Applications of β-Himalayene synthase mutants and its product cedrol

Abstract

This invention discloses a β-Himalayan cedrene synthase mutant and the application of its product, cedrol, belonging to the fields of enzyme engineering, genetic engineering, and pharmaceutical technology. The β-Himalayan cedrene synthase mutant disclosed in this invention is S434V, and its amino acid sequence is shown in SEQ ID NO. 5. This invention not only provides an efficient biosynthetic method for producing cedrol, but also offers potential candidate compounds for the development of novel anti-liver cancer drugs, possessing significant theoretical and practical value.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering, genetic engineering and pharmaceutical technology, and more specifically to the application of β-Himalayene synthase mutants and their product cedrol. Background Art

[0002] Cedrol is a naturally occurring sesquiterpene alcohol with the molecular formula C6H2O. 15 H 26 O, which has two isomers, is widely distributed throughout the plant kingdom, and is commonly found in conifers such as Atlantic cedar (Cedrus atlantica). Cedarol has been reported to possess various biological activities, such as antibacterial, anti-inflammatory, hair growth promoting, analgesic, and anti-anxiety activities. In recent years, many researchers have focused on its anti-tumor activity, which has been validated in various tumor models, including colorectal cancer, lung cancer, glioblastoma, and breast cancer.

[0003] Currently, cedrol is mainly obtained through organic chemical synthesis and plant extraction; however, both methods have drawbacks. Plant extraction typically yields low concentrations, and overexploitation of natural resources leads to excessive resource depletion, posing a significant challenge and threat to the ecological environment. Chemical synthesis often utilizes strong acids and bases, requires specific conditions, and inevitably involves high temperatures and pressures, resulting in high costs and a high risk of environmental problems, making it difficult to adhere to green production principles. Compared to these two methods, microbial fermentation has become a promising alternative for industrial application. Compared to the two methods mentioned above, enzymatic synthesis of cedrol offers advantages such as safety, efficiency, low cost, and environmental friendliness, but relevant literature is scarce. Therefore, developing a more potent catalytic enzyme for the production of these two sesquiterpenes would greatly improve the industrial production efficiency of β-Himalayene and cedrol, significantly impacting their market development.

[0004] Liver cancer is a major disease posing a serious threat to human health worldwide, with persistently high incidence and mortality rates. According to the latest statistics from the International Agency for Research on Cancer (IARC) of the World Health Organization, liver cancer has become the sixth most common cancer and the third leading cause of cancer death globally, with over 900,000 new cases worldwide in 2024. This severe epidemiological situation underscores the urgency of strengthening liver cancer prevention and control. In the field of anticancer drug development, cedrol has attracted significant attention due to its broad-spectrum antitumor activity, making it a promising candidate for liver cancer treatment.

[0005] Therefore, providing β-Himalayene synthase mutants and the application of its product cedrol is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of β-Himalayene synthase mutant and its product cedrol.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The β-Himalayene synthase mutant is S434V;

[0009] The amino acid sequence of the β-Himalayan synthase mutant is shown in SEQ ID NO. 5;

[0010] The amino acid sequence of the β-Himalayan synthase is shown in SEQ ID NO.1.

[0011] Furthermore, the relevant biomaterials of the β-Himalayan synthase mutant are at least one of the following (1)-(4):

[0012] (1) Nucleic acid molecules encoding β-Himalayene synthase mutants;

[0013] (2) An expression cassette containing the nucleic acid molecule described in (1);

[0014] (3) A recombinant vector containing the nucleic acid molecule described in (1) or a recombinant vector containing the expression cassette described in (2);

[0015] (4) A recombinant microorganism containing the nucleic acid molecule described in (1), a recombinant microorganism containing the nucleic acid molecule of the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3).

[0016] Furthermore, the application of the β-Himalayene synthase mutant or the related biomaterials in increasing cedrol production.

[0017] Furthermore, the application of the β-Himalayene synthase mutant or the related biomaterials in the production of cedrol.

[0018] Furthermore, the application of cedrol in the preparation of antitumor drugs.

[0019] Furthermore, the tumor is liver cancer.

[0020] As can be seen from the above technical solution, compared with the prior art, this invention discloses a β-hemicerin synthase mutant and the application of its product cedrol. By performing site-directed mutagenesis (SER434→VAL434) on a key site of the wild-type PoTPS1 enzyme, a novel catalytic mutant PoTPS1 (S434V) was successfully constructed. This mutant was efficiently expressed in the *Saccharomyces cerevisiae* CEN.PK113-5D expression system using the pSPGM1 yeast protein expression plasmid as a vector. Notably, this mutant significantly altered the product specificity, changing the main metabolite from β-hemicerin to the more valuable cedrol. Through the establishment of an efficient separation and purification process, high-purity cedrol was obtained from the culture of the recombinant strain YCP00 / pSPGM1-PoTPS1 (S434V). In vitro antitumor experiments showed that this compound exhibited significant antiproliferative activity against both HepG2 and SK-Hep-1 human liver cancer cell lines. Further mechanistic studies revealed that cedrol can promote apoptosis in liver cancer cells. This invention not only provides an efficient biosynthetic method for producing cedrol, but also offers a potential candidate compound for the development of novel anti-liver cancer drugs, possessing significant theoretical and practical value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The diagram shows the molecular docking results of β-Himalayan synthase PoTPS1 with the substrate small molecule FPP; where A: 2D display image, B: 3D display image.

[0023] Figure 2 YCP-00 / pSP-GM1-PoTPS1 Mutant DNA agarose gel chromatography pattern of recombinant yeast strain (target band size 1746 bp); where M: standard DNA molecular ruler; lanes 1-20 are wild-type PoTPS1 and PoTPS2, respectively. S434A PoTPS1 S434C PoTPS1 S434D PoTPS1 S434E PoTPS1 S434F PoTPS1 S434G PoTPS1S434H PoTPS1 S434I PoTPS1 S434K PoTPS1 S434L PoTPS1 S434M PoTPS1 S434N PoTPS1 S434P PoTPS1 S434Q PoTPS1 S434R PoTPS1 S434T PoTPS1 S434V PoTPS1 S434W and PoTPS1 S434Y Gene DNA sample.

[0024] Figure 3 The recombinant strains YCP-00 / pSP-GM1-PoTPS1 and YCP-00 / pSP-GM1-PoTPS1 Mutant GC spectra of fermentation products; where Figure A represents wild-type PoTPS1; Figure B represents PoTPS1. S434D PoTPS1 S434E and PoTPS1 S434G Figure C shows PoTPS1. S434A PoTPS1 S434C PoTPS1 S434F and PoTPS1 S434H Figure D shows PoTPS1. S434I PoTPS1 S434L and PoTPS1 S434N Figure E shows PoTPS1. S434K PoTPS1 S434M PoTPS1 S434P PoTPS1 S434Q PoTPS1 S434R PoTPS1 S434W and PoTPS1 S434Y Figure F shows PoTPS1. S434T ; G diagram is PoTPS1 S434V .

[0025] Figure 4 The image shows the GC detection result of pure cedrol; the retention time of cedrol is 9.15 min.

[0026] Figure 5 The image shows the NMR spectrum of cedrol; the upper image is the proton NMR spectrum, which is analyzed in detail below. 1¹H NMR (600MHz, CDCl₃) δ 1.90 – 1.82 (m, 2H), 1.82 – 1.78 (m, 1H), 1.72 – 1.68 (m, 1H), 1.68 – 1.64 (m, 1H), 1.64 – 1.61 (m, 1H), 1.55 – 1.49 (m, 1H), 1.47 – 1.41 (m, 1H), 1.41 – 1.33 (m, 3H), 1.32 (s, 3H), 1.30 – 1.24 (m, 1H), 1.26 (s, 3H), 1.00 (s, 3H), 0.84 (d, J = 7.1 Hz, 3H); The following figure is the carbon spectrum, detailed analysis is as follows: 13 C NMR (151MHz, CDCl3) δ 75.30, 61.19, 56.68, 54.26, 43.55, 42.13, 41.62, 37.17, 35.52,31.76, 30.33, 29.08, 27.79, 25.52, 15.74.

[0027] Figure 6 Infrared spectrum of cedrol; detailed resolution at 3458 cm⁻¹ -1 The absorption peak at 2954 cm⁻¹ is due to the stretching vibration of OH. -1 The absorption peaks at 2932 and 2866 cm⁻¹ are due to the stretching vibration of the methyl group. -1 The absorption peak at 1624 cm⁻¹ corresponds to the antisymmetric and symmetric stretching vibrations of the methylene group. -1 The absorption peak at 1459 cm⁻¹ is due to the in-plane bending vibration of water OH. -1 The absorption peaks at 1459 and 1369 cm⁻¹ are due to the antisymmetric variable-angle vibration of the methylene group. -1 The absorption peak at 1333 cm⁻¹ is due to the antisymmetric and symmetric angular vibrations of the methyl group. -1 The absorption peak at 1229 cm⁻¹ is due to the angular vibration of CH. -1 The absorption peak at 1130 cm⁻¹ is due to the rocking vibration of the methylene group. -1 The absorption peak at that point is due to the stretching vibration of CO.

[0028] Figure 7 The effect of cedrol on the activity of two types of human liver cancer cells; where A represents the IC50 of the two types of liver cancer cells. 50 Value; B represents the proliferation rate of two types of liver cancer cells.

[0029] Figure 8 The apoptosis rate of cedrol on two types of human liver cancer cells is shown in Figure 1; where A represents the number of apoptotic cells detected by TUNEL assay, and B represents the apoptosis rate.

[0030] Figure 9 This study detects the expression levels of key apoptosis proteins in two types of human liver cancer cells after treatment with cedrol; the left figure shows the Western blot (WB) analysis of the proteins, and the right figure shows the relative expression levels of the proteins. Detailed Implementation

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] The amino acid sequence of β-Himalayene is shown in SEQ ID NO.1.

[0033] MSNLMGDHISSLSSIPSNAFNQWDDAFIQSMETPYGEPEYRERAETLAKEIKIFLKDMQSGGGDGDLIERLEIVDALQCLGIDRYFQAEIKAALDYVYNCWDESVGIG LGSQSATKDLNATALALRVFRLNRYDVSADTLKYFKDNNGRFVLCGDNKDNNDEDNSKEEKVMRSMLNLLRLSSLAFPAEIVMEEAKAFSSRYLKELLGKSGDTSKKS FLKEVEYALIYEWPRTFIRWEARNFIEIYELDNERLKDKRILELAKLDFNILQFHYKLEMKNLSSWWVESEISKLIATRERSIEYLLWAISSMDELEHSSSRIALAKI TSLITILDDIFDDYATFEQLKCIRDAIFKGWDVSIIENIPNNWKRCMEFVFKTIHQLTIDATDYQGRDMMPFVSKAWEDYVEACFEQARWKLKGYFPTYNEYIKIAGKS S SEQ ID NO.1.

[0034] The nucleotide sequence of β-Himalayene is shown in SEQ ID NO.2.

[0035] tct ggcttcggtccattttttttgcactctgctattctggcttctccaaatttgagggacgatgacattcaaaagatttacttggacaagtctaggttctaccagttgatgagag tcgctatgagattgattgatgacattcacgactttgaggaggaaaggttgcacggtaaaatggcttctgctatttcttgttatatggccgaccacccaaattgttctgagaaa gaagctatgaaccacattattgagttgaacaacgaggtcctgaaggagttgactagggaatttttgaagccctctatgattttccatgagtgggagaaaattttcgtcaactc tactaggggtgtccagttcttttatgttcatggtgatggtttcacttacactcacaaagaaattaagcatcagattttgaagattattgtcgacccaattaaggtctga; SEQ ID NO.2.

[0036] Example 1: Homology modeling and molecular docking of β-Cedrusene synthase derived from Platycladus orientalis

[0037] The crystal structure of β-heliocedrene protein (as shown in SEQ ID NO.1) was predicted using AlphaFold3. The obtained protein crystals were processed using the Protein Preparation Wizard module of Schrödinger software for protein preprocessing, regenerating states of native FPP, H-bond assignment optimization, protein energy minimization, and water removal. The 2D SDF structure file of the compound farnesyl pyrophosphate (FPP) was processed using the LigPrep module in Schrödinger to generate all its 3D chiral conformations. The ReceptorGrid Generation module in Schrödinger was used to set the optimal enclosing box to perfectly encapsulate the ligand molecules in the protein crystal, and the active site of β-heliocedrene protein was obtained based on this. The processed ligand compound FPP was then molecularly docked with the active site of the target protein (using the highest precision XP docking). The docking results are shown below. Figure 1As shown, the results indicate that FPP penetrates deep into the active pocket of β-Himalayene protein, where residues such as PHE554 and TYR555 form hydrophobic interactions with FPP. This ligand forms one salt bridge and one hydrogen bond with residue LYS491, three salt bridges and one hydrogen bond with residue ARG475, and one metal coordination bond and two salt bridges with magnesium ions. Furthermore, the active pocket of β-Himalayene protein contains four polar amino acids, including THR325, THR329, SER433, and SER434 (…). Figure 1 A is labeled as a light blue amino acid.

[0038] Example 2: Construction of pSP-GM1-PoTPS1 Mutants Recombinant yeast plasmid

[0039] The positions of the four polar amino acids in the active pocket of β-Himalayene protein were analyzed. The SER434 site was closest to the substrate; therefore, a saturation mutagenesis was performed on this site. Primers were designed based on the β-Himalayene gene sequence (as shown in SEQ ID NO.2) to amplify the target gene. The primer sequences are as follows:

[0040] Primer pSP-GM1-PoTPS1 Mutants -F: TGGTAAGTCTNNKGGCTTCGGTCCATTTTTTTTTGCAC; SEQ ID NO.3.

[0041] Primer pSP-GM1-PoTPS1 Mutants -R: CCGAAGCCNNNKAGACTTACCAGCAATCTTAATATACT; SEQID NO.4.

[0042] Using the plasmid pSP-GM1-URA-PoTPS1 containing the encoding gene of the arborvitae sesquiterpene synthase PoTPS1 from the published patent (patent publication number: CN118853643A) as a template, PCR amplification was performed using two degenerate primers (SEQ ID NO.3 and SEQ ID NO.4) to saturate-mutate the SER434 site of PoTPS1. The specific procedure was as follows: the total reaction volume was 50 μL, with 25 μL of 2×PrimerStar Mix, 20 μL of ddH2O, and primer pSP-GM1-PoTPS1 added. Mutants -F 2 μL, primer pSP-GM1-PoTPS1 Mutants-R 2 μL, template 1 μL. Reaction conditions: 98 ℃ pre-denaturation for 3 min; 98 ℃ denaturation for 10 s, 60 ℃ annealing for 15 s, 72 ℃ extension for 2 min, for a total of 35 cycles from denaturation to extension; 72 ℃ for a final extension for 10 min. Finally, store at 4 ℃. After PCR, the products were detected by 1% agarose gel electrophoresis. After gel excision, the target fragment was recovered. The mutant plasmid fragment obtained after gel recovery was self-ligated using the Gibson homologous recombination method. The ligation system was 5 μL of the target mutant plasmid fragment and 5 μL of 2× Gibson Assemble Mix, ligated at 50 ℃ for 15 min.

[0043] The transformation was carried out using the chemical transformation method with E. coli, and the steps are as follows:

[0044] (1) Take Trans 10 competent cells and place them on ice to thaw. Add all 10 μL of the above ligation product and incubate on ice for 30 min.

[0045] (2) Heat shock at 42 ℃ for 30 s, followed immediately by ice bath for 2 min.

[0046] (3) Add 500 μL of antibiotic-free LB medium that has been pre-ice bathed, and revive at 37 ℃ and 180 rpm for 1 h.

[0047] (4) After the resuscitation is completed, centrifuge at 6000 rpm for 2 min, remove the supernatant, keep 100 μL, resuspend the bacterial cells, and spread them all on LB plates containing Amp resistance.

[0048] (5) Place in an incubator at 37 ℃ and incubate overnight for about 12 to 16 hours.

[0049] From the cultured plates, select all uniformly shaped single colonies from the solid medium and transfer them to liquid LB medium containing Amp resistance. Incubate at 37 °C and 180 rpm for approximately 12 h. Take 1 mL of the bacterial culture and verify it using first-generation sequencing. Select 19 mutants of SER434 with correct sequencing results for subsequent fermentation.

[0050] Nineteen mutants of SER434, whose amino acid sequence is SEQ ID NO.1, were obtained through the following mutations:

[0051] S434A, S434C, S434D, S434E, S434F, S434G, S434H, S434I, S434K, S434L, S434M, S434N, S434P, S434Q, S434R, S434T, S434V, S434W, S434Y.

[0052] Nineteen mutants of SER434 have nucleotide sequences in which the 1300-1302bp “tct” of SEQ ID NO.2 is replaced with the following: gca, tgc, gac, gag, ttt, gga, cac, att, aaa, ctg, atg, aat, cct, cag, aga, act, gtt, tgg, tac.

[0053] Example 3: Construction of YCP-00 / pSP-GM1-PoTPS1 (Mutants) recombinant yeast strain

[0054] The conversion was carried out using the electroconversion method with brewer's yeast, and the steps are as follows:

[0055] (1) Inoculate the Saccharomyces cerevisiae CEN.PK113-5D into 5 mL of sterile YPD medium and culture overnight at 30 °C and 200 rpm.

[0056] (2) The pre-cultured cells were seeded into 30 mL Erlenmeyer flasks containing YPD medium, and the initial OD was determined. 600 =0.3, incubated at 30 ℃ for about 9 h, until OD 600 =1.2 to 1.6 is sufficient.

[0057] (3) When the OD reaches the set value, pour the bacterial solution into a 50 mL sterile centrifuge tube and centrifuge at 4 ℃ and 3000 rpm for 3 min.

[0058] (4) Rinse once with 20 mL of pre-cooled sterile water and discard the supernatant.

[0059] (5) Wash once with 20 mL of pre-cooled sorbitol and discard the supernatant.

[0060] (6) Resuspend the cells in 16 mL of 1M sorbitol, add 2 mL of 10×TE and 2 mL of 10×LiAc, and revive at 30 °C and 200 rpm for 30 min.

[0061] (7) After adding 200 µL of 1M DTT, continue resuscitation for 15 min.

[0062] (8) After the recovery is complete, collect the bacterial cells by centrifugation at 3000 rpm for 3 min and wash them twice with 20 mL of sorbitol (to wash away DTT, otherwise it will affect the subsequent transformation efficiency).

[0063] (9) After centrifugation, remove all liquid as much as possible, resuspend the yeast cells in 200 μL of 1M sorbitol, and then dispense 50 μL into sterile EP tubes and place them on ice for later use.

[0064] (10) Gently add 1 μL of the recombinant plasmid constructed above into the prepared competent state, and transfer the liquid into a pre-cooled electroporation cup (0.2 cm gap).

[0065] (11) Place the electroporation cup in the electroporation apparatus, select program Sc2, 1.5 KV for electroporation, and immediately add 1 mL of 1M sorbitol to the electroporation cup after electroporation. After mixing the liquid, transfer the cells from the electroporation cup to 6 mL of mixture (3 mL YPD + 3 mL 1M sorbitol) and revive at 30 °C and 200 rpm for 1 h.

[0066] (12) After the resuscitation was completed, centrifuge at 3000 rpm at 4 ℃, discard the supernatant, resuspend in 1 mL of sterile water, take 100 µL and spread it on SC-Ura (ammonia-free yeast nitrogen source (YNB) 1.7 g / L, (NH4)2SO4 5 g / L, amino acid mixture 1.655 g / L, His 0.043 g / L, Leu 0.173 g / L, glucose 20 g / L, agar powder 25 g / L) plate, and incubate in a 30℃ incubator for 48-72 h.

[0067] The amino acid mixture consists of: adenine 0.5g, alanine 2.0g, arginine 2.0g, asparagine 2.0g, aspartic acid 2.0g, cysteine ​​2.0g, glutamine 2.0g, glutamic acid 2.0g, glycine 2.0g, inositol 2.0g, isoleucine 2.0g, lysine 2.0g, methionine 2.0g, para-aminobenzoic acid 0.2g, phenylalanine 2.0g, proline 2.0g, serine 2.0g, threonine 2.0g, tryptophan 2.0g, tyrosine 2.0g, and valine 2.0g.

[0068] Remove the cultured plate and randomly select uniformly shaped single colonies for colony PCR. The products are then analyzed by 1% agarose gel electrophoresis to check the band size. Figure 2 As shown, the results indicate that the selected transformants have a single band around 1800 bp, proving that the transformants are positive clones.

[0069] Example 4 Induced Fermentation

[0070] (1) The verified positive clones were inoculated into 5 mL of SC-Ura liquid medium and cultured overnight to obtain seed culture.

[0071] (2) OD 600 The seed culture with a concentration of 1.6 was inoculated into 50 mL of SC-Ura medium at a ratio of 1:50 and cultured at 30 °C and 200 rpm for 24 h.

[0072] (3) 24 h after inoculation, add 20% organic phase n-nonane to the fermentation broth and then continue to culture under the above conditions for 96 h.

[0073] (4) After fermentation is complete, take 200 μL of the organic phase n-nonane from the upper layer of the fermentation broth for GC-MS detection.

[0074] Example 5: GC-MS detection of fermentation and catalytic products

[0075] (1) GC-MS detection method

[0076] Column: HP-5 ms; Ion source: EI, 230 eV; Injection volume: 1 μL; Injection temperature: 250 ℃; Detector temperature: 325 ℃; Column temperature: 250 ℃; Temperature program: Initial temperature: 100 ℃, increase to 240 ℃ at 10 ℃ / min, hold for 5 min, then increase to 300 ℃ at 30 ℃ / min, hold for 5 min.

[0077] (2) Calculate the yield using the internal standard method. Prepare a 50 mg / L standard using tetradecane as the internal standard. Take 135 μL of the sample and 15 μL of 50 mg / L tetradecane to make the final concentration of tetradecane 5 mg / L. Calculate the sample yield based on the ratio of the peak area of ​​the product to that of the standard.

[0078] Test results as follows Figure 3 As shown, YCP-00 / pSP-GM1-PoTPS1 Mutant GC-MS images of recombinant yeast strain products; where A is the GC spectrum of fermentation products from wild-type strain YCP-00 / pSP-GM1-PoTPS1, producing two products: β-himachalene with a retention time of 7.63 min and cedrol with a retention time of 9.15 min. B is the GC spectrum of mutant strain YCP-00 / pSP-GM1-PoTPS1. S434D / E / G GC spectrum of fermentation product; strain inactivated, no product generated. C represents mutant strain YCP-00 / pSP-GM1-PoTPS1. S434A / C / F / H The GC spectrum of the fermentation product showed no change in product specificity, but the yield was significantly reduced. D represents the mutant strain YCP-00 / pSP-GM1-PoTPS1. S434I / L / N The GC chromatograms of the fermentation products showed product-specific alterations. In addition to the original two sesquiterpenes, two new sesquiterpenes were generated: β-farnesene (retention time 7.23 min) and α-farnesene (retention time 7.94 min). E represents the mutant strain YCP-00 / pSP-GM1-PoTPS1.S434K / M / P / Q / R / W / Y The GC spectrum of the fermentation products shows a change in product specificity, producing only α-Farnesene and β-Farnesene. F represents the mutant strain YCP-00 / pSP-GM1-PoTPS1. S434T The GC chromatograms of the fermentation products showed product-specific changes. In addition to the original two sesquiterpenes, six new sesquiterpenes were generated: β-farnesene (7.23 min retention), α-farnesene (7.94 min retention), α-cedrene (6.92 min retention), β-cedrene (7.11 min retention), β-curcumene (8.02 min retention), and acoradiene (7.56 min retention). G represents the mutant strain YCP-00 / pSP-GM1-PoTPS1. S434V The GC spectrum of the fermentation product showed a change in product specificity, producing only one sesquiterpene, cedrol, and the yield was significantly increased, rising from 0.56 mg / L in the wild type to [a much higher concentration in the mutant]. S434V 7.78 mg / L.

[0079] sesquiterpene synthase PoTPS1 mutant PoTPS1 S434V The amino acid sequence is shown in SEQ ID NO.5.

[0080] MSNLMGDHISSLSSIPSNAFNQWDDAFIQSMETPYGEPEYRERAETLAKEIKIFLKDMQSGGGDGDLIERLEIVDALQCLGIDRYFQAEIKAALDYVYNCWDESVGIGLGSQSATKDLNATALALRVFRLNRYDVSADTLKYFKDNNGRFVLCGDNKDNNDEDNSKEEKVMRSMLNLLRLSSLAFPAEIVMEEAKAFSSRYLKELLGKSGDTSKKSFLKEVEYALIYEWPRTFIRWEARNFIEIYELDNERLKDKRILELAKLDFNILQFHYKLEMKNLSSWWVESEISKLIATRERSIEYLLWAISSMDELEHSSSRIALAKITSLITILDDIFDDYATFEQLKCIRDAIFKGWDVSIIENIPNNWKRCMEFVFKTIHQLTIDATDYQGRDMMPFVSKAWEDYVEACFEQARWKLKGYFPTYNEYIKIAGKS V GFGPFFLHSAILASPNLRDDDIQKIYLDKSRFYQLMRVAMRLIDDIHDFEEERLHGKMASAISCYMADHPNCSEKEAMNHIIELNNEVLKELTREFLKPSMIFHEWEKIFVNSTRGVQFFYVHGDGFTYTHKEIKHQILKIIVDPIKV; SEQ ID NO.5。

[0081] The nucleotide sequence of the sesquiterpene synthase PoTPS1 mutant PoTPS1 S434V is shown in SEQ ID NO.6。

[0082] gtt ggcttcggtccattttttttgcactctgctattctggcttctccaaatttgagggacgatgacattcaaaagatttacttggacaagtctaggttctaccagttgatgagag tcgctatgagattgattgatgacattcacgactttgaggaggaaaggttgcacggtaaaatggcttctgctatttcttgttatatggccgaccacccaaattgttctgagaaa gaagctatgaaccacattattgagttgaacaacgaggtcctgaaggagttgactagggaatttttgaagccctctatgattttccatgagtgggagaaaattttcgtcaactc tactaggggtgtccagttcttttatgttcatggtgatggtttcacttacactcacaaagaaattaagcatcagattttgaagattattgtcgacccaattaaggtctga; SEQ ID NO.6.

[0083] Example 6 Preparation of pure cedrol

[0084] The fermentation broth and organic phase mixture from Example 4 were poured into a centrifuge tube and centrifuged at 4000 rpm to separate the fermentation broth from the organic phase n-nonane. After centrifugation, the upper organic phase was collected and evaporated using rotary evaporation at 50 °C to obtain a mixture of sesquiterpenes containing Cedrol (pale yellow liquid) for later use.

[0085] The separated sesquiterpene products (mixture) were purified. First, a silica gel column was prepared and pretreated with silver nitrate. Then, silver ion silica gel was packed into the column. The column was first flushed with n-nonane to flatten it and remove air. Next, the oily sesquiterpene liquid was added to the column, and the mixed sesquiterpene compounds were eluted with n-nonane. The liquid flowing down the column outlet was collected in a 1.5 mL centrifuge tube, and a sample was taken for GC-MS analysis to determine the product purity. The GC chromatogram of the pure product is shown below. Figure 4 As shown, the purity is greater than 99%.

[0086] Example 7 Characterization of cedrol structure

[0087] (1) Nuclear magnetic resonance detection

[0088] Nuclear magnetic resonance (NMR) data were recorded using CDCl3 as solvent on a 600 MHz NMR spectrometer. Chemical shifts were referenced to tetramethylsilane (TMS) and reported in ppm. Proton chemical shifts (…) 1 (H NMR) reference to the proton resonance of CHCl3, carbon chemical shift ( 13 C10 NMR (referencing CDCl3 carbon resonance). Data include chemical shifts, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplicity, br = broad), coupling constant (Hz), and integral values. Results are as follows: Figure 5 As shown.

[0089] (2) Infrared spectroscopy characterization

[0090] The absorption and reflectance spectra of the target sesquiterpenes were analyzed using Fourier transform infrared spectroscopy (FTIR) on a Nicolet 6700 spectrometer. The chemical composition and structure of the purified samples were directly determined. Results are as follows: Figure 6 As shown.

[0091] Example 8: Drug toxicity assay of cedrol against human hepatocellular carcinoma cells HepG2 and SK-Hep-1

[0092] The CCK8 assay was used to detect the toxicity of the samples to experimental cells and the IC50. 50 The values ​​and cedrol concentrations are grouped as shown in Table 1.

[0093] Table 1

[0094]

[0095] The experimental procedure is as follows:

[0096] (1) Cells in the logarithmic growth phase (HepG2 and SK-Hep-1) were digested with trypsin, and then seeded into 96-well plates and cultured in an incubator at 37 ℃.

[0097] (2) After the cells adhere to the wall, change the culture medium (to MEM (containing NEAA) medium with 10% FBS) and starve the cells for 12 h.

[0098] (3) Replace the culture medium with different concentration gradients of cedrol (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM) diluted with the above culture medium, and culture for 72 h.

[0099] (4) After 72 h of incubation, 10 μL of CCK-8 solution was added to each well of each experimental group in turn, and then they were incubated in a 37 ℃ incubator for no less than 1 h and no more than 4 h.

[0100] (5) The absorbance was measured at a wavelength of 450 nm using an ELISA reader, and the effect of the drug on cell viability was calculated using the following formula:

[0101] Proliferation rate % = (Experimental group - blank) / (Control group - blank) × 100%

[0102] blank: absorbance of the culture medium; experimental group: absorbance of cells after intervention with different concentrations of cedrol (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM); control group: absorbance of cells after intervention with 0 μM cedrol.

[0103] Experimental results are as follows Figure 7 As shown, the results indicate that the viability of both types of human liver cancer cells decreased with increasing drug concentration, suggesting drug dependence. The IC50 values ​​for the drug were also determined for both types of liver cancer cells. 50 The values ​​were 20.19 and 23.86 μM, respectively. Figure 7 A). Simultaneously select IC 50 Cell viability tests were conducted at a concentration of one-quarter of the recommended concentration, and it was found that cell proliferation treated with cedrol was significantly inhibited in a concentration- and time-dependent manner, and both concentrations reduced the activity of two types of human liver cancer cells. Figure 7 B).

[0104] Example 9

[0105] TUNEL staining was used to detect cell apoptosis rate. Experimental groups are shown in Table 2.

[0106] Table 2

[0107]

[0108] After pretreatment, cells and tissue sections from each group were washed once with PBS, fixed with 4% paraformaldehyde for 30 min, washed again with PBS, and then incubated at room temperature for 5 min with PBS solution containing 0.3% Triton X-100. They were then washed three times with PBS, and 50 μl of TUNEL assay solution (TdT enzyme and fluorescent labeling solution prepared at a 1:9 ratio, Beyotime One-Step TUNEL Apoptosis Detection Kit - C1088) was added and incubated at 37 ℃ in the dark for 1 h. After washing three times with PBS, the slides were mounted with anti-fluorescence quenching mounting medium and observed and photographed under a fluorescence microscope. Experimental results are as follows: Figure 8 As shown, the results indicated that the number of apoptotic cells was significantly increased in the two types of liver cancer cells treated with cedrol, suggesting that cedrol promotes apoptosis. Figure 8A). Cedarol increased the apoptosis rate of HepG-2 cells from 3.18% to 15.68%; and increased the apoptosis rate of SK-Hep-1 cells from 2.61% to 17.08%. Figure 8 B).

[0109] Example 10: Western blot detection of the expression of key apoptosis proteins in cells.

[0110] Perform the experimental procedures for each group of cells in Table 2 according to the following steps:

[0111] (1) Total protein extraction

[0112] Total cell protein extraction: Cells in the logarithmic growth phase were extracted using a 1×10⁻⁶ protein solution. 6 Cells were seeded into each well of a 6-well plate. After cell attachment, the cells were treated according to their assigned groups. After the intervention, the 6-well plates were removed, cells were collected, and the cell samples were washed twice with pre-chilled PBS. 0.5 mL of RIPA lysis buffer with a final concentration of 1 mM PMSF was added to each well. After complete lysis, the cells were centrifuged at 12000 g for 5 min at 4 °C. The supernatant was immediately transferred to a pre-chilled EP tube, which contained the extracted cell proteins. The extracted proteins were stored at -80 °C for later use.

[0113] (2) Protein quantification using the BCA method

[0114] Based on the sample quantity, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mixing thoroughly. Completely dissolve the protein standard, and dilute 10 μL of the protein standard to 100 μL with PBS to achieve a final concentration of 0.5 mg / mL. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the wells of a 96-well plate, and then add dilution solution to bring the total volume to 20 μL. Add an appropriate volume of sample to the sample wells of the 96-well plate. If the sample volume is less than 20 μL, add standard diluent to bring the total volume to 20 μL. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance of each well at 562 nm, plot a standard curve, and finally calculate the protein concentration of the sample using a formula, in mg / mL. Finally, add 5× Loading Buffer (to make the final concentration 1×) and boil in a water bath for 10 min. The sample preparation is complete and can be stored at -20℃.

[0115] (3) Gel electrophoresis, membrane transfer, blocking and antibody incubation

[0116] Prepare a 12% separating gel and a 5% stacking gel, and pour the SDS-PAGE gel. Add an appropriate amount of pre-chilled 1× electrophoresis buffer, then add the previously extracted total cell protein extract and pre-stained protein markers to the lanes. Electrophoresis is performed at a constant voltage of 80 V for approximately 30 min. After the sample enters the separating gel, the voltage is adjusted to 120 V and electrophoresis continues. Electrophoresis is stopped when the target band reaches the appropriate position (refer to the position of the pre-stained protein markers). Cut a PVDF membrane to the size of the gel, activate it in methanol for 1 min, and then soak it in transfer buffer. Filter paper is also soaked in transfer buffer for 15 min. Prepare a transfer "sandwich" (i.e., gel on the negative electrode side, membrane on the positive electrode side) according to the principle of filter paper ≥ PVDF membrane ≥ gel ≥ filter paper. Ensure air bubbles are removed before starting constant voltage transfer. After transfer, wash with TBST for 2 min. At room temperature, the membrane was blocked by shaking with rapid blocking buffer on a shaker for 15 min, followed by overnight incubation with primary antibody at 4°C. The membrane was washed three times with TBST for 15 min each time. Then, secondary antibody was added, and the membrane was incubated at room temperature for 2 h. The membrane was washed three times with TBST for 15 min each time.

[0117] (4) Chemiluminescence, development, and fixing

[0118] Equal volumes of chemiluminescence reagents A and B (China); ECL luminescence solution AB (P2100) (NCMBiotech, China) were mixed, and the PVDF membrane protein side down was brought into full contact with this mixture. After 3 min, the PVDF membrane was detected using a JP-K6000 chemiluminescence imager. Protein expression levels were analyzed using ImageJ software based on optical density values, and the relative protein expression level was calculated as the gray value of the target protein / the gray value of the internal reference protein.

[0119] The antibody dilution factors are shown in Table 3.

[0120] Table 3

[0121]

[0122] Experimental results are as follows Figure 9 As shown, the results indicate that the expression levels of the three apoptosis proteins were significantly upregulated in both HepG2 and SK-Hep-1 liver cancer cells, suggesting that cedrol promotes apoptosis in liver cancer cells.

[0123] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A β-Himalayene synthase mutant, characterized in that, The amino acid sequence of the β-Himalayene synthase mutant is shown in SEQ ID NO.

5.

2. The biomaterials related to the β-Himalayene synthase mutant according to claim 1, characterized in that, It is at least one of the following (1)-(4): (1) Nucleic acid molecules encoding β-Himalayene synthase mutants; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1) or a recombinant vector containing the expression cassette described in (2); (4) A recombinant microorganism containing the nucleic acid molecule described in (1), a recombinant microorganism containing the nucleic acid molecule of the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3).

3. The application of the β-Himalayene synthase mutant of claim 1 or the related biomaterials of claim 2 in increasing cedrol production.

4. The application of the β-Himalayene synthase mutant of claim 1 or the related biomaterial of claim 2 in the production of cedrol.

5. The application according to claim 3 or 4, characterized in that, The cedrol is used to prepare an anti-tumor drug; the tumor is liver cancer.

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