strategy to maintain the DO value above 15%. Fermentation continued for 120 hours before being discharged from the tank. During fermentation, samples were taken periodically to determine the yield of L213A-BgaS (R233K / R234K / W288Y) in the fermentation broth supernatant (BCA protein concentration assay kit; Beyotime) and the cell wet weight (…). Figure 5 ).

[0199] 1.3 Enzyme Preparation

[0200] a) Centrifuge the fermentation broth in step 1.2 at 10,000 rpm for 10 min. Retain the supernatant and remove the precipitate.

[0201] b) The pre-frozen fermentation supernatant, which had been frozen overnight in a -80°C freezer, was freeze-dried in a freeze dryer to obtain the freeze-dried enzyme powder.

[0202] Example 4: Enzyme conversion reaction mediated by biological enzymes

[0203] 1.1 Weigh an appropriate amount of ginsenoside Rb1 raw material and mix it with the lyophilized enzyme powder prepared in Example 3 at a weight ratio of 1:1. Add the mixture to an acetate-sodium acetate buffer solution (pH 5.5). Place the mixture in a metal bath at 85°C and allow it to shake for 2 hours before stopping the reaction.

[0204] 1.2 Shake the reaction solution thoroughly, take 200 μL of the reaction solution, add 800 μL of anhydrous ethanol, and vortex for 5 min to mix thoroughly. Centrifuge at 8,000 rpm for 5 min.

[0205] 1.3 After centrifugation, the supernatant was filtered into a sample vial using a 0.22 μm filter membrane for HPLC analysis. The chromatographic column used was a Symmetry® C18 5 μm, 4.6 × 250 mm; wavelength: 203 nm; mobile phase A was water, and mobile phase B was acetonitrile, with a flow rate of 1 mL / min. Elution conditions were: 0 min, 70% A; 10 min, 70% A; 35 min, 45% A; 50 min, 45% A; 60 min, 70% A.

[0206] 1.4 Test results are as follows Figure 6 As shown, the retention time of ginsenoside CK was 39.9 min, and the retention time of Rb1 was 21.2 min. This indicates that the glucosinolate hydrolase bio-enzyme preparation produced using Pichia pastoris can specifically convert ginsenoside Rb1 into the rare ginsenoside CK, with almost no residual intermediate product Rd. Peak area normalization calculations showed that over 95% of the ginsenoside Rb1 substrate was converted into the rare ginsenoside CK.

[0207] The above embodiments use GRAS-grade Pichia pastoris GS115 as the host, alleviating food safety issues related to endotoxin residues in the E. coli system; through sRNA targeted regulation ( The gene significantly enhances the expression level of recombinant glycoside hydrolase, alleviating the problem of low expression levels. The prepared recombinant enzyme exhibits strong heat resistance and high catalytic specificity; a reaction at 85℃ for 2 hours can achieve a conversion rate of over 95% for ginsenoside Rb1, producing a single, impurity-free product, thus solving the problems of insufficient substrate conversion efficiency and poor product controllability. Simultaneously, the accompanying high-density fermentation and enzyme preparation process facilitates industrial scale-up, overcoming the bottleneck of existing technologies that hinder industrialization.

[0208] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0209] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A recombinant Pichia pastoris expressing recombinant glucosinolate hydrolase, characterized in that, The recombinant Pichia pastoris expresses recombinant glucosinolate hydrolase; and... Expression Target The MicC sRNA and Hfq protein of the gene.

2. The recombinant Pichia pastoris expressing recombinant glucosinolate hydrolase according to claim 1, characterized in that, The nucleic acid molecule encoding the recombinant glucosinolate hydrolase, and the nucleic acid molecule encoding the target At least one of the nucleic acid molecules of the MicC sRNA of the gene and the nucleic acid molecule encoding the Hfq protein is integrated into the genome of the recombinant Pichia pastoris; Optionally, the nucleic acid molecule encoding the recombinant glucosinolate hydrolase is integrated into the HIS4 site of yeast; Optionally, the target is encoded. The nucleic acid molecules of the MicC sRNA of the gene and the nucleic acid molecules encoding the Hfq protein are integrated into the AOX1 promoter region of yeast; Optionally, the genome of the recombinant Pichia pastoris integrates a first nucleic acid molecule, the first nucleic acid molecule containing a coding sequence encoding the recombinant glucosinolate hydrolase; Optionally, the genome of the recombinant Pichia pastoris integrates a second nucleic acid molecule, the second nucleic acid molecule containing the target The coding sequence of the MicC sRNA of the gene and the coding sequence of the Hfq protein; Optionally, the second nucleic acid molecule contains, in tandem, the following coding sequence: Gene-binding sequence, MicC backbone coding sequence, yeast terminator sequence, yeast promoter sequence, and Hfq coding sequence; Optionally, the first nucleic acid molecule is a coding sequence encoding the recombinant glucosinolate hydrolase, the amino acid sequence of which is as shown in SEQ ID NO.1, or has at least 90% identity with the sequence shown in SEQ ID NO.1, and the 233rd amino acid residue is K, the 234th amino acid residue is K, and the 288th amino acid residue is Y; Optionally, the The gene-binding sequence is as shown in SEQ ID NO.4, or has at least 90% identity with the sequence shown in SEQ ID NO.4; Optionally, the MicC skeleton coding sequence is as shown in SEQ ID NO.5, or has at least 90% identity with the sequence shown in SEQ ID NO.5; Optionally, the yeast terminator sequence includes the AOX1 terminator sequence; more preferably, the AOX1 terminator sequence is as shown in SEQ ID NO.6, or has at least 90% identity with the sequence shown in SEQ ID NO.

6. Optionally, the yeast promoter sequence includes the TEF1 promoter sequence; more preferably, the TEF1 promoter sequence is as shown in SEQ ID NO.7, or has at least 90% identity with the sequence shown in SEQ ID NO.

7. Optionally, the amino acid sequence encoded by the Hfq coding sequence is as shown in SEQ ID NO.8, or has at least 90% identity with the sequence shown in SEQ ID NO.8; Optionally, the Hfq encoded sequence is as shown in SEQ ID NO.3, or has at least 90% identity with the sequence shown in SEQ ID NO.3; Optionally, the amino acid sequence encoded by the second nucleic acid molecule is as shown in SEQ ID NO.2, or has at least 90% identity with the sequence shown in SEQ ID NO.2; Optionally, the starting strain of the recombinant Pichia pastoris is Pichia pastoris strain GS115.

3. A method for preparing recombinant Pichia pastoris expressing recombinant glucosinolate hydrolase as described in claim 1 or 2, characterized in that, This includes the nucleic acid molecule encoding the recombinant glucosinolate hydrolase, and the nucleic acid molecule encoding the target... The nucleic acid molecules of the MicC sRNA gene and the nucleic acid molecules encoding the Hfq protein were transformed into the starting strain to construct the recombinant Pichia pastoris. Optionally, the preparation method includes preparing a nucleic acid molecule encoding the recombinant glucosidase and a nucleic acid molecule encoding the target... At least one of the nucleic acid molecules of the MicC sRNA of the gene and the nucleic acid molecule encoding the Hfq protein is integrated into the genome of the originating strain; Optionally, the preparation method includes integrating a nucleic acid molecule encoding the recombinant glucosinolate hydrolase into the HIS4 site of yeast; Optionally, the preparation method includes encoding the target The nucleic acid molecules of the MicC sRNA of the gene and the nucleic acid molecules encoding the Hfq protein are integrated into the AOX1 promoter region of yeast; Optionally, the preparation method includes first transforming the nucleic acid molecule encoding the recombinant glucosinolate hydrolase into the starting strain, and screening for positive strains expressing the recombinant glucosinolate hydrolase; then transforming the nucleic acid molecule encoding the target... The nucleic acid molecules of the MicC sRNA gene and the nucleic acid molecules encoding the Hfq protein were transformed into the positive strain to construct the recombinant Pichia pastoris. Optionally, the preparation method includes transforming the first nucleic acid molecule and the second nucleic acid molecule described in claim 2 into the starting strain to construct the recombinant Pichia pastoris; Optionally, the starting strain includes Pichia pastoris strain GS115.

4. A recombinant glucosinolate hydrolase, characterized in that, The recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in claim 1 or 2, or the recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the preparation method described in claim 3.

5. A method for preparing a recombinant glucosinolate hydrolase, characterized in that, This includes culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in claim 1 or 2, or recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the preparation method described in claim 3; Then the recombinant glucosinolate hydrolase expressed by the recombinant Pichia pastoris was isolated.

6. A recombinant glucosinolate hydrolase preparation, characterized in that, The product comprises the recombinant glucosyl hydrolase of claim 4; or a culture of recombinant Pichia pastoris expressing the recombinant glucosyl hydrolase of claim 1 or 2; or a culture of recombinant Pichia pastoris expressing the recombinant glucosyl hydrolase prepared by the preparation method of claim 3. Optionally, the culture includes fermentation broth, fermentation supernatant, bacterial cells, or lysates; Optionally, the recombinant glucosidase preparation further includes a crude extract or solid preparation of the culture after treatment; Optionally, the process includes drying; Optionally, the recombinant glucosinolate hydrolase preparation is a solid preparation of the fermentation broth supernatant that has been freeze-dried.

7. A method for preparing a recombinant glucosinolate hydrolase preparation, characterized in that, The process includes culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in claim 1 or 2, or culturing recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the preparation method described in claim 3; and then collecting the culture. Optionally, the process also includes separating the culture supernatant from the culture and then lyophilizing it.

8. A product for constructing the recombinant Pichia pastoris according to claim 1 or 2, characterized in that, Includes at least one of (i) to (ii): (i) A nucleic acid composition comprising the first nucleic acid molecule as described in claim 2 and the second nucleic acid molecule as described in claim 2; (ii) comprising a first carrier and a second carrier, wherein the first carrier carries the first nucleic acid molecule as described in claim 2, and the second carrier carries the second nucleic acid molecule as described in claim 2; Optionally, the first vector is a pPIC9K vector that integrates the first nucleic acid molecule; Optionally, the second vector is a pPICZA vector that integrates the second nucleic acid molecule.

9. The recombinant Pichia pastoris expressing recombinant glucosyl hydrolase as described in claim 1 or 2, the recombinant Pichia pastoris expressing recombinant glucosyl hydrolase prepared by the preparation method of claim 3, the recombinant glucosyl hydrolase as described in claim 4, the recombinant glucosyl hydrolase preparation as described in claim 6, or the product as described in claim 8, used in at least one of (a) to (b): (a) Synthetic ginsenoside CK; and, (b) Preparation of products for the synthesis of ginsenoside CK.

10. A method for synthesizing ginsenoside CK, characterized in that, This includes mixing (Ⅰ) and (Ⅱ) below, and preparing ginsenoside CK after reaction; (I) The reaction substrate, wherein the reaction substrate comprises at least one of ginsenoside Rb1, ginsenoside Rb2 and ginsenoside Rd; (II) The recombinant glucosidase according to claim 4, or the recombinant glucosidase preparation according to claim 6; Optionally, the reaction conditions include at least one of the following: the pH of the reaction environment is 5 to 5.5, the reaction temperature is 80°C to 85°C, and the reaction time is 3 to 5 hours. Optionally, the scheme includes using ginsenoside Rb1 as a reaction substrate, and mixing the ginsenoside Rb1 with the freeze-dried solid preparation of the fermentation broth supernatant of claim 6 at a weight ratio of (1~2):(1~2) and then reacting.

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