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 the first to 25th amino acids at the N terminal of the amino acid sequence of wild squalene epoxidase SgSQE1 from momordica grosvenori, and then connecting a dissolution-promoting tag sequence to the N terminal and / or the C terminal. The squalene epoxidase mutant provided by the invention can be used for remarkably improving the yield of 2, 3, 22, 23-diepoxy squalene.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering and enzyme engineering, in particular to a squalene epoxidase mutant and application thereof. Background Art

[0002] Mogroside exists in mature monk fruit, and its sweetness is about 300 times that of sucrose, and it is a low-calorie sweetener. Mogroside has no peculiar smell or bitterness, and its sweetness is basically the same as sucrose. It is almost not absorbed into the intestines and does not cause blood sugar fluctuations. It can also be consumed by diabetics and people who control their blood sugar. In addition, mogroside also has the effects of moistening the lungs and relieving cough, anti-inflammatory and antibacterial, and enhancing immunity.

[0003] In the biosynthetic pathway of mogroside, squalene epoxidase catalyzes the epoxidation of the carbon-carbon double bonds between C2=C3 and C22=C23 of squalene to generate 2,3,22,23-diepoxysqualene. This is the first step in the synthesis of triterpenoids from squalene and is also a rate-limiting step. Subsequently, under the action of cucurbitadienol synthase, epoxide hydrolase, cytochrome P450 reductase and glucosyltransferase, diepoxysqualene undergoes multiple steps to form mogroside.

[0004] According to existing research, the commonly used squalene epoxidase ERG1 of brewer's yeast 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 of squalene is extremely low, which is not conducive to the biosynthesis of mogroside, limits the synthesis efficiency, and hinders industrial production and application. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a squalene epoxidase mutant capable of greatly improving the yield of 2,3,22,23-diepoxysqualene.

[0006] In a first aspect, the present invention provides a squalene epoxidase mutant, the amino acid sequence of the squalene epoxidase mutant being: the amino acid sequence obtained by deleting the 1st to 25th amino acids at the N-terminus of the amino acid sequence of wild-type squalene epoxidase SgSQE1 derived from Momordica grosvenori, and then connecting a solubility-promoting tag sequence to the N-terminus and / or C segment.

[0007] Preferably, the lytic tag sequence includes one or more of a GB1 sequence, a SUMO sequence, a MBP sequence, a 3×FLAG sequence, and a 3×Myc sequence.

[0008] Preferably, the N-terminally connected lytic tag is selected from one or more of a GB1 sequence, a SUMO sequence, and an MBP sequence.

[0009] Preferably, the lytic tag connected to the C-terminus is selected from 3×FLAG sequence and / or 3×Myc sequence.

[0010] Preferably, the amino acid sequence of the squalene epoxidase mutant has:

[0011] (1) the amino acid sequence shown in SEQ ID NO.14, or

[0012] (2) A sequence that has at least 90% (e.g., at least 95%, at least 98%, at least 99%) sequence identity with the amino acid sequence of SEQ ID NO. 14 and retains its biological activity.

[0013] Preferably, the amino acid sequence of the squalene epoxidase 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.

[0014] Preferably, the amino acid sequence of the squalene epoxidase mutant is an amino acid sequence shown in SEQ ID NO.14 having at least one mutation selected from the following group:

[0015] (1) the arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine;

[0016] (2) valine at position 106 mutated to leucine;

[0017] (3) mutation of glycine at position 107 to tryptophan;

[0018] (4) mutation of glycine at position 365 to glutamine;

[0019] (5) mutation of glycine at position 226 to alanine or tryptophan;

[0020] (6) The arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine; the glycine at position 226 is mutated to alanine or tryptophan.

[0021] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the squalene epoxidase mutant described in any one of the above items.

[0022] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described above.

[0023] In a fourth aspect, the present invention provides a host cell, wherein the host cell comprises the expression vector described above, or the genome of the host cell is integrated with the nucleic acid molecule described above.

[0024] Preferably, the host cell also expresses NADPH cytochrome P450 reductase, or contains a nucleotide sequence encoding the enzyme or an expression vector thereof.

[0025] Preferably, the NADPH cytochrome P450 reductase is derived from rat.

[0026] Preferably, the nucleotide sequence encoding the NADPH cytochrome P450 reductase is shown as SEQ ID NO.12.

[0027] In a fifth aspect, the present invention provides 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.

[0028] In a sixth aspect, the present invention provides a method for producing 2,3,22,23-diepoxysqualene, the method comprising the following steps:

[0029] (1) culturing the host cell described above to produce 2,3,22,23-diepoxysqualene;

[0030] (2) Optionally, isolating 2,3,22,23-diepoxysqualene from the culture solution obtained in (1).

[0031] Effects of the Invention

[0032] The invention transforms wild-type Momordica squalene epoxidase SgSQE1 by genetic engineering, so that the solubility, catalytic activity and biosynthesis efficiency of the transformed Momordica squalene epoxidase mutant are significantly improved, thereby meeting the requirements of industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a bar graph showing the yield of 2,3,22,23-diepoxysqualene produced by squalene epoxidase catalyzed by SEQ ID NO.1 to SEQ ID NO.9 in Example 3;

[0034] Figure 2 Plasmid maps constructed for the present invention;

[0035] Figure 3 This is a bar graph showing the yield of 2,3,22,23-diepoxysqualene produced by squalene epoxidase as shown in SEQ ID NO.10 to SEQ ID NO.11 in Example 4. DETAILED DESCRIPTION

[0036] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The 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 belongs.

[0037] In order to increase the yield of 2,3,22,23-diepoxysqualene, the current solution is to increase the number of copies of squalene epoxidase. However, the yield increase is not significant, and it increases the metabolic pressure of chassis cells. The inventors have found that the squalene epoxidase SgSQE1 derived from monk fruit can catalyze the carbon-carbon double bond between C22=C23, but the protein has poor solubility, and direct expression in Escherichia coli will form inclusion bodies, the enzyme activity is low, and the yield of 2,3,22,23-diepoxysqualene is obviously insufficient, making it difficult to carry out industrial production. After extensive and in-depth research, the inventors unexpectedly discovered a variety of mutants of squalene epoxidase SgSQE1. These mutants include amino acids 1 to 25 at the N-terminus of the amino acid sequence of wild-type monk fruit-derived squalene epoxidase SgSQE1, and then the amino acid sequence obtained by connecting a solubility-promoting tag sequence to the N-terminus and / or the C segment. In particular, on this basis, point mutations are further performed on the amino acids in the catalytic pocket of the sequence shown in SEQ ID NO.14, for example, one or more mutations at positions 104, 106, 107, 365, and 226. These mutants promote the binding of substrates squalene and 2,3-epoxysqualene to the enzyme, significantly improve the efficiency of catalytic synthesis of 2,3,22,23-diepoxysqualene, improve the water solubility of the protein, greatly increase the yield of 2,3,22,23-diepoxysqualene, and provide substrates and precursors for the subsequent production of mogrosides or other terpenoids.

[0038] 2,3,22,23-Bis(epoxysqualene) (CAS No.: 31063-19-1), also known as 2,3,22,23-epoxysqualene, has the following structural formula:

[0039]

[0040] In a specific embodiment, the nucleotide sequence of the 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: the amino acid sequence obtained by deleting the 1st to 25th amino acids at the N-terminus of the amino acid sequence of the wild-type squalene epoxidase SgSQE1 derived from Momordica grosvenori, and then connecting a solubility tag sequence to the N-terminus and / or C segment.

[0041] In certain embodiments, the lytic tag sequence includes one or more of a GB1 sequence, a SUMO sequence, a MBP sequence, a 3×FLAG sequence, and a 3×Myc sequence.

[0042] In certain embodiments, the N-terminally connected lytic tag is selected from one or more of a GB1 sequence, a SUMO sequence, and an MBP sequence.

[0043] In certain embodiments, the C-terminus-connected lytic tag is selected from a 3×FLAG sequence and / or a 3×Myc sequence.

[0044] In certain embodiments, the amino acid sequence of the squalene epoxidase mutant has:

[0045] (1) the amino acid sequence shown in SEQ ID NO.14, or

[0046] (2) A sequence that has at least 90% (e.g., at least 95%, at least 98%, at least 99%) sequence identity with the amino acid sequence of SEQ ID NO. 14 and retains its biological activity.

[0047] In a specific embodiment, the homology or sequence identity may be greater than 90%, preferably greater than 95%, more preferably 96%, 97%, 98%, or 99%.

[0048] In a specific embodiment, the nucleotide sequence encoding the squalene epoxidase mutant is shown as SEQ ID NO.2.

[0049] In certain embodiments, the amino acid sequence of the squalene epoxidase 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.

[0050] In certain embodiments, the amino acid sequence of the squalene epoxidase mutant is an amino acid sequence shown in SEQ ID NO.14 having at least one mutation selected from the following group:

[0051] (1) the arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine;

[0052] (2) valine at position 106 mutated to leucine;

[0053] (3) mutation of glycine at position 107 to tryptophan;

[0054] (4) mutation of glycine at position 365 to glutamine;

[0055] (5) mutation of glycine at position 226 to alanine or tryptophan;

[0056] (6) The arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine; the glycine at position 226 is mutated to alanine or tryptophan.

[0057] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that the arginine at position 104 in the amino acid sequence shown in SEQ ID NO.14 is mutated to aspartic acid, and the nucleotide sequence encoding it is shown in SEQ ID NO.3.

[0058] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that the arginine at position 104 in the amino acid sequence shown in SEQ ID NO.14 is mutated to glycine, and the nucleotide sequence encoding it is shown in SEQ ID NO.4.

[0059] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that the arginine at position 104 in the amino acid sequence shown in SEQ ID NO.14 is mutated to asparagine, and the nucleotide sequence encoding it is shown in SEQ ID NO.5.

[0060] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that the arginine at position 104 in the amino acid sequence shown in SEQ ID NO.14 is mutated to glutamine, and the nucleotide sequence encoding it is shown in SEQ ID NO.6.

[0061] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that the valine at position 106 in the amino acid sequence shown in SEQ ID NO.14 is mutated to leucine, and the nucleotide sequence encoding it is shown in SEQ ID NO.7.

[0062] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that the glycine at position 107 in the amino acid sequence shown in SEQ ID NO.14 is mutated to tryptophan, and the nucleotide sequence encoding it is shown in SEQ ID NO.8.

[0063] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that glycine at position 365 in the amino acid sequence shown in SEQ ID NO.14 is mutated to glutamine, and the nucleotide sequence encoding it is shown in SEQ ID NO.9.

[0064] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that the arginine at position 104 is mutated to aspartic acid and the glycine at position 226 is mutated to alanine in the amino acid sequence shown in SEQ ID NO.14, and the nucleotide sequence encoding it is shown in SEQ ID NO.10.

[0065] In certain preferred embodiments, the amino acid sequence of the squalene epoxidase mutant is that the arginine at position 104 is mutated to aspartic acid, and the glycine at position 226 is mutated 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.11.

[0066] In addition, it is not difficult for a person skilled in the art to know that changing a few amino acid residues in certain regions of a polypeptide, such as non-critical regions, will not substantially change the biological activity. For example, a sequence obtained by appropriately replacing certain amino acids will not affect its activity (see Watson et al., Molecular Biology of The Gene, 4th edition, 1987, The Benjamin / Cummings Pub. Co. P224). Therefore, a person skilled in the art can implement such replacements and ensure that the resulting molecule still has the desired biological activity.

[0067] Therefore, it is obvious that the squalene epoxidase mutant of the present invention can be further mutated to obtain a further mutant that still has the function and activity of squalene epoxidase, especially the function and activity of catalyzing squalene to synthesize 2,3,22,23-diepoxysqualene. For example, it is well known to those skilled in the art that adding or reducing a number of amino acid residues, such as preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue at either end of the polypeptide will not affect the function of the obtained mutant. For example, for the convenience of purification, technicians often carry a 6×His tag at either end of the obtained protein, and this protein has the same function as the protein without the 6×His tag.

[0068] Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, JM, ed., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program 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.

[0069] The present invention also provides a polynucleotide sequence encoding any of the above-mentioned squalene epoxidase mutants. The polynucleotide sequence encoding the squalene epoxidase mutant may include a polynucleotide sequence encoding the squalene epoxidase mutant, or may also include additional coding and / or non-coding polynucleotide sequences.

[0070] Therefore, “containing”, “having” or “including” used in this article include “comprising”, “mainly consisting of…”, “substantially consisting of…”, and “consisting of…”; “mainly consisting of…”, “substantially consisting of…” and “consisting of…” are subordinate concepts of “containing”, “having” or “including”.

[0071] The present invention also provides an expression vector, which comprises the nucleic acid molecule described above.

[0072] In a preferred embodiment, the expression vector is a plasmid.

[0073] In a specific and preferred embodiment, the expression vector is pMAL.

[0074] The present invention also provides a host cell, wherein the host cell comprises the expression vector described above, or the genome of the host cell is integrated with the nucleic acid molecule described above.

[0075] The term "host cell" used herein has the meaning generally understood by those of ordinary skill in the art, i.e., 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 the host cell.

[0076] In a preferred embodiment, the host cell is Escherichia coli (E. coli).

[0077] The host cell of the present invention can be used to prepare 2,3,22,23-diepoxysqualene; that is, 2,3,22,23-diepoxysqualene is synthesized by culturing the host cell of the present invention, so the host cell of the present invention also expresses NADPH cytochrome P450 reductase, or contains a nucleotide sequence encoding the enzyme or an expression vector thereof to produce squalene, and then under the catalysis of the squalene epoxidase mutant, squalene synthesizes 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.

[0078] The present invention also provides use of any of the above-mentioned squalene epoxidase mutants, or the above-mentioned nucleic acid molecules, or the above-mentioned expression vectors, or the above-mentioned host cells in the production of 2,3,22,23-diepoxysqualene.

[0079] The present invention also provides a method for producing 2,3,22,23-diepoxysqualene, the method comprising the following steps:

[0080] (1) culturing the host cell described above to produce 2,3,22,23-diepoxysqualene;

[0081] (2) Optionally, isolating 2,3,22,23-diepoxysqualene from the culture solution obtained in (1).

[0082] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0083] Example 1

[0084] The nucleotide sequence of the wild-type squalene epoxidase SgSQE1 from Momordica grosvenori was artificially synthesized (SEQ ID NO.1, primer SQE-F1 / R1), and the wild-type fruit squalene epoxidase SgSQE1 was sequenced and sequence analysis was performed to confirm that the first 25 amino acids of the SgSQE1 amino acid sequence (SEQ ID NO.13) were transmembrane domains, which were truncated, and then the GB1 sequence was designed to be added to the N-terminus, and the 3×FLAG sequence was added to the C-terminus. The new sequence was codon optimized to make it suitable for Escherichia coli expression. The SEQ ID NO.2 sequence (amino acid sequence as shown in SEQ ID NO.14) was obtained, and the gene sequence was artificially synthesized (primer SQE-F2 / R2), and the amplification primer information is shown in Table 1.

[0085] SEQ ID NO.1:

[0086]

[0087] SEQ ID NO.13:

[0088] MVDQCALGWILASVLGAAALYFLFGRKNGGVSNERRHESIKNIATTNGEVKSSNSDGGDIIIVGAGVAGSALAYTLGKDGRRVHVIERDLTEPDRIVGELLQPGGYLKLTELGLEDCVDIDAQRVYGYALFKDGKDTRLSYPLEKFHSDVAGRSFHNGRFIQRMREKAASLPNVSLEQGTVTSLLEENGIIKGVRYKTKGQEMTAYAPLTIVCDGCFSNLRRSLCNPKVDVPSCFVGLVLENCDLPYANHGHVILADPSPILFYRISSTEIRCLVDVPGQKVPSISNGEMANYLKNVVAPQIPSQLYDSFVAAIDKGNIRTMPNRSMPADPYPTPGALLMGDAFNMRHPLTGGGMTVALSDVVVLRDLLKPLRDLNDAPTLSKYLEAFYTLRKPVASTINTLAGALYKVFCASPDQARKEMRQACFDYLSLGGIFSNGPVSLLSGLNPRPISLVLHFFAVAIYGVGRLLIPFPSPKRVWIGARIISGASAIIFPIIKAEGVRQMFFPATVAAYYRAPRVVKGR

[0089] SEQ ID NO.2:

[0090]

[0091] SEQ ID NO.14:

[0092] MDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTESSIELGEWDGDIIIVGAGVAGSALAYTLGKDGRRVHVIERDLTEPDRIVGELLQPGGYLKLTELGLEDCVDDIDAQRVYGYAL FKDGKDTRLSYPLEKFHSDVAGRSFHNGRFIQRMREKAASLPNVSLEQGTVTSLLEENGIIKGVRYKTKTGQEMTAYAPLTIVCDGCFSNLRRSLCNPKVDVPSCFVGLVLENCDLPYANHGHVILADPSPILFYRISSTE IRCLVDVPGQKVPSISNGEMANYLKNVVAPQIPSQLYDSFVAAIDKGNIRTMPNRSMPADPYPTPGALLMGDAFNMRHPLTGGGMTVALSDVVVLRDLLKPLRDLNDAPTLSKYLEAFYTLRKPVASTINTLAGALYKVFC ASPDQARKEMRQACFDYLSLGGIFSNGPVSLLSGLNPRPISLVLHFFAVAIYGVGRLLIPFPSPKRVWIGARIISGASAIIFPIIKAEGVRQMFFPATVAAYYRAPRSSSNNNNNDYKDDDDKDYKDDDDKDYKDDDDKGS

[0093] Example 2

[0094] Based on the sequence of SEQ ID NO.2, primers for the point mutation of arginine (Arg) at position 104 (corresponding to the arginine at position 94 of the wild-type SgSQE1) were designed, and it 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), respectively. The amplification primer information is shown in Table 1. Through PCR amplification and DNA gel recovery and purification, the corresponding gene fragments (SEQ ID NO.3 to SEQ ID NO.6) were obtained.

[0095] Based on the sequence of SEQ ID NO.2, primers R106-F1 / R1 (see Table 1) were designed to mutate the valine (Val) at position 106 (corresponding to the valine at position 96 of the wild-type SgSQE1) to leucine (Leu). Through PCR amplification and DNA gel recovery and purification, the corresponding gene fragment SEQ ID NO.7 was obtained.

[0096] Based on the sequence of SEQ ID NO.2, primers R107-F1 / R1 (see Table 1) were designed to mutate the glycine (Gly) at position 107 (corresponding to the glycine at position 97 of the wild-type SgSQE1) to tryptophan (Trp). Through PCR amplification and DNA gel recovery and purification, the corresponding gene fragment SEQ ID NO.8 was obtained.

[0097] Based on the sequence of SEQ ID NO.2, primers R365-F1 / R1 (see Table 1) were designed to mutate glycine (Gly) at position 365 (corresponding to glycine at position 355 of wild-type SgSQE1) to glutamine (Gln). Through PCR amplification and DNA gel recovery and purification, the corresponding gene fragment SEQ ID NO.9 was obtained.

[0098] Table 1

[0099]

[0100]

[0101] The specific operations for obtaining gene fragments are as follows:

[0102] Using SEQ ID NO.2 as a template, SQE-F2 / R2 and the above-mentioned mutant primers were firstly amplified by segmented PCR, and then the two obtained fragments were used as templates for fusion PCR amplification to obtain gene fragments of SEQ ID NO.3 to SEQ ID NO.9. PCR reaction system:

[0103] 2×PhantaFlashMasterMix(DyePlus) 25μL F Primer 1μL R Primer 1μL SEQ ID NO.2Gene sequence 20ng <![CDATA[ddH2O]]> Make up to 50 μL

[0104] SEQ ID NO.3 (R104D):

[0105] ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC GAC

[0106] SEQ ID NO.4(R104G):

[0107] ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC GGC

[0108] SEQ ID NO.5(R104N):

[0109] ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC AAC

[0110] SEQ ID NO.6(R104Q):

[0111] ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC CAG

[0112] SEQ ID NO.7(V106L):

[0113] ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGACCGTATT CTG

[0114] SEQ ID NO.8(G107W):

[0115] ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGACCGTATTGTT TGG

[0116] SEQ ID NO.9(G365Q):

[0117] CAG ATGACCGTGGCACTGAGCGACGTGGTCGTACTGCGCGATCTGCTGAAACCGCTGCGTGATCTGAACGATGCTCCGACCCTGAGCAAATACCTGGAAGCGTTCTATACCCTGCGTAAACCGGTCGCGTCCACTATCAATACCCTGGCCGG CGCTCTGTACAAAGTATTCTGCGCGTCTCCGGATCAGGCTCGTAAGGAGATGCGCCAGGCATGCTTCGACTACCTGTCTCTGGGTGGTATCTTCTCCAACGGTCCAGTGTCCCTGCTGAGCGGTCTGAATCCGCGTCCGATCTCTCTGG TCCTGCACTTCTTCGCTGTTGCCATCTACGGCGTCGGCCGTCTGCTGATCCCATTTCCGAGCCCGAAGCGTGTCTGGATCGGTGCACGTATTATCTCCGGCGCGAGCGCCATCATCTTTCCGATCATCAAAGCGGAAGGCGTACGTCAA ATGTTCTTCCCTGCCACTGTAGCGGCTTACTACCGTGCTCCACGTTCGTCCAGCAACAACAAATAACAATGACTACAAAGACGACGATGATAAAGACTACAAAGATGACGATGACAAGGACTACAAAGACGATGACGACAAGGGCAGCTAA

[0118] Example 3

[0119] Design and synthesize pMAL-line-F / R primers (see Table 1) to linearize the pMAL vector. The PCR reaction system is as follows:

[0120] 2×PhantaFlashMasterMix(DyePlus) 25μL pMAL-line-F Primer 1μL pMAL-line-R Primer 1μL pMAL-c5X Plasmid 20ng <![CDATA[ddH2O]]> Make up to 50 μL

[0121] After gel recovery and purification, a linearized pMAL vector was obtained, and the linearized pMAL vector was assembled with the fragments of SEQ ID NO.1 to SEQ ID NO.9 by Gibson assembly to obtain a recombinant plasmid. The plasmid was transformed into E. coli DH5α competent cells, cultured overnight on an ampicillin plate, and a single clone was selected for sequencing identification.

[0122] The samples with correct sequencing were expanded and cultured, and sufficient amounts of pMAL-SEQ ID NO.1 to pMAL-SEQ ID NO.9 plasmids were extracted.

[0123] A recombinant Escherichia coli expression strain for producing squalene is prepared, and NADPH cytochrome P450 reductase (RatCPR) from rat (Rattus norvegicus) is recombinantly expressed in advance in the strain, the N-terminal transmembrane domain of the protein (corresponding to the 1st to 63rd amino acids of the wild type) is truncated, and the start codon at the 1st position is retained, and its encoding nucleic acid sequence is obtained as SEQ ID NO.12 sequence.

[0124] SEQ ID NO.12:

[0125]

[0126] The pMAL-SEQ ID NO.1 to pMAL-SEQ ID NO.9 plasmids were transformed into the above-constructed squalene-producing Escherichia coli cells by electroporation, and then spread on ampicillin plates for overnight culture. Positive clones were selected and cultured overnight in LB (Amp+) liquid medium. The culture was expanded with LB (Amp+) liquid medium, and the bacteria were inoculated into shake flasks and cultured until the OD value was about 0.8. The IPTG induction concentration of the recombinant strain was 200 μM, the culture temperature was 25°C, and ampicillin was added to maintain the presence of the plasmid. The working concentration of ampicillin was 50 μg / mL. After fermentation and culture in 50 mL of culture medium for 72 hours, samples were taken and the production of 2,3,22,23-diepoxysqualene was detected by high performance liquid chromatography. The results are as follows Figure 1 shown.

[0127] The results showed that the wild-type SgSQE1 (SEQ ID NO.1) had extremely poor solubility and formed inclusion bodies when directly expressed in Escherichia coli. The yield of 2,3,22,23-diepoxysqualene was approximately 0.1 mg / L, which was almost undetectable. The mutant shown in SEQ ID NO.2 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 6.4 mg / L, the mutant shown in SEQ ID NO.3 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 9.4 mg / L, the mutant shown in SEQ ID NO.4 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 8.8 mg / L, the mutant shown in SEQ ID NO.5 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 9.1 mg / L, the mutant shown in SEQ ID NO.6 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 8.5 mg / L, the mutant shown in SEQ ID NO.7 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 9.2 mg / L, and the mutant shown in SEQ ID NO.8 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 9.6 mg / L. The mutant shown in NO.8 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 8.6 mg / L, and the mutant shown in SEQ ID NO.9 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a yield of about 8.9 mg / L.

[0128] In summary, the yield of soluble mutant form of 2,3,22,23-diepoxysqualene shown in SEQ ID NO.2 provided by the present invention is about 6.4 mg / L, which is more than sixty times higher than the yield of the wild type (SEQ ID NO.1) that forms inclusion bodies; the mutants formed by single-point mutations at multiple sites on the basis of SEQID NO.2 have their 2,3,22,23-diepoxysqualene yields increased by more than 30%.

[0129] Example 4

[0130] 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 primer G226-F2 / R1 was designed to mutate to tryptophan (Trp). The amplification primer sequence information is shown in Table 1.

[0131] Using SEQ ID NO.3 as a template, SQE-F2 / R2 and the above-mentioned mutant primers were firstly amplified by segmented PCR, and then the two obtained fragments were used as templates for fusion PCR amplification to obtain SEQ ID NO.10 and SEQ ID NO.11 gene fragments. PCR reaction system:

[0132] 2×PhantaFlashMasterMix(DyePlus) 25μL F Primer 1μL R Primer 1μL SEQ ID NO.3Gene sequence 20ng <![CDATA[ddH2O]]> Make up to 50 μL

[0133] SEQ ID NO.10 (R104D / G226A):

[0134] ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGA CCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC GACATTGTTGGTGAACTGCTGCAGCCAGGTGGCTACCTGAAACTGACCGAGCTGGGCCTGGAGGACTGTGTGGACGATATCGACGCTCAGCGTGTCTACGGTTACGCTCTGTTCAAAGACGGTAAAGATACCCGCCTGTCCTATCCGCTGGAGAAATTTCACAGCGATGTAGCAGGCCGTAGCTTCCATAACGGTCGCTTCATCCAGCGTATGCGTGAGAAAGCGGCCAGCCTGCCGAACGTTAGCCTGGAACAGGGCACCGTAACCTCTCTGCTGGAAGAGAACGGTATCATCAAAGGCGTGCGCTACAAAACTAAGACCGGCCAAGAAATGACGGCGTATGCACCACTGACCATCGTGTGCGAC GCG

[0135] SEQ ID NO.11(R104D / G226W):

[0136] ATGGACACTTACAAACTGATCCTGAATGGTAAGACATTGAAAGGCGAAACGACCACTGAAGCTGTTGATGCTGCTACTGCAGAGAAAGTCTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTCACAGTTACTGAATCTAGCATCGAACTGGGTGAATGGGATGGTGACATCATCATCGTGGGTGCAGGTGTTGCAGGTTCCGCTCTGGCGTACACTCTGGGCAAAGATGGTCGTCGCGTGCATGTCATCGAACGCGATCTGACCGAACCGGAC GAC ATTGTTGGTGAACTGCTGCAGCCAGGTGGCTACCTGAAACTGACCGAGCTGGGCCTGGAGGACTGTGTGGACGATATCGACGCTCAGCGTGTCTACGGTTACGCTCTGTTCAAAGACGGTAAAGATACCCGCCTGTCCTATCCGCTGGAGAAATTTCACAGCGATGTAGCAGGCCGTAGCTTCCATAACGGTCGCTTCATCCAGCGTATGCGTGAGAAAGCGGCCAGCCTGCCGAACGTTAGCCTGGAACAGGGCACCGTAACCTCTCTGCTGGAAGAGAACGGTATCATCAAAGGCGTGCGCTACAAAACTAAGACCGGCCAAGAAATGACGGCGTATGCACCACTGACCATCGTGTGCGAC TGG

[0137] Design and synthesize pMAL-line-F / R primers (see Table 1), perform linear amplification on the pMAL vector, and obtain the linearized pMAL vector after gel recovery and purification (see Figure 3 ). PCR reaction system:

[0138] 2×PhantaFlashMasterMix(DyePlus) 25μL pMAL-line-F Primer 1μL pMAL-line-R Primer 1μL pMAL-c5X Plasmid 20ng ddH2O Make up to 50 μL

[0139] The linearized pMAL vector was assembled with the fragments of SEQ ID NO.10 and SEQ ID NO.11 by Gibson assembly to obtain the recombinant plasmid. The plasmid was transformed into E. coli DH5α competent cells, cultured overnight on ampicillin plates, and single clones were selected for sequencing identification. The samples with correct sequencing were expanded and sufficient pMAL-SEQ ID NO.10 and pMAL-SEQ ID NO.11 plasmids were extracted.

[0140] According to the method of Example 3, a recombinant Escherichia coli expression strain for producing squalene expressing NADPH cytochrome P450 reductase (RatCPR) from rats (Rattus norvegicus) was prepared, and the pMAL-SEQ ID NO.10 and pMAL-SEQ ID NO.11 plasmids were transformed into the above-constructed squalene-producing Escherichia coli cells by electroporation, and then spread on ampicillin plates for overnight culture. Positive clones were selected and cultured overnight in LB (Amp+) liquid culture medium. The culture was expanded with LB (Amp+) liquid culture medium, and the bacteria were inoculated into shake flasks for culture until the OD value was about 0.8. The IPTG induction concentration of the recombinant strain was 200 μM, the culture temperature was 25°C, and ampicillin was added to maintain the presence of the plasmid, and the working concentration of ampicillin was 50 μg / mL. After fermentation and culture 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. The results are as follows Figure 3 shown.

[0141] After testing, the mutant shown in SEQ ID NO.10 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with an output of about 13.1 mg / L, and the mutant shown in SEQ ID NO.11 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with an output of about 12.9 mg / L. Compared with the wild-type SgSQE1 (SEQ ID NO.1), the mutant shown in SEQ ID NO.10 catalyzes the synthesis of 2,3,22,23-diepoxysqualene and the mutant shown in SEQ ID NO.11 catalyzes the synthesis of 2,3,22,23-diepoxysqualene with a catalytic efficiency of hundreds of times, and compared with the mutant form after soluble modification (the mutant shown in SEQ ID NO.2), the catalytic efficiency is also increased by 100%. This shows that after the wild-type SgSQE1 is subjected to soluble modification and amino acid point mutation, the mutant form provided by the present invention has excellent catalytic efficiency.

[0142] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A squalene epoxidase mutant, characterized in that: The amino acid sequence of the squalene epoxidase mutant is obtained by deleting the 1st to 25th amino acids at the N-terminus of the amino acid sequence of the wild-type squalene epoxidase SgSQE1 derived from Momordica grosvenori, and then connecting a solubilizing tag sequence to the N-terminus and / or C segment.

2. The squalene epoxidase mutant according to claim 1, characterized in that The lytic tag sequence includes one or more of a GB1 sequence, a SUMO sequence, an MBP sequence, a 3×FLAG sequence, and a 3×Myc sequence; Preferably, the N-terminally connected lytic tag is selected from one or more of a GB1 sequence, a SUMO sequence, and an MBP sequence; Preferably, the lytic tag connected to the C-terminus is selected from 3×FLAG sequence and / or 3×Myc sequence.

3. The squalene epoxidase mutant according to claim 1 or 2, characterized in that: 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 that has at least 90% (e.g., at least 95%, at least 98%, at least 99%) sequence identity with the amino acid sequence of SEQ ID NO. 14 and retains its biological activity.

4. The squalene epoxidase mutant according to claim 3, characterized in that The amino acid sequence of the squalene epoxidase 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.

5. The squalene epoxidase mutant according to claim 4, characterized in that: The amino acid sequence of the squalene epoxidase mutant is an amino acid sequence shown in SEQ ID NO.14 having at least one mutation selected from the following group: (1) the arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine; (2) valine at position 106 mutated to leucine; (3) mutation of glycine at position 107 to tryptophan; (4) mutation of glycine at position 365 to glutamine; (5) mutation of glycine at position 226 to alanine or tryptophan; (6) The arginine at position 104 is mutated to aspartic acid, glycine, asparagine or glutamine, preferably aspartic acid or asparagine; the glycine at position 226 is mutated to alanine or tryptophan.

6. An isolated nucleic acid molecule encoding the squalene epoxidase mutant according to any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A host cell, the host cell comprising the expression vector of claim 7, or the genome of the host cell is integrated with the nucleic acid molecule of claim 6; Preferably, the host cell also expresses NADPH cytochrome P450 reductase, or contains a nucleotide sequence encoding the enzyme or an expression vector thereof; Preferably, the NADPH cytochrome P450 reductase is derived from rat; Preferably, the nucleotide sequence encoding the NADPH cytochrome P450 reductase is shown as SEQ ID NO.

12.

9. Use of the squalene epoxidase mutant according to any one of claims 1 to 5, or the nucleic acid molecule according to claim 6, or the expression vector according to claim 7, or the host cell according to claim 8 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 cell according to claim 8 to produce 2,3,22,23-diepoxysqualene; (2) Optionally, isolating 2,3,22,23-diepoxysqualene from the culture solution obtained in (1).

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