Squalene epoxidase mutant and application thereof

Abstract

The invention relates to a squalene epoxidase mutant and application thereof. The amino acid sequence of the squalene epoxidase mutant is an amino acid sequence obtained by deleting amino acids from the first site to the 55th site at the N end of an amino acid sequence of wild squalene epoxidase SgSQE1 from momordica grosvenori, and then connecting a dissolution-promoting tag sequence to the N end and / or the C segment. The squalene epoxidase mutant provided by the invention can be used for remarkably improving the yield of 2, 3, 22, 23-diepoxy squalene.

Description

[0001] This application is a divisional application of the Chinese Patent Application No. 202411865500.1, with the title of "A squalene epoxidase mutant and its application", filed on December 18, 2024. TECHNICAL FIELD

[0002] The present application relates to the technical field of genetic engineering and enzyme engineering, in particular to a squalene epoxidase mutant and its application. BACKGROUND

[0003] Mogrosides exist in mature Siraitia grosvenorii, with a sweetness about 300 times that of sucrose, belonging to a low-calorie sweetener. Mogrosides have no odor or bitter taste, and the sweetness characteristics are basically consistent with sucrose. They are hardly absorbed in the intestine and do not cause blood sugar fluctuations, so they can be consumed by diabetics and sugar control groups. In addition, mogrosides also have the effects of soothing the lungs and relieving cough, anti-inflammatory and antibacterial, and enhancing immunity.

[0004] In the biosynthesis pathway of mogrosides, squalene epoxidase catalyzes the epoxidation of carbon-carbon double bonds between C2=C3 and C22=C23 of squalene to generate 2,3,22,23-epoxysqualene. This is the first step in the synthesis of triterpenoids from squalene, and also a rate-limiting step. Subsequently, under the action of cucurbitadienol synthase, epoxide hydrolase, cytochrome P450 reductase and glucosyltransferase, 2,3,22,23-epoxysqualene undergoes a multi-step reaction to form mogrosides.

[0005] According to existing research, the commonly used squalene epoxidase ERG1 of Saccharomyces cerevisiae mainly catalyzes the epoxidation of the double bond between C2=C3, and the product is 2,3-epoxysqualene. However, the efficiency of catalyzing the carbon-carbon double bond between C22=C23 is extremely low, which is not conducive to the biosynthesis of mogrosides, limits the synthesis efficiency, and hinders the industrialized production and application. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application aims to provide a squalene epoxidase mutant capable of greatly increasing the yield of 2,3,22,23-epoxysqualene.

[0007] In a first aspect, the present application provides a squalene epoxidase mutant, wherein the amino acid sequence of the squalene epoxidase mutant is: the amino acid sequence obtained after deleting the amino acids at positions 1-55 in the N-terminal of the amino acid sequence of wild-type squalene epoxidase SgSQE1 from Siraitia grosvenorii, and then connecting a solubility tag sequence to the N-terminal and / or C-terminal.

[0008] Preferably, the solubilization tag sequence includes one or more of the following: GB1 sequence, SUMO sequence, MBP sequence, 3×FLAG sequence, and 3×Myc sequence.

[0009] Preferably, the N-terminal lysing tag is selected from one or more of the GB1 sequence, SUMO sequence, and MBP sequence.

[0010] Preferably, the C-terminal ligation tag is selected from 3×FLAG sequence and / or 3×Myc sequence.

[0011] Preferably, the amino acid sequence of the squalene epoxidase mutant has: (1) The amino acid sequence shown in SEQ ID NO.14, or (2) A sequence having at least 90% (e.g., at least 95%, at least 98%, at least 99%) sequence identity with the amino acid sequence described in SEQ ID NO.14 and retaining its biological activity.

[0012] Preferably, the amino acid sequence of the squalene cyclooxygenase mutant has one or more mutations at positions 104, 106, 107, 365, and 226 of the amino acid sequence shown in SEQ ID NO.14.

[0013] Preferably, the amino acid sequence of the squalene cyclooxygenase mutant has at least one mutation selected from the group consisting of the amino acid sequence shown in SEQ ID NO. 14: (1) Arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine; (2) Valine at position 106 is mutated to leucine; (3) Glycine at position 107 is mutated to tryptophan; (4) Glycine at position 365 is mutated to glutamine; (5) The glycine at position 226 is mutated to alanine or tryptophan; (6) Arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine; glycine at position 226 is mutated to alanine or tryptophan.

[0014] In a second aspect, the present invention provides an isolated nucleic acid molecule that encodes a squalene epoxidase mutant as described in any of the preceding claims.

[0015] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecules described above.

[0016] Fourthly, the present invention provides a host cell comprising the expression vector described above, or the genome of the host cell having the nucleic acid molecules described above integrated.

[0017] Preferably, the host cell also expresses NADPH cytochrome P450 reductase, or contains the encoding nucleotide sequence of the enzyme or its expression vector.

[0018] Preferably, the NADPH cytochrome P450 reductase is derived from rats.

[0019] Preferably, the nucleotide sequence encoding the NADPH cytochrome P450 reductase is shown in SEQ ID NO.12.

[0020] Fifthly, the present invention provides the use of any of the above-described squalene epoxidase mutants, or the above-described nucleic acid molecules, or the above-described expression vectors, or the above-described host cells in the production of 2,3,22,23-diepoxysqualene.

[0021] In a sixth aspect, the present invention provides a method for producing 2,3,22,23-diepoxysqualene, the method comprising the following steps: (1) Culture the host cells described above to produce 2,3,22,23-diepoxysqualene; (2) Optionally isolate 2,3,22,23-diepoxysqualene from the culture medium obtained in (1).

[0022] The effects of the invention This invention modifies the squalene epoxidase SgSQE1 derived from wild-type monk fruit using genetic engineering techniques, significantly improving the solubility, catalytic activity, and biosynthetic efficiency of the modified monk fruit squalene epoxidase mutant, thereby meeting the requirements for industrial application. Attached Figure Description

[0023] Figure 1 The bar chart shows the yield of 2,3,22,23-diepoxysqualene catalyzed by squalene epoxidase as shown in SEQ ID NO.1 ~ SEQ ID NO.9 in Example 3; Figure 2 The plasmid map constructed for this invention; Figure 3 The bar chart shows the yield of 2,3,22,23-diepoxysqualene catalyzed by squalene epoxidase as shown in SEQ ID NO.10~SEQ ID NO.11 in Example 4. Detailed Implementation

[0024] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0025] To increase the yield of 2,3,22,23-diepoxysqualene, the current solution is to increase the copy number of squalene epoxidase. However, the yield increase is not significant and it increases the metabolic stress on the chakra cells. Our research found that the squalene epoxidase SgSQE1 derived from *Siraitia grosvenorii* can catalyze the carbon-carbon double bond between C22 and C23, but this protein has poor solubility. Direct expression in *E. coli* leads to inclusion bodies and low enzyme activity, resulting in a significantly insufficient yield of 2,3,22,23-diepoxysqualene, making industrial-scale production difficult. Through extensive and in-depth research, the inventors unexpectedly discovered multiple mutants of squalene epoxidase SgSQE1. These mutants include the deletion of amino acids 1-55 from the N-terminus of the amino acid sequence of wild-type monk fruit-derived squalene epoxidase SgSQE1, followed by the attachment of a solubilizing tag sequence to the N-terminus and / or C-segment to obtain the resulting amino acid sequence. In particular, based on this, further point mutations were made to the amino acids in the catalytic pocket of the sequence shown in SEQ ID NO. 14, for example, mutations at one or more sites such as amino acids 104, 106, 107, 365, and 226. These mutants promoted the binding of the substrates squalene and 2,3-epoxysqualene to the enzyme, significantly improved the efficiency of catalytic synthesis of 2,3,22,23-diepoxysqualene, enhanced the water solubility of the protein, and greatly increased the yield of 2,3,22,23-diepoxysqualene, providing substrates and precursors for the subsequent production of monk fruit glycosides or other terpenoids.

[0026] 2,3,22,23-diepoxysqualene (CAS No.: 31063-19-1), also known as 2,3,22,23-epoxysqualene, has the following structural formula:

[0027] In a specific embodiment, the nucleotide sequence of wild-type squalene epoxidase SgSQE1 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.13. The amino acid sequence of the squalene epoxidase mutant of the present invention is obtained by deleting amino acids 1-55 from the N-terminus of the amino acid sequence of wild-type squalene epoxidase SgSQE1 derived from *Siraitia grosvenorii*, and then linking a solubilization tag sequence to the N-terminus and / or C-segment.

[0028] In some embodiments, the solubilization tag sequence includes one or more of the following: GB1 sequence, SUMO sequence, MBP sequence, 3×FLAG sequence, and 3×Myc sequence.

[0029] In some embodiments, the N-terminal lysosomal tag is selected from one or more of the GB1 sequence, SUMO sequence, and MBP sequence.

[0030] In some embodiments, the C-terminal ligation tag is selected from 3×FLAG sequences and / or 3×Myc sequences.

[0031] In some embodiments, the amino acid sequence of the squalene epoxidase mutant has: (1) The amino acid sequence shown in SEQ ID NO.14, or (2) A sequence having at least 90% (e.g., at least 95%, at least 98%, at least 99%) sequence identity with the amino acid sequence described in SEQ ID NO.14 and retaining its biological activity.

[0032] In specific implementations, the homology or sequence similarity can be 90% or more, preferably 95% or more, and more preferably 96%, 97%, 98%, or 99% homology.

[0033] In a specific embodiment, the nucleotide sequence encoding the squalene epoxidase mutant is shown in SEQ ID NO.2.

[0034] In some embodiments, the amino acid sequence of the squalene cyclooxygenase mutant has one or more mutations at positions 104, 106, 107, 365, and 226 of the amino acid sequence shown in SEQ ID NO. 14.

[0035] In some embodiments, the amino acid sequence of the squalene epoxidase mutant has at least one mutation selected from the group consisting of the amino acid sequence shown in SEQ ID NO. 14: (1) Arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine; (2) Valine at position 106 is mutated to leucine; (3) Glycine at position 107 is mutated to tryptophan; (4) Glycine at position 365 is mutated to glutamine; (5) The glycine at position 226 is mutated to alanine or tryptophan; (6) Arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine; glycine at position 226 is mutated to alanine or tryptophan.

[0036] In some preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that arginine at position 104 is mutated to aspartic acid in the amino acid sequence shown in SEQ ID NO. 14, and the nucleotide sequence encoding it is shown in SEQ ID NO. 3.

[0037] In some preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that arginine at position 104 is mutated to glycine in the amino acid sequence shown in SEQ ID NO. 14, and the nucleotide sequence encoding it is shown in SEQ ID NO. 4.

[0038] In some preferred embodiments, the amino acid sequence of the squalene cyclooxygenase mutant is that arginine at position 104 is mutated to asparagine in the amino acid sequence shown in SEQ ID NO. 14, and the nucleotide sequence encoding it is shown in SEQ ID NO. 5.

[0039] In some preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that arginine at position 104 is mutated to glutamine in the amino acid sequence shown in SEQ ID NO. 14, and the nucleotide sequence encoding it is shown in SEQ ID NO. 6.

[0040] In some preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is a mutation of valine to leucine at position 106 in the amino acid sequence shown in SEQ ID NO. 14, and the nucleotide sequence encoding it is shown in SEQ ID NO. 7.

[0041] In some preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is a mutation of glycine at position 107 to tryptophan in the amino acid sequence shown in SEQ ID NO. 14, and the nucleotide sequence encoding it is shown in SEQ ID NO. 8.

[0042] In some preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that glycine at position 365 is mutated to glutamine in the amino acid sequence shown in SEQ ID NO. 14, and the nucleotide sequence encoding it is shown in SEQ ID NO. 9.

[0043] In some preferred embodiments, the amino acid sequence of the squalene cyclooxygenase mutant is such that arginine at position 104 is mutated to aspartic acid and glycine at position 226 is mutated to alanine, as shown in SEQ ID NO. 10.

[0044] In some preferred embodiments, the amino acid sequence of the squalene cyclooxygenase mutant is such that arginine at position 104 is mutated to aspartic acid and glycine at position 226 is mutated to tryptophan, as shown in SEQ ID NO. 14, and the nucleotide sequence encoding it is shown in SEQ ID NO. 11.

[0045] Furthermore, it will be readily apparent to those skilled in the art that altering a few amino acid residues in certain regions of a polypeptide, such as non-critical regions, will not substantially change its biological activity. For example, appropriately substituting certain amino acids will not affect the resulting sequence (see Watson et al., Molecular Biology of The Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224). Therefore, those skilled in the art can implement such substitutions and ensure that the resulting molecule retains the desired biological activity.

[0046] Therefore, it is obvious that further mutations of the squalene epoxidase mutant of the present invention can yield further mutants that still possess the function and activity of squalene epoxidase, particularly the function and activity of catalyzing the synthesis of 2,3,22,23-diepoxysqualene from squalene. For example, it is known to those skilled in the art that adding or removing several amino acid residues at either end of a polypeptide, such as preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue, will not affect the function of the resulting mutant. For example, for ease of purification, those skilled in the art often add a 6×His tag to either end of the obtained protein, and such a protein has the same function as a protein without a 6×His tag.

[0047] Methods for determining sequence homology or identity known to those skilled in the art include, but are not limited to: Computational Molecular Biology, edited by Lesk, A.M., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, D.W., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, edited by Griffin, A.M. and Griffin, H.G., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J.M., Stockton Press, New York, 1991; and Carillo, H. See Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0048] The present invention also provides a polynucleotide sequence encoding a squalene epoxidase mutant as described in any of the preceding claims. The polynucleotide sequence encoding the squalene epoxidase mutant may include a polynucleotide sequence encoding a squalene epoxidase mutant, or it may include additional coding and / or non-coding polynucleotide sequences.

[0049] Therefore, the terms “containing,” “having,” or “including” used in this article include “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0050] The present invention also provides an expression vector comprising the nucleic acid molecules described above.

[0051] In a preferred embodiment, the expression vector is a plasmid.

[0052] In a specific and preferred embodiment, the expression vector is pMAL.

[0053] The present invention also provides a host cell comprising the expression vector described above, or the genome of the host cell having the nucleic acid molecules described above integrated.

[0054] As used herein, the term "host cell" has the meaning commonly understood by those skilled in the art, namely, a host cell capable of producing the squalene epoxidase mutant of the present invention. In other words, the present invention can utilize any host cell, as long as the squalene epoxidase mutant of the present invention can be expressed in that host cell.

[0055] In a preferred embodiment, the host cell is Escherichia coli (E. coli).

[0056] The host cells of this invention can be used to prepare 2,3,22,23-diepoxysqualene; that is, 2,3,22,23-diepoxysqualene is synthesized by culturing the host cells of this invention. Therefore, the host cells of this invention also express NADPH cytochrome P450 reductase, or contain the encoding nucleotide sequence of said enzyme or its expression vector to produce squalene, and then, under the catalysis of a squalene epoxidase mutant, squalene is synthesized into 2,3,22,23-diepoxysqualene. In a preferred embodiment, the NADPH cytochrome P450 reductase is derived from rats. In a specific and preferred embodiment, the nucleotide sequence encoding the NADPH cytochrome P450 reductase is shown in SEQ ID NO. 12.

[0057] The present invention also provides the use of any of the above-described squalene epoxidase mutants, or the above-described nucleic acid molecules, or the above-described expression vectors, or the above-described host cells in the production of 2,3,22,23-diepoxysqualene.

[0058] The present invention also provides a method for producing 2,3,22,23-diepoxysqualene, the method comprising the following steps: (1) Culture the host cells described above to produce 2,3,22,23-diepoxysqualene; (2) Optionally isolate 2,3,22,23-diepoxysqualene from the culture medium obtained in (1).

[0059] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0060] Example 1 The nucleotide sequence of wild-type squalene epoxidase SgSQE1 derived from *Siraitia grosvenorii* (SEQ ID NO. 1, primer SQE-F1 / R1) was synthesized artificially. Sequence analysis of wild-type fruit squalene epoxidase SgSQE1 confirmed that the first 55 amino acids of the SgSQE1 amino acid sequence (SEQ ID NO. 13) constitute a transmembrane domain. This domain was truncated, and a GB1 sequence was designed and added to the N-terminus, and a 3×FLAG sequence was added to the C-terminus. The codons of the new sequence were optimized to suit expression in *E. coli*. The sequence SEQ ID NO. 2 (amino acid sequence shown in SEQ ID NO. 14) was obtained, and the gene sequence was synthesized artificially (primer SQE-F2 / R2). The amplification primer information is shown in Table 1.

[0061] SEQ ID NO.1: SEQ ID NO.13: MVDQCALGWILASVLGAAALYFLFGRKNGGVSNERRHESIKNIATTNGEVKSSNSDGGDIIIVGAGVAGSALAYTLGKDGRRVHVIERDLTEPDRIVGELLQPGGYLKLTELGLEDCVDIDAQRVYGYALFKDGKDTRLSYPLEKFHSDVAGRSFHNGRFIQRMREKAASLPNVSLEQGTVTSLLEENGIIKGVRYKTKGQEMTAYAPLTIVCDGCFSNLRRSLCNPKVDVPSCFVGLVLENCDLPYANHGHVILADPSPILFYRISSTEIRCLVDVPGQKVPSISNGEMANYLKNVVAPQIPSQLYDSFVAAIDKGNIRTMPNRSMPADPYPTPGALLMGDAFNMRHPLTGGGMTVALSDVVVLRDLLKPLRDLNDAPTLSKYLEAFYTLRKPVASTINTLAGALYKVFCASPDQARKEMRQACFDYLSLGGIFSNGPVSLLSGLNPRPISLVLHFFAVAIYGVGRLLIPFPSPKRVWIGARIISGASAIIFPIIKAEGVRQMFFPATVAAYYRAPRVVKGR SEQ ID NO.2: SEQ ID NO.14: MDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTESSIELGEWDGDIIIVGAGVAGSALAYTLGKDGRRVHVIERDLTEPDRIVGELLQPGGYLKLTELGLEDCVDDIDAQRVYGYAL FKDGKDTRLSYPLEKFHSDVAGRSFHNGRFIQRMREKAASLPNVSLEQGTVTSLLEENGIIKGVRYKTKTGQEMTAYAPLTIVCDGCFSNLRRSLCNPKVDVPSCFVGLVLENCDLPYANHGHVILADPSPILFYRISSTE IRCLVDVPGQKVPSISNGEMANYLKNVVAPQIPSQLYDSFVAAIDKGNIRTMPNRSMPADPYPTPGALLMGDAFNMRHPLTGGGMTVALSDVVVLRDLLKPLRDLNDAPTLSKYLEAFYTLRKPVASTINTLAGALYKVFC ASPDQARKEMRQACFDYLSLGGIFSNGPVSLLSGLNPRPISLVLHFFAVAIYGVGRLLIPFPSPKRVWIGARIISGASAIIFPIIKAEGVRQMFFPATVAAYYRAPRSSSNNNNNDYKDDDDKDYKDDDDKDYKDDDDKGS

[0062] Example 2 Based on the sequence of SEQ ID NO.2, primers were designed to target the point mutation of arginine (Arg) at position 104 (corresponding to arginine at position 94 in wild-type SgSQE1), which was mutated to aspartic acid (Asp, primer 104-F1 / R1), glycine (Gly, primer 104-F2 / R1), asparagine (Asn, primer 104-F3 / R1), and glutamine (Gln, primer 104-F4 / R1). The primer information is shown in Table 1. The corresponding gene fragments (SEQ ID NO.3~SEQ ID NO.6) were obtained by PCR amplification and DNA gel recovery and purification.

[0063] Based on the sequence of SEQ ID NO.2, primers R106-F1 / R1 (see Table 1) were designed to target the mutation of valine (Val) at position 106 (corresponding to valine at position 96 of wild-type SgSQE1) to leucine (Leu). The corresponding gene fragment SEQ ID NO.7 was obtained by PCR amplification and DNA gel recovery and purification.

[0064] Based on the sequence of SEQ ID NO.2, primers R107-F1 / R1 (see Table 1) were designed to target the mutation of glycine (Gly) at position 107 (corresponding to glycine at position 97 of wild-type SgSQE1) to tryptophan (Trp). The corresponding gene fragment SEQ ID NO.8 was obtained by PCR amplification and DNA gel recovery and purification.

[0065] Based on the sequence of SEQ ID NO.2, primers R365-F1 / R1 (see Table 1) were designed to target the mutation of glycine (Gly) at position 365 (corresponding to glycine at position 355 of wild-type SgSQE1) to glutamine (Gln). The corresponding gene fragment SEQ ID NO.9 was obtained by PCR amplification and DNA gel recovery and purification.

[0066] Table 1

[0067] The specific steps for obtaining gene fragments are as follows: Using SEQ ID NO.2 as a template, SQE-F2 / R2 and the above-mentioned mutant primers were first used for segmental PCR amplification, and then the two obtained fragments were used as templates for fusion PCR amplification to obtain gene fragments SEQ ID NO.3~SEQ ID NO.9. PCR reaction system:

[0068] SEQ ID NO.3 (R104D): ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC GAC SEQ ID NO.4(R104G): ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC GGC SEQ ID NO.5(R104N): ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC AAC SEQ ID NO.6(R104Q): ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC CAG SEQ ID NO.7(V106L): ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGACCGTATT CTG SEQ ID NO.8(G107W): ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGACCGTATTGTT TGG SEQ ID NO.9(G365Q): CAG ATGACCGTGGCACTGAGCGACGTGGTCGTACTGCGCGATCTGCTGAAACCGCTGCGTGATCTGAACGATGCTCCGACCCTGAGCAAATACCTGGAAGCGTTCTATACCCTGCGTAAACCGGTCGCGTCCACTATCAATACCCTGGCCGG CGCTCTGTACAAAGTATTCTGCGCGTCTCCGGATCAGGCTCGTAAGGAGATGCGCCAGGCATGCTTCGACTACCTGTCTCTGGGTGGTATCTTCTCCAACGGTCCAGTGTCCCTGCTGAGCGGTCTGAATCCGCGTCCGATCTCTCTGG TCCTGCACTTCTTTCGCTGTTGCCATCTACGGCGTCGGCCGTCTGCTGATCCCATTTCCGAGCCCGAAGCGTGTCTGGATCGGTGCACGTATTATCTCCGGCGCGAGCGCCATCATCTTTCCGATCATCAAAGCGGAAGGCGTACGTCAA ATGTTCTTCCCTGCCACTGTAGCGGCTTACTACCGTGCTCCACGTTCGTCCAGCAACAACAAATAACAATGACTACAAAGACGACGATGATAAAGACTACAAAGATGACGATGACAAGGACTACAAAGACGATGACGACAAGGGCAGCTAA

[0069] Example 3 pMAL-line-F / R primers were designed and synthesized (see Table 1) to linearize the pMAL vector. The PCR reaction system is as follows:

[0070] After gel recovery and purification, linearized pMAL vectors were obtained. These linearized pMAL vectors were then assembled with fragments SEQ ID NO.1 through SEQ ID NO.9 using Gibson assembly to obtain recombinant plasmids. The plasmids were transformed into *E. coli* DH5α competent cells, cultured overnight on ampicillin plates, and single clones were selected for sequencing identification.

[0071] Samples with correct sequencing were cultured and sufficient amounts of pMAL-SEQ ID NO.1~pMAL-SEQ ID NO.9 plasmids were extracted.

[0072] A recombinant Escherichia coli expression strain was prepared to produce squalene, and the NADPH cytochrome P450 reductase (RatCPR) from rat (Rattus norvegicus) was pre-expressed in this strain. The N-terminal transmembrane domain of this protein (corresponding to amino acids 1-63 in the wild type) was truncated, and the start codon at position 1 was retained, resulting in its encoding nucleic acid sequence as shown in SEQ ID NO. 12.

[0073] SEQ ID NO. 12: The pMAL-SEQ ID NO.1~pMAL-SEQ ID NO.9 plasmids were transformed into the aforementioned squalene-producing *E. coli* cells via electroporation, and then plated onto ampicillin plates for overnight culture. Positive clones were selected and cultured overnight in LB (Amp+) liquid medium. The culture was then expanded using LB (Amp+) liquid medium, and the cells were transferred to shake flasks and cultured until the OD value reached approximately 0.8. The IPTG induction concentration for the recombinant strain was 200 μM, the culture temperature was 25℃, and ampicillin was added to maintain plasmid presence (working concentration: 50 μg / mL). After fermentation in 50 mL of culture medium for 72 hours, samples were taken and the yield of 2,3,22,23-diepoxysqualene was determined by high-performance liquid chromatography (HPLC). The results are as follows: Figure 1 As shown.

[0074] The results showed that wild-type SgSQE1 (SEQ ID NO.1) had extremely poor solubility, and direct expression in E. coli would form inclusion bodies, with a yield of approximately 0.1 mg / L of 2,3,22,23-diepoxysqualene, which was almost undetectable. The mutant shown in SEQ ID NO.2 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 6.4 mg / L; the mutant shown in SEQ ID NO.3 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 9.4 mg / L; the mutant shown in SEQ ID NO.4 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 8.8 mg / L; the mutant shown in SEQ ID NO.5 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 9.1 mg / L; the mutant shown in SEQ ID NO.6 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 8.5 mg / L; and the mutant shown in SEQ ID NO.7 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 9.2 mg / L. The mutant shown in NO.8 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 8.6 mg / L, and the mutant shown in SEQ ID NO.9 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 8.9 mg / L.

[0075] In summary, the soluble mutant form of 2,3,22,23-diepoxysqualene provided by this invention has a yield of approximately 6.4 mg / L, which is more than sixty times higher than the wild type (SEQ ID NO.1) that forms inclusion bodies. Mutants formed by single-point mutations at multiple sites based on SEQ ID NO.2 all showed an increase in 2,3,22,23-diepoxysqualene yield of more than 30%.

[0076] Example 4 Based on the sequence of SEQ ID NO.2, primers G226-F1 / R1 were designed to mutate glycine (Gly) at position 226 (corresponding to glycine at position 216 of wild-type SgSQE1) to alanine (Ala), and primers G226-F2 / R1 were designed to mutate it to tryptophan (Trp). The amplification primer sequence information is shown in Table 1.

[0077] Using the sequence SEQ ID NO.3 as a template, SQE-F2 / R2 and the above-mentioned mutant primers were first used for segmental PCR amplification, and then the two obtained fragments were used as templates for fusion PCR amplification to obtain gene fragments SEQ ID NO.10 and SEQ ID NO.11. PCR reaction system:

[0078] SEQ ID NO.10 (R104D / G226A): ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGA CCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC GACATTGTTGGTGAACTGCTGCAGCCAGGTGGCTACCTGAAACTGACCGAGCTGGGCCTGGAGGACTGTGTGGACGATATCGACGCTCAGCGTGTCTACGGTTACGCTCTGTTCAAAGACGGTAAAGATACCCGCCTGTCCTATCCGCTGGAGAAATTTCACAGCGATGTAGCAGGCCGTAGCTTCCATAACGGTCGCTTCATCCAGCGTATGCGTGAGAAAGCGGCCAGCCTGCCGAACGTTAGCCTGGAACAGGGCACCGTAACCTCTCTGCTGGAAGAGAACGGTATCATCAAAGGCGTGCGCTACAAAACTAAGACCGGCCAAGAAATGACGGCGTATGCACCACTGACCATCGTGTGCGAC GCG SEQ ID NO.11(R104D / G226W): ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC GAC ATTGTTGGTGAACTGCTGCAGCCAGGTGGCTACCTGAAACTGACCGAGCTGGGCCTGGAGGACTGTGTGGACGATATCGACGCTCAGCGTGTCTACGGTTACGCTCTGTTCAAAGACGGTAAAGATACCCGCCTGTCCTATCCGCTGGAGAAATTTCACAGCGATGTAGCAGGCCGTAGCTTCCATAACGGTCGCTTCATCCAGCGTATGCGTGAGAAAGCGGCCAGCCTGCCGAACGTTAGCCTGGAACAGGGCACCGTAACCTCTCTGCTGGAAGAGAACGGTATCATCAAAGGCGTGCGCTACAAAACTAAGACCGGCCAAGAAATGACGGCGTATGCACCACTGACCATCGTGTGCGAC TGG pMAL-line-F / R primers were designed and synthesized (see Table 1) to linearize the pMAL vector. After gel purification, the linearized pMAL vector was obtained (see Table 1). Figure 3 PCR reaction system:

[0079] The linearized pMAL vector was assembled with fragments SEQ ID NO.10 and SEQ ID NO.11 using Gibson assembly to obtain recombinant plasmids. The plasmids were transformed into *E. coli* DH5α competent cells and cultured overnight on ampicillin plates. Single clones were selected for sequencing identification. Samples with correct sequencing results were amplified and sufficient quantities of pMAL-SEQ ID NO.10 and pMAL-SEQ ID NO.11 plasmids were extracted.

[0080] A recombinant *E. coli* strain expressing NADPH cytochrome P450 reductase (RatCPR) from rat (Rattus norvegicus) to produce squalene was prepared according to the method in Example 3. The pMAL-SEQ ID NO.10 and pMAL-SEQ ID NO.11 plasmids were transformed into the aforementioned *E. coli* cells producing squalene via electroporation. The cells were then plated on ampicillin plates and cultured overnight. Positive clones were selected and cultured overnight in LB (Amp+) liquid medium. The culture was then expanded using LB (Amp+) liquid medium, and the cells were inoculated into shake flasks and cultured until the OD value reached approximately 0.8. The IPTG induction concentration for the recombinant strain was 200 μM, the culture temperature was 25°C, and ampicillin was added to maintain plasmid presence (working concentration: 50 μg / mL). After fermentation in 50 mL of culture medium for 72 hours, samples were taken and the yield of 2,3,22,23-diepoxysqualene was detected using high-performance liquid chromatography (HPLC). The results are as follows: Figure 3 As shown.

[0081] Testing revealed that the mutant shown in SEQ ID NO. 10 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 13.1 mg / L, and the mutant shown in SEQ ID NO. 11 catalyzed the synthesis of 2,3,22,23-diepoxysqualene with a yield of approximately 12.9 mg / L. Compared to wild-type SgSQE1 (SEQ ID NO. 1), the catalytic efficiency of the mutants shown in SEQ ID NO. 10 and SEQ ID NO. 11 for the synthesis of 2,3,22,23-diepoxysqualene was increased by hundreds of times. Compared to the soluble modified mutant form (the mutant shown in SEQ ID NO. 2), the catalytic efficiency was also increased by 100%. This indicates that after soluble modification and amino acid point mutation of wild-type SgSQE1, the mutant form provided by this invention has excellent catalytic efficiency.

[0082] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A squalene epoxidase mutant, characterized in that, The amino acid sequence of the squalene cyclooxygenase mutant has one or more site mutations at positions 106, 107, and 365 of the amino acid sequence shown in SEQ ID NO.

14.

2. The squalene epoxidase mutant according to claim 1, characterized in that, The amino acid sequence of the squalene epoxidase mutant is such that it has at least one mutation selected from the group consisting of the amino acid sequence shown in SEQ ID NO. 14: (1) Valine at position 106 is mutated to leucine; (2) Glycine at position 107 is mutated to tryptophan; (3) Glycine at position 365 is mutated to glutamine.

3. An isolated nucleic acid molecule, said nucleic acid molecule encoding the squalene epoxidase mutant of claim 1 or 2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the expression vector of claim 4, or the genome of the host cell having the nucleic acid molecule of claim 3 integrated therein.

6. The host cell according to claim 5, characterized in that, The host cell also expresses NADPH cytochrome P450 reductase, or contains the encoding nucleotide sequence of the enzyme or its expression vector.

7. The host cell according to claim 6, characterized in that, The NADPH cytochrome P450 reductase was derived from rats.

8. The host cell according to claim 7, characterized in that, The nucleotide sequence encoding the NADPH cytochrome P450 reductase is shown in SEQ ID NO.

12.

9. The use of the squalene epoxidase mutant of claim 1 or 2, or the nucleic acid molecule of claim 3, or the expression vector of claim 4, or the host cell of claim 5 in the production of 2,3,22,23-diepoxysqualene.

10. A method for producing 2,3,22,23-diepoxysqualene, the method comprising the following steps: (1) Culturing the host cells of claim 5 to produce 2,3,22,23-diepoxysqualene; (2) Optionally isolate 2,3,22,23-diepoxysqualene from the culture medium obtained in (1).

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