Cucurbitadienol synthase mutant and application thereof

Abstract

The invention discloses a cucurbitadienol synthase mutant and application thereof, and relates to the technical field of protein engineering and genetic engineering. The inventor carries out random mutation on CDS, one or more CDS mutants are obtained through a large number of screening, and the mutants can improve the catalytic activity and / or substrate selectivity of cucurbitadienol synthase. Therefore, the cucurbitadienol synthase mutant provided by the invention provides a good foundation for the industrial production of the 24, 25-epoxy cucurbitadienol and the mogroside V. The invention further provides a preparation method of the cucurbitadienol synthase mutant.

Description

Technical Field

[0001] The invention relates to the technical field of protein engineering and genetic engineering, and in particular to a cucurbitadienol synthase mutant and application thereof. Background Art

[0002] Mogroside is mainly found in the fruit of Luo Han Guo. As a medicinal and edible plant, Luo Han Guo has been used in China for more than 300 years and is included in the Chinese Pharmacopoeia as a traditional Chinese medicine [1]. The main active ingredient of Luo Han Guo, Mogroside, is a tetracyclic triterpenoid compound formed by the addition of sugar chains of varying lengths to the -OH of the C3 and C24 positions of the parent nucleus mogroside after glycosyltransferase modification. Mogroside has good effects in anti-cancer, anti-inflammatory, antioxidant, cough and expectorant, and lowering blood sugar and blood lipids. The main active ingredient in Mogroside is Mogroside V, which accounts for about 20% of the total saponins. In terms of sweetness, siamenoside, Mogroside IV and Mogroside V have the highest sweetness, which are 563 times, 392 times and 425 times that of sucrose, respectively [2]. Because of its high sweetness and low calorie characteristics, it has great commercial potential in the development of new natural non-sugar sweeteners with health and therapeutic functions. Due to the complex structure of mogrosides and the difficulty of chemical synthesis, the main way to obtain them is to extract them from fruits. However, even the content of mogroside V, the most abundant mogroside, in monk fruit is only about 1% [3]. The cost of directly extracting mogrosides from fruits is high and the economic benefits are small. Compared with the traditional extraction from fruits, the use of microbial cell factories to produce natural compounds has the advantages of short cycle, green and pollution-free, and simple extraction process. At present, the biosynthetic pathway of mogrosides has been completely analyzed [4]. In general, its synthesis pathway can be divided into three steps: the first step is to generate squalene through the MVA pathway. Squalene undergoes two-step cyclization under the action of squalene epoxidase to generate 2,3-oxisqualene and 2,3;22,23-dioxidoqualene in sequence; then 2,3;22,23-dioxidoqualene is converted into 24,25-cucurbitadienol by 24,25-epoxy-cucurbitadienol synthase (CDS), and then 24,25-cucurbitadienol is converted into 24,25-cucurbitadienol by epoxide hydrolases (EPH) and cytochrome Under the catalysis of P450, the mother nucleus of mogroside, mogroside alcohol, is generated. Finally, mogroside alcohol undergoes multi-step reactions of different glycosyltransferases at positions C3 and C24 to ultimately generate different types of mogrosides.

[0003] Saccharomyces cerevisiae can provide the precursor skeleton of mogroside, squalene, through its endogenous MVA pathway. In addition, Squalene epoxidase ERG1[5] of S.cerevisiae itself can also catalyze squalene to produce 2,3-epoxysqualene and 2,3;22,23-diepoxysqualene. Therefore, S.cerevisiae is considered to be an ideal chassis for the synthesis of mogroside. However, when constructing a de novo synthesis pathway for mogroside in Saccharomyces cerevisiae, the inventors found that CDS preferentially catalyzes 2,3-epoxysqualene to produce the byproduct cucurbitadienol rather than catalyzing 2,3;22,23-diepoxysqualene to produce 24,25-epoxycucurbitadienol. In addition, the byproduct cucurbitadienol produced by CDS catalyzing 2,3-epoxysqualene can also compete with the downstream p450 Cytochrome P450, thereby affecting the synthesis of the parent nucleus mogroside. It can be seen that CDS is the first rate-limiting enzyme for the de novo synthesis of mogrosides in yeast. Therefore, in order to efficiently synthesize mogrosides from scratch, the substrate selectivity and activity of CDS must be solved first.

[0004] Protein engineering is the use of molecular biology and bioinformatics to conduct directed evolution or rational design of target proteins, thereby obtaining protein mutants with improved performance or new functions. Directed evolution is widely used in protein modification engineering because of its wide sequence coverage and high success rate.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a cucurbitadienol synthase mutant and its application to solve the substrate selectivity and / or activity problem of cucurbitadienol synthase. After random mutation of cucurbitadienol synthase (CDS) by directed evolution, one or more mutants of CDS are provided, and these mutants are improved in catalytic activity or substrate selectivity, or the catalytic activity and substrate selectivity are improved at the same time. The mutant provided by the present invention is expressed in a yeast chassis and can be further applied to the production of 24,25-epoxycucurbitadienol and mogroside V.

[0007] the term

[0008] As used herein, "CDS" or "wild-type cucurbitadienol synthase" refers to a naturally occurring, unmodified cucurbitadienol synthase, the nucleotide sequence of which can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and the amino acid sequence of which can be derived from the nucleotide sequence. The amino acid sequence of the wild-type cucurbitadienol synthase is shown in SEQ ID NO.1.

[0009] The identifiers of CDS mutants all use "amino acid replaced at the original amino acid position" to indicate the mutated amino acid in the cucurbitadienol synthase mutant, such as W258H, which means that the amino acid at position 258 is replaced by H from W of the parent CDS.

[0010] As used herein, the terms "mutant", "cucurbitacadienol synthase mutant", "mutant of the present invention", and "CDS mutant" are used interchangeably, all referring to non-naturally occurring cucurbitacadienol synthase mutant proteins, and the mutant protein is an artificially modified protein based on the protein shown in SEQ ID NO. 1. The mutant protein of the present invention has the enzyme activity of catalyzing 2,3;22,23-diepoxysqualene to form 24,25-epoxycucurbitacadienol.

[0011] The identity comparison method is as follows: input the amino acid sequence of a species into the Blast website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for ProteinBlast comparison, and find the identity between this species and other species that need to be compared from the comparison results.

[0012] The present invention is achieved in that:

[0013] In a first aspect, the present invention provides a cucurbitadienol synthase mutant having the activity of synthesizing 24,25-epoxycucurbitadienol, as shown in (1) or (2):

[0014] (1): It is obtained by mutating at least one amino acid of a wild-type cucurbitadienol synthase from a plant of the Cucurbitaceae family, wherein the amino acid position of the wild-type cucurbitadienol synthase to be mutated is determined by the following method: the wild-type cucurbitadienol synthase is aligned with a reference sequence, and the amino acid position of the wild-type cucurbitadienol synthase to be mutated corresponds to at least one of the following positions of the reference sequence: position 258, position 535 and position 372, wherein the amino acid sequence of the reference sequence is shown in SEQ ID NO.1;

[0015] The amino acid at position 258 of the cucurbitadienol synthase mutant is D, Y, H, S, N or E;

[0016] The amino acid at position 535 of the cucurbitadienol synthase mutant is Q or C;

[0017] The amino acid at position 372 of the cucurbitadienol synthase mutant is A;

[0018] (2): It has at least 85% identity with the cucurbitadienol synthase mutant shown in (1), and is identical to the cucurbitadienol synthase mutant shown in (1) at amino acid position 258.

[0019] The inventors used error-prone PCR technology to randomly mutate the CDS of wild-type Momordica grosvenori. From the random mutation library, three mutants, W258A, Y535A and N372A, were screened to increase the ratio of 24,25-epoxycucurbitadienol to cucurbitadienol in the yeast chassis. Subsequently, saturation mutations were performed on positions 258 and 535, respectively, and when the amino acid at position 258 was mutated to D, Y, H, S, N, E or R, the mutants had a lower catalytic activity of 2,3-epoxysqualene than the CDS of wild-type Momordica grosvenori. Therefore, these mutants can be used to produce 24,25-epoxycucurbitadienol, which can significantly reduce the accumulation of the byproduct cucurbitadienol.

[0020] When the amino acid at position 258 is mutated to D, Y or H, the catalytic activity for 2,3;22,23-diepoxysqualene is significantly improved compared to the wild type, so these mutants can be used to produce 24,25-epoxycucurbitadienol, which can significantly increase the target product 24,25-epoxycucurbitadienol. When the amino acid at position 258 is mutated to Y or H, it can not only improve the catalytic activity of 2,3;22,23-diepoxysqualene and obtain more target product 24,25-epoxycucurbitadienol, but also significantly reduce the accumulation of byproduct cucurbitadienol.

[0021] When the amino group at position 535 is mutated to Q or C, the catalytic activity for 2,3;22,23-diepoxysqualene is significantly improved compared to the wild type. Therefore, these mutants can be used to produce 24,25-epoxycucurbitadienol, which can significantly increase the target product 24,25-epoxycucurbitadienol.

[0022] When amino acid 372 is A, it has lower catalytic activity for 2,3-epoxysqualene than CDS of wild-type Momordica grosvenori, so these mutants can be used to produce 24,25-epoxycucurbitadienol and can significantly reduce the accumulation of byproduct cucurbitadienol.

[0023] The amino acid sequence of the reference sequence is from the cucurbitadienol synthase of the wild type Momordica grosvenori.

[0024] The sequence alignment method can use the Blast website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to perform Protein Blast alignment; the same results can be obtained using other sequence alignment methods or tools well known in the art.

[0025] It should be noted that the amino acid position to be mutated in the wild-type cucurbitadienol synthase may also be the 258th position in its own sequence, but it may not be the 258th position (for example, the cucurbitadienol synthase derived from zucchini corresponds to the 263rd position, the cucurbitadienol synthase derived from cucumber corresponds to the 284th position, and the cucurbitadienol synthase derived from bitter melon corresponds to the 261st position). The specific position of the amino acid to be mutated is determined according to the aforementioned sequence alignment. As long as it corresponds to the 258th, 535th or 372nd position of the reference sequence after alignment with the reference sequence, it is the amino acid position to be mutated in the present invention, that is, the mutation site.

[0026] The active site of the wild-type cucurbitadienol synthase of the Cucurbitaceae plant may be offset from the reference sequence of Momordica grosvenori in its own sequence. The specific position is determined based on the aforementioned sequence alignment. As long as it is aligned with the reference sequence, the active site corresponding to the reference sequence is the mutation site.

[0027] All wild-type cucurbitadienol synthases of Cucurbitaceae plants have homology and have basically the same functions and domains in Cucurbitaceae plants. Therefore, the cucurbitadienol synthase mutants obtained by making the above mutations at position 258, 535 or 372 of the wild-type cucurbitadienol synthase from any Cucurbitaceae plant have the activity of synthesizing 24,25-epoxycucurbitadienol. Therefore, the cucurbitadienol synthase mutants obtained by making the above mutations of the wild-type cucurbitadienol synthase from any Cucurbitaceae plant belong to the protection scope of the present invention.

[0028] In other embodiments, the 258th and 535th positions of the wild-type cucurbitadienol synthase are mutated at the same time, or the 258th and 372th positions are mutated at the same time, or the 535th and 372th positions are mutated at the same time, or the 258th, 535th or 372th positions are mutated, and those skilled in the art can also expect that the obtained cucurbitadienol synthase mutants all have the activity of synthesizing 24,25-epoxycucurbitadienol. For example, the 258th, 535th or 372th positions of the wild-type cucurbitadienol synthase are mutated at the same time, which can not only produce 24,25-epoxycucurbitadienol, but also significantly reduce the accumulation of the byproduct cucurbitadienol.

[0029] In addition, those skilled in the art know and can easily implement simple amino acid substitution, deletion or addition in the non-conserved region of the cucurbitacadienol synthase mutant shown in (1) and maintain the amino acid after the mutation at the nth position, and make the cucurbitacadienol synthase mutant obtained by further mutation have at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identity with the cucurbitacadienol synthase mutant shown in (1), and its functions include enzyme catalysis of 2,3; 22,23-diepoxysqualene activity and / or catalysis of 2,3-epoxysqualene activity equivalent to or slightly decreased or slightly increased or greatly increased as the cucurbitacadienol synthase mutant shown in (1). Therefore, such cucurbitacadienol synthase should also fall within the scope of protection of the present invention.

[0030] Mutants of the present invention are synthetic proteins or recombinant proteins, i.e., they can be the product of chemical synthesis, or they can be produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production scheme, the mutagen of the present invention can be glycosylated, or it can be non-glycosylated. The mutagen of the present invention may also include or not include an initial methionine residue.

[0031] The present invention also includes fragments, derivatives and analogs of the mutant protein. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity of the mutant protein.

[0032] The mutant protein fragment, derivative or analog of the present invention can be (i) a mutant protein with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a mutant protein with a substitution group in one or more amino acid residues, or (iii) a mutant protein formed by fusion of a mature mutant protein with another compound (such as a compound that prolongs the half-life of the mutant protein, such as polyethylene glycol), or (iv) a mutant protein formed by fusion of an additional amino acid sequence to the mutant protein sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify the mutant protein, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0033] In a preferred embodiment of the present invention, the Cucurbitaceae plants include but are not limited to Momordica grosvenori, Trichosanthes kirilowii, watermelon, wax gourd, bitter melon or cucumber.

[0034] In addition, the mutant protein of the present invention can also be modified. Modifications (usually without changing the primary structure) include: chemical derivatization forms of mutant proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those mutant proteins produced by glycosylation modification during the synthesis and processing of mutant proteins or in further processing steps. This modification can be accomplished by exposing the mutant protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are mutant proteins that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0035] In a second aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned cucurbitadienol synthase mutant.

[0036] The term "nucleic acid molecule encoding a cucurbitadienol synthase mutant" may be a polynucleotide encoding a mutant protein of the present invention, or may be a polynucleotide further comprising additional coding and / or non-coding sequences.

[0037] In the case where the present invention provides the above amino acid sequence, those skilled in the art can easily obtain the nucleic acid sequence encoding the above cucurbitadienol synthase mutant according to the degeneracy of the codons. For example, the nucleic acid sequence encoding the above cucurbitadienol synthase mutant can be obtained by making corresponding nucleotide mutations on the nucleic acid sequence encoding the wild-type cucurbitadienol synthase. This is easy to achieve for those skilled in the art.

[0038] The mutant proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0039] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0040] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0041] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0042] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0043] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0044] In an alternative embodiment, the nucleic acid molecule is selected from DNA, RNA, or a combination thereof.

[0045] In a third aspect, the present invention also provides a vector, which contains the above-mentioned nucleic acid molecule. The vector includes but is not limited to an expression vector, a shuttle vector and an integration vector.

[0046] Expression vector

[0047] The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, host cells produced by genetic engineering using the vectors of the present invention or the coding sequences of the mutant proteins of the present invention, and methods for producing the mutant proteins of the present invention by recombinant technology.

[0048] The polynucleotide sequences of the present invention can be used to express or produce recombinant mutant proteins by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0049] (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a mutant protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0050] (2) host cells cultured in a suitable culture medium;

[0051] (3) Isolate and purify proteins from culture medium or cells.

[0052] In the present invention, the polynucleotide sequence encoding the mutant protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0053] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the mutant protein of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; λ phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0054] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0055] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0056] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

[0057] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0058] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0059] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0060] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0061] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0062] In a fourth aspect, the present invention further provides a recombinant bacterium or a recombinant cell, which contains the above-mentioned nucleic acid molecule or vector. The above-mentioned nucleic acid molecule or the above-mentioned vector is integrated into the genome of the recombinant bacterium or the recombinant cell.

[0063] In an optional embodiment, the recombinant bacteria is Escherichia coli. The recombinant cells are selected from yeast cells.

[0064] In a fifth aspect, the present invention also provides the use of a cucurbitadienol synthase mutant, a cucurbitadienol synthase, a nucleic acid molecule, a vector or a recombinant bacterium or a recombinant cell in synthesizing 24,25-epoxycucurbitadienol or synthesizing mogroside V (Mogroside V, M5).

[0065] In a preferred embodiment of the present invention, the above application also includes at least one of the following application modes:

[0066] (1) Integrating nucleic acid molecules into strains or cells;

[0067] (2) integrating the vector into the strain or cell;

[0068] (3) Cultivating the recombinant bacteria or cells to synthesize 24,25-epoxycucurbitadienol.

[0069] On the basis of the cucurbitadienol synthase mutant provided by the present invention, those skilled in the art can easily think of using conventional technical means in the art to make strains or cells express the cucurbitadienol synthase mutant, thereby obtaining 24,25-epoxycucurbitadienol, or further producing mogroside V. Therefore, no matter what technology is used, as long as it utilizes the cucurbitadienol synthase mutant provided by the present invention to endow strains or cells with catalytic synthesis of 24,25-epoxycucurbitadienol, it belongs to the protection scope of the present invention.

[0070] In an alternative embodiment, gene editing technology is used to integrate nucleic acid molecules or vectors into strains or cells.

[0071] The gene editing technology is selected from zinc finger nuclease (ZFN, zinc-fingernucleases) technology, transcription activator-likeeffector nuclease (TALEN, transcription activator-likeeffector nucleases) technology or CRISPR / Cas9.

[0072] In a preferred embodiment of the present invention, the endogenous cucurbitadienol synthase in the target strain or cell is modified to encode a cucurbitadienol synthase mutant. The modification means are, for example, conventional transgenic technology, gene editing technology (such as zinc finger nuclease (ZFN, zinc-finger nucleases) technology, transcription activator-like effector nuclease (TALEN, transcription activator-like effector nucleases) technology or CRISPR / Cas9) and the like. The target plant is transformed to have a gene encoding the above cucurbitadienol synthase mutant, thereby improving the catalytic activity and / or substrate selectivity of the cucurbitadienol synthase.

[0073] In an alternative embodiment, the target strain or cell is Escherichia coli, yeast cell or Cucurbitaceae plant.

[0074] In a preferred embodiment of the present invention, the application includes: mutagenizing and screening strains or cells so that the strains or cells encode cucurbitadienol synthase mutants. The mutagen includes but is not limited to physical and chemical mutagenesis.

[0075] In a preferred embodiment of the present invention, the above-mentioned strain or cell is Escherichia coli, yeast cell or Cucurbitaceae plant.

[0076] In a sixth aspect, the present invention further provides a method for synthesizing 24,25-epoxycucurbitadienol, comprising the following steps:

[0077] The cucurbitadienol synthase mutant is contacted with a reaction substrate; or the recombinant bacteria or recombinant cells are cultured; and then the target product is extracted and separated from the recombinant bacteria or recombinant cells.

[0078] The reaction substrate is 2,3;22,23-diepoxysqualene.

[0079] The present invention has the following beneficial effects:

[0080] The present invention has discovered the catalytic active site of cucurbitadienol synthase through a large number of screenings and modifications.

[0081] Mutation of the active site can improve the catalytic activity and / or substrate selectivity of cucurbitadienol synthase. When the amino acid at position 258 is mutated to D, Y, H, S, N, E or R, the mutants have a lower catalytic activity of 2,3-epoxysqualene than the CDS of wild-type Momordica grosvenori, so these mutants can be used to produce 24,25-epoxycucurbitadienol, which can significantly reduce the accumulation of the byproduct cucurbitadienol.

[0082] When the amino acid at position 258 is mutated to D, Y or H, the catalytic activity for 2,3;22,23-diepoxysqualene is significantly improved compared to the wild type, so these mutants can be used to produce 24,25-epoxycucurbitadienol, which can significantly increase the target product 24,25-epoxycucurbitadienol. When the amino acid at position 258 is mutated to Y or H, it can not only improve the catalytic activity of 2,3;22,23-diepoxysqualene and obtain more target product 24,25-epoxycucurbitadienol, but also significantly reduce the accumulation of byproduct cucurbitadienol.

[0083] When the amino acid at position 535 is mutated to Q or C, the catalytic activity for 2,3;22,23-diepoxysqualene is significantly improved compared to the wild type. Therefore, these mutants can be used to produce 24,25-epoxycucurbitadienol, which can significantly increase the target product 24,25-epoxycucurbitadienol.

[0084] When the amino acid at position 372 is mutated to A, it has lower catalytic activity for 2,3-epoxysqualene than the CDS of wild-type Momordica grosvenori, so these mutants can be used to produce 24,25-epoxycucurbitadienol, which can significantly reduce the accumulation of the by-product cucurbitadienol.

[0085] The cucurbitadienol synthase mutant provided by the invention provides a good basis for the industrial production of 24,25-epoxycucurbitadienol and mogroside V. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0087] Figure 1 This is the result of HPLC analysis of the products of 2,3-epoxysqualene and 2,3;22,23-diepoxysqualene catalyzed by CDS in vitro;

[0088] Figure 2 Figure 1. Results of in vitro catalytic liquid phase assay of CDS Y535H mutant;

[0089] Figure 3 Production of 24,25-epoxycucurbitadienol and cucurbitadienol by CDS mutants in yeast. DETAILED DESCRIPTION

[0090] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0091] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0092] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0093] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0094] The culture medium and culture medium formula involved in the following examples are as follows:

[0095] LB liquid medium (1 L): NaCl 5 g, Yeast extract 10 g, Tryptone 10 g, 121°C, high temperature sterilization for 20 min.

[0096] LB solid medium (1L): NaCl 5g, Yeast Extract 10g, Tryptone 10g, agar powder 20g. 121℃, high temperature sterilization for 20min.

[0097] LB solid screening medium (1L): NaCl 5g, Yeast Extract 10g, Tryptone 10g, agar powder 20g. Sterilize at 121℃ for 20min. Add kanamycin at a final concentration of 100μM before it completely solidifies.

[0098] YPD liquid medium (1L): Glucose 22g, Bacter-peptone 20g, Yeast extract 10g. Sterilize at 115℃ for 20min.

[0099] YPD liquid induction medium: Glucose 22g, Bacter-peptone 20g, Yeast extract 10g. Sterilize at 115℃ for 20min. After sterilization and cooling, add CuSO4 at a final concentration of 500μM.

[0100] YPD solid screening medium (1L): Glucose 22g, Bacter-peptone 20g, Yeast extract 10g. Sterilize at 115℃ for 20min. Add 0.6% (V / V) Hygromycin B before the medium solidifies.

[0101] The CDS in vitro catalytic reaction system involved in the following examples is as follows:

[0102] Preparation of substrate solution: 2,3-epoxysqualene and 2,3;22,23-diepoxysqualene reaction substrate solutions were prepared with methanol, with the concentration of both being 25 μg / mL.

[0103] Reaction fluid: Take 500 μL of the squeezed CDS crude enzyme solution, add 2 μL of 2,3-epoxysqualene and 2,3;22,23-diepoxysqualene substrate solution respectively, and react for 16 h at room temperature with the Ferris wheel rotating.

[0104] Product extraction: The crude enzyme solution after the reaction was treated with an equal volume of ethyl acetate, rotated on a Ferris wheel for 3 h, centrifuged, and the ethyl acetate in the upper organic phase was drawn, concentrated and evaporated to dryness, resuspended with 70 μL of methanol, and detected by HPLC.

[0105] Example 1

[0106] In vitro expression of CDS and establishment of its catalytic system

[0107] The wild-type CDS gene was amplified from the fruit cDNA library of Momordica grosvenori using primers F: 5'-ATGTGGAGGTTAAAGGTCGGA-3' and R: 5'-TTATTCAGTCAAAACCCGATGG-3' and constructed into the BamHI site of the expression plasmid pET28a, and then transformed into the expression host Roseta competent state, coated with LB solid screening medium plate, selected positive clones and sequenced for verification. After sequencing is correct, select clones to LB liquid test tubes and culture at 37℃ overnight, then transfer to LB liquid triangular flasks, culture at 37℃ when OD grows to about 0.6, add IPTG and induce at 16℃ overnight. Collect the induced cells, squeeze, take the crude enzyme solution and add 2,3-epoxysqualene and 2,3;22,23-diepoxysqualene substrate solution at the same time, react at room temperature for 16h, extract the product with ethyl acetate and detect it by HPLC.

[0108] Results Figure 1 ,from Figure 1 It can be seen that compared with the blank control (empty plasmid without CDS gene), cucurbitacadienol (catalyzed product of 2,3-squalene) and 24,25-epoxycucurbitacadienol (catalyzed product of 2,3; 22,23-diepoxysqualene) were detected in the crude enzyme solution of the expression strain containing CDS gene at the same time. The above data show that the inventors have successfully established a CDS in vitro catalytic system.

[0109] Example 2

[0110] CDS random mutation library construction

[0111] Using the pET28a plasmid containing the wild-type CDS gene in Example 1 as a template, CDS was randomly mutated using error-prone PCR technology. The specific reaction system of error-prone PCR is as follows: H20 33.7μL, 10X MutazymeⅡbuffer 5μL; 40mMdNTP 1μL; primer mix 2μL; MutazymeⅡDNA polymerase (2.5U / μL) 1μL, Template 7.3μL (total concentration is 500ng). Annealing temperature 55℃, annealing time 30s, extension temperature 72℃, extension time 78s, 25 cycles. Subsequently, the CDS mutant gene was amplified using a high-fidelity DNA polymerase, and the Saccharomyces cerevisiae competent cells were transformed by the lithium acetate method to construct a random mutation library of CDS. The mutation library was induced and screened by the 96-well plate method. From the mutant library, we screened and obtained three mutants, namely W228A, Y535A and N372A, whose ratio of target product (24,25-epoxycucurbitadienol) to byproduct (cucurbitadienol) in yeast was significantly higher than that of wild type (see Table 1 for details). As can be seen from Table 1, the ratio of target product to byproduct of W258A, Y535A and N372A mutants was 1.8:1, 1.6:1 and 1.5:1, respectively, which was higher than 1.3:1 of wild type. Therefore, the inventors selected W258 and Y535 for further saturation mutation.

[0112] Table 1 The ratio of target products and byproducts of CDS mutants in yeast

[0113]

[0114] Example 3

[0115] This example is directed to a saturation mutation at position 535 of CDS.

[0116] The inventors carried out saturation mutation on position 535 of CDS, and substituted Y535 with Y535G, Y535V, Y535L, Y535I, Y535M, Y535F, Y535W, Y535S, Y535P, Y535T, Y535C, Y535N, Y535Q, Y535K, Y535H, Y535R, Y535D and Y535E, respectively. According to the in vitro catalytic system established in Specific Example 1, the in vitro catalytic efficiency of the above mutants to 2,3-epoxysqualene and 2,3;22,23-epoxysqualene was determined, and the specific results are shown in Table 2.

[0117] Table 2 Relative catalytic activity of CDS 535 saturation mutants in vitro

[0118]

[0119] Table 2 is a statistical table of the activities of each mutant relative to the wild type, where the activity of the wild type on 2,3-epoxysqualene and 2,3;22,23-diepoxysqualene is set as 100%. It can be seen that the conversion rates of Y535Q and Y535C to the target product are significantly higher than those of the wild type, the activities of Y535T and Y535F mutants are almost the same as those of the wild type, the conversion rate of the target product of the Y535H mutant is reduced to 21.4% of that of the wild type, and the conversion rate of the byproducts is also reduced to about 15%. No activity was detected for all other mutants, whether the target product or the byproduct.

[0120] In addition, the inventors found that the Y535H mutant not only produced the target product 24,25-epoxycucurbitadienol, but also produced an obvious new peak ( Figure 3 ), and then the inventors conducted mass spectrometry on the catalytic sample and found that the new peak molecular weight was consistent with the 24,25-epoxycucurbitadienol standard, but the retention time was inconsistent, indicating that the new product was not 24,25-epoxycucurbitadienol. It was initially speculated that it might be an isomer of 24,25-epoxycucurbitadienol. The inventors will further analyze its specific structure after purification. The discovery of the new product of the Y535H mutant provided new evidence for the inventors to analyze the catalytic mechanism of CDS later.

[0121] Example 4

[0122] In this example, saturation mutation was performed at position 258 of CDS.

[0123] The inventors carried out saturation mutation on position 258 of CDS, and substituted W258 with W258G, W258V, W258L, W258I, W258M, W258F, W258Y, W258S, W258P, W258T, W258C, W258N, W258Q, W258K, W258H, W258R, W258D and W258E, respectively. According to the in vitro catalytic system established in Specific Example 1, the in vitro catalytic efficiency of the above mutants on 2,3-epoxysqualene and 2,3;22,23-epoxysqualene was determined, and the specific results are shown in Table 3.

[0124] Table 3 Relative catalytic activity of CDS 258 saturation mutant in vitro

[0125]

[0126] Table 3 is a statistical table of the activities of each mutant relative to the wild type, wherein the activities of the wild type on 2,3-epoxysqualene and 2,3;22,23-diepoxysqualene are set as 100%. It can be seen from Table 3 that compared with the wild type, the catalytic activities of the mutants W258Y, W258F, W258H, W258S, W258N, W258E and W258R on 2,3-epoxysqualene are significantly reduced, so these mutants can be used to produce 24,25-epoxycucurbitadienol, which can significantly reduce the accumulation of the byproduct cucurbitadienol.

[0127] Compared with the wild type, the catalytic activity of the W258D, W258Y and W258H mutants towards 2,3;22,23-diepoxysqualene was significantly improved, so these mutants can be used to produce 24,25-epoxycucurbitadienol and can significantly increase the yield of the target product 24,25-epoxycucurbitadienol.

[0128] In particular, the W258Y and W258H mutants can not only improve the catalytic activity of 2,3;22,23-diepoxysqualene and obtain more target product 24,25-epoxycucurbitadienol, but also significantly reduce the accumulation of by-product cucurbitadienol.

[0129] Example 5

[0130] This example is a practical application example of CDS mutants for efficiently synthesizing 24,25-epoxycucurbitadienol in a yeast cell factory.

[0131] The inventors selected CDS preferred mutant N372A in Specific Example 2; CDS preferred mutants Y535Q and Y535C in Specific Example 3; CDS preferred mutants W258H, W258D and W258Y in Specific Example 4, and tested their respective activities in the Saccharomyces cerevisiae chassis. It should be noted that in practical applications, the above six preferred mutants are included but not limited to the above six mutants.

[0132] The inventors integrated the Y535Q, Y535C, N372A, W258H, W258D and W258Y genes of CDS into the W15 site of the Saccharomyces cerevisiae genome by the Cas9 method, and the promoter used was the gal 1 / 10 promoter. After transforming the competent cells of Saccharomyces cerevisiae by the lithium acetate method, the plates were coated with YPD solid screening medium, and after incubation at 30°C for 2 days, the monoclonal clones with correct sequencing were selected and inoculated into YPD liquid medium test tubes. After incubation at 30°C overnight, the yeast solution in the test tube was aspirated into a YPD liquid induction medium triangular flask. After induction at 30°C for 3 days, 500ul of the fermentation liquid was aspirated and extracted with the same volume of ethyl acetate, and the supernatant was aspirated and the product was detected by HPLC (see Figure 3 ).

[0133] Figure 3 The results showed that in yeast cells, the production of 24,25-epoxycucurbitadienol in Y535Q, Y535C, W258H, W258D and W258Y mutants increased by 18%, 22%, 25%, 150% and 200% respectively compared with the wild type; the production of byproduct cucurbitadienol in W258H and W258Y mutants decreased by 66% and 33% respectively compared with the wild type. Although the production of 24,25-epoxycucurbitadienol in N372A mutant decreased by 37% compared with the wild type, the production of byproduct cucurbitadienol decreased by 60%, which is also suitable for the de novo synthesis of mogroside V.

[0134] In summary, the present invention adopts protein engineering to randomly mutate CDS, and obtains one or more CDS mutants through a large number of screenings, and the mutants can improve the catalytic activity and / or substrate selectivity of cucurbitadienol synthase. The cucurbitadienol synthase mutants provided by the present invention provide a good basis for the industrial production of 24,25-epoxycucurbitadienol and mogroside V.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0136] References

[0137] [1]LI C, LIN LM, SUI F, et al. Chemistry and pharmacology of Siraitiagrosvenorii: a review[J]. Chin J Nat Med, 2014, 12(2):89-102.

[0138] [2]LUO Z, SHI H, ZHANG K, et al. Liquid chromatography with tandem massspectrometry method for the simultaneous determination of multiple sweetmogrosides in the fruits of Siraitia grosvenorii and its marketed sweeteners[J]. J Sep Sci, 2016, 39(21): 4124-35.

[0139] [3]MAKAPUGAY HC,NANAYAKKARA NPD,SOEJARTO DD,et al.6.High-Performance Liquid-ChromatographicAnalysis of the Major Sweet Principle of Lo Han Kuo Fruits[J].J Agr FoodChem,1985,33(3):348-50.

[0140] Proc Natl Acad Sci USA,2016,113(47):E7619-E28 [4]ITKIN M,DAVIDOVICH-RIKANATI R,COHEN S,et al.

[0141] [5]LANDL KM,KLOSCH B,TURNOWSKY F.ERG1,encoding squalene epoxidase,islocated on the right arm of chromosome VII of Saccharomyces cerevisiae[J].Yeast,1996,12(6):609-1

Claims

1. A cucurbitadienol synthase mutant having the activity of synthesizing 24,25-epoxycucurbitadienol, characterized in that: It is as follows (1) or (2): (1): It is obtained by mutating at least one amino acid of a wild-type cucurbitadienol synthase from a Cucurbitaceae plant, wherein the amino acid position to be mutated of the wild-type cucurbitadienol synthase is determined by the following method: the wild-type cucurbitadienol synthase is aligned with a reference sequence, and the amino acid position to be mutated of the wild-type cucurbitadienol synthase corresponds to at least one of the following positions of the reference sequence: position 258, position 535 and position 372, wherein the amino acid sequence of the reference sequence is shown in SEQ ID NO.1; The amino acid at position 258 of the cucurbitadienol synthase mutant is D, Y, H, S, N, E or R; The amino acid at position 535 of the cucurbitadienol synthase mutant is Q and C; The amino acid at position 372 of the cucurbitadienol synthase mutant is A; (2): It has at least 85% identity with the cucurbitadienol synthase mutant shown in (1), and is identical to the cucurbitadienol synthase mutant shown in (1) at amino acid position 258.

2. The cucurbitadienol synthase mutant according to claim 1, characterized in that The cucurbitaceae plant is selected from momordica grosvenori, trichosanthes kirilowii, watermelon, wax gourd, bitter gourd or cucumber.

3. A nucleic acid molecule, characterized in that It encodes the cucurbitadienol synthase mutant according to any one of claims 1-2.

4. A carrier, characterized in that It contains the nucleic acid molecule according to claim 3.

5. A recombinant bacterium or recombinant cell, characterized in that: It contains the nucleic acid molecule according to claim 3 or the vector according to claim 4; Preferably, the recombinant bacteria is Escherichia coli; Preferably, the recombinant cell is selected from yeast cells.

6. Use of the cucurbitadienol synthase mutant, cucurbitadienol synthase according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, or the recombinant bacterium or recombinant cell according to claim 5 in synthesizing 24,25-epoxycucurbitadienol or synthesizing mogroside V (Mogroside V, M5).

7. The use according to claim 6, characterized in that: The application method includes at least one of the following: (1) integrating the nucleic acid molecule into a strain or cell; (2) integrating the vector into a strain or cell; (3) culturing the recombinant bacteria or recombinant cells to synthesize 24,25-epoxycucurbitadienol; Preferably, the nucleic acid molecule or the vector is integrated into the strain or cell using gene editing technology.

8. The use according to claim 6, characterized in that: Modifying the endogenous cucurbitadienol synthase in the target strain or cell so that it encodes the cucurbitadienol synthase mutant; Preferably, the target strain or cell is Escherichia coli, yeast cell or Cucurbitaceae plant.

9. The use according to claim 7 or 8, characterized in that: The application comprises: mutagenizing and screening the strain or cell so that the strain or cell encodes the cucurbitadienol synthase mutant; Preferably, the strain or cell is Escherichia coli, yeast cell or Cucurbitaceae plant.

10. A method for synthesizing 24,25-epoxycucurbitadienol, characterized in that: It includes the following steps: contacting the cucurbitadienol synthase mutant according to any one of claims 1 to 2 with a reaction substrate; or culturing the recombinant bacterium or recombinant cell according to claim 5; The reaction substrate is 2,3;22,23-diepoxysqualene.

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