Application of beta-camptothecin synthase mutant and product cedrol thereof

Abstract

The invention discloses an application of a beta-limakedarene synthase mutant and a product cedrol thereof, and belongs to the technical fields of enzyme engineering, gene engineering and medicines. The invention discloses a beta-limadeodar alkene synthase mutant, which is S434V, and the amino acid sequence of the beta-limadeodar alkene synthase mutant is as shown in SEQ ID NO. 5. The invention not only provides a biosynthesis method for efficiently producing cedrol, but also provides a potential candidate compound for developing a novel anti-hepatoma drug, and has important theoretical significance and application value.

Description

Technical Field

[0001] The present invention relates to the fields of enzyme engineering, genetic engineering and medical technology, and more particularly to applications of a beta-himacedrene synthase mutant and its product, cedrol. Background Art

[0002] Cedrol is a naturally occurring sesquiterpene alcohol with the molecular formula C 15 H 26 O, with two isomers, is widely distributed throughout the plant kingdom and is prevalent in conifers such as Atlantic cedar (Cedrus atlantica). Cedrol has been reported to possess a variety of biological activities, including antibacterial, anti-inflammatory, hair growth promoting, analgesic, and anxiolytic activities. In recent years, numerous 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] At present, the acquisition of cedarol mainly relies on organic chemical synthesis and plant extraction, but both methods have some defects and shortcomings. The content of plant extracts is usually low, and the over-exploitation of natural resources will lead to excessive resource consumption, which poses a great challenge and threat to the ecological environment. Chemical synthesis methods usually use strong acids and strong bases, and the conditions are special. High temperature and high pressure conditions are inevitable, the cost is high, and it is very easy to cause environmental problems, making it difficult to practice the principle of green production. Compared with the above two methods, microbial fermentation has become an alternative method with great prospects for industrial application. Compared with the above two methods, the bioenzymatic method for synthesizing cedarol has the advantages of safety, efficiency, low cost and environmental friendliness, but there are few relevant literature disclosures. Therefore, if a bioenzyme with stronger catalytic ability can be developed for the production of these two sesquiterpenoid compounds, it will greatly improve the industrial preparation efficiency of β-hima-cedrene and cedarol, which is of great significance to their market development.

[0004] Liver cancer is a major global health threat, with persistently high morbidity and mortality rates. According to the latest statistics from the World Health Organization's International Agency for Research on Cancer (IARC), liver cancer has become the sixth most common cancer and the third leading cause of cancer-related death worldwide, with over 900,000 new cases expected worldwide in 2024. This dire epidemiological situation highlights the urgency of strengthening liver cancer prevention and treatment. In the field of anticancer drug research and development, cedarwood has attracted considerable attention due to its broad-spectrum antitumor activity, offering promising application prospects in liver cancer treatment.

[0005] Therefore, providing a β-himacadrene synthase mutant and the application of its product cedrol is an urgent problem to be solved by those skilled in the art. Summary of the Invention

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

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The mutant of β-himacadrene synthase is S434V; The amino acid sequence of the β-himacadrene synthase mutant is shown in SEQ ID NO.5; The amino acid sequence of the β-himacadrene synthase is shown in SEQ ID NO.1.

[0008] Furthermore, the biological material related to the β-himacladusin synthase mutant is at least one of the following (1)-(4): (1) a nucleic acid molecule encoding a mutant of β-himacladus synthase; (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 nucleic acid molecule 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).

[0009] Furthermore, the β-himacadrene synthase mutant or the related biological material is used to increase the yield of cedarol.

[0010] Furthermore, the β-himacadrene synthase mutant or the related biological material is used in the production of cedar.

[0011] Furthermore, the cedrol is used in the preparation of anti-tumor drugs.

[0012] Furthermore, the tumor is liver cancer.

[0013] As can be seen from the above technical solutions, compared to the prior art, the present invention provides a mutant β-himacadrene synthase and its product, cedrol. By performing site-directed mutagenesis (SER434→VAL434) on a key site in the wild-type PoTPS1 enzyme, a mutant PoTPS1 (S434V) with novel catalytic function 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, converting the primary metabolite from the original β-himacadrene to the more valuable cedrol. By establishing an efficient separation and purification process, high-purity cedrol was obtained from cultures of the recombinant strain YCP00 / pSPGM1-PoTPS1 (S434V). In vitro anti-tumor experiments demonstrated that the compound exhibited significant anti-proliferative activity against both HepG2 and SK-Hep-1 human liver cancer cell lines. Further mechanistic studies revealed that cedarol can promote apoptosis in liver cancer cells. This invention not only provides a biosynthetic method for the efficient production of cedarol but also provides a potential candidate compound for the development of new anti-liver cancer drugs, possessing significant theoretical and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0015] Figure 1 The diagram shows the molecular docking results of β-himacladus synthase PoTPS1 and the substrate small molecule FPP; A: 2D display diagram, B: 3D display diagram.

[0016] Figure 2 YCP-00 / pSP-GM1-PoTPS1 Mutant DNA agarose gel map of recombinant yeast strain verification (target band size is 1746 bp); where M is a standard DNA molecular ruler; lanes 1-20 are wild-type PoTPS1, PoTPS1 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 Genetic DNA samples.

[0017] Figure 3 For the recombinant strains YCP-00 / pSP-GM1-PoTPS1 and YCP-00 / pSP-GM1-PoTPS1 Mutant GC spectra of fermentation products; Figure A is the wild-type PoTPS1; Figure B is 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 Figure G shows PoTPS1 S434V .

[0018] Figure 4 This is the GC detection chart of pure cedarol; the retention time of cedarol is 9.15 min.

[0019] Figure 5 This is the nuclear magnetic resonance characterization diagram of cedarol; the upper figure is the hydrogen spectrum, and the detailed analysis is 1H 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 figure below is the carbon spectrum, with detailed analysis 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.

[0020] Figure 6 This is the infrared spectrum of cedarol; the detailed analysis is 3458cm -1 The absorption peak at 2954 cm is the stretching vibration of OH. -1 The absorption peaks at 2932 and 2866 cm are the stretching vibrations of methyl groups. -1 The absorption peaks at 1624 cm-1 are the antisymmetric and symmetric stretching vibrations of methylene. -1 The absorption peak at 1459 cm is the in-plane bending vibration of water OH. -1 The absorption peaks at 1459 and 1369 cm are the antisymmetric vibrations of methylene. -1 The absorption peaks at 1333 cm are the antisymmetric and symmetric vibrations of methyl groups. -1 The absorption peak at 1229 cm is the angle-shifting vibration of CH. -1 The absorption peak at 1130 cm is the rocking vibration of methylene. -1 The absorption peak at is the stretching vibration of CO.

[0021] Figure 7 is the effect of cedrol on the activity of two human liver cancer cells; A is the IC of two liver cancer cells 50 Value; B is the proliferation rate of two types of liver cancer cells.

[0022] Figure 8 is the apoptosis rate of two human liver cancer cells treated with cedrol; A is the number of apoptotic cells detected by TUNEL method; B is the apoptosis rate.

[0023] Figure 9 The expression levels of key apoptosis proteins in two human liver cancer cells after cedrol treatment are detected; the left picture is the protein WB detection picture; the right picture is the relative expression content of the protein. DETAILED DESCRIPTION

[0024] 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.

[0025] The amino acid sequence of β-himacadrene is shown in SEQ ID NO.1.

[0026] MSNLMGDHISSLSSIPSNAFNQWDDAFIQSMETPYGEPEYRERAETLAKEIKIFLKDMQSGGGDGDLIERLEIVDALQCLGIDRYFQAEIKAALDYVYNCWDESVGIG LGSQSATKDLNATALALRVFRLNRYDVSADTLKYFKDNNGRFVLCGDNKDNNDEDNSKEEKVMRSMLNLLRLSSLAFPAEIVMEEAKAFSSRYLKELLGKSGDTSKKS FLKEVEYALIYEWPRTFIRWEARNFIEIYELDNERLKDKRILELAKLDFNILQFHYKLEMKNLSSWWVESEISKLIATRERSIEYLLWAISSMDELEHSSSRIALAKI TSLITILDDIFDDYATFEQLKCIRDAIFKGWDVSIIENIPNNWKRCMEFVFKTIHQLTIDATDYQGRDMMPFVSKAWEDYVEACFEQARWKLKGYFPTYNEYIKIAGKS S SEQ ID NO.1.

[0027] The nucleotide sequence of β-himacadrene is shown in SEQ ID NO.2.

[0028] tct ggcttcggtccattttttttgcactctgctattctggcttctccaaatttgagggacgatgacattcaaaagatttacttggacaagtctaggttctaccagttgatgagag tcgctatgagattgattgatgacattcacgactttgaggaggaaaggttgcacggtaaaatggcttctgctatttcttgttatatggccgaccacccaaattgttctgagaaa gaagctatgaaccacattattgagttgaacaacgaggtcctgaaggagttgactagggaatttttgaagccctctatgattttccatgagtgggagaaaattttcgtcaactc tactaggggtgtccagttcttttatgttcatggtgatggtttcacttacactcacaaagaaattaagcatcagattttgaagattattgtcgacccaattaaggtctga; SEQ ID NO.2.

[0029] Example 1 Homology Modeling and Molecular Docking of β-Himaecedrene Synthase from Platycladus orientalis AlphaFold3 was used to predict the crystal structure corresponding to β-himacefedrine protein (as shown in SEQ ID NO.1). The obtained protein crystals were processed by Protein Preparation Wizard module of Schrödinger software for protein preprocessing, regenerate states of native FPP, H-bond assignment optimization, protein energy minimization, and remove waters. The 2D sdf structure file of the compound farnesyl pyrophosphate (FPP) was processed by LigPrep module in Schrödinger to generate all its 3D chiral conformations. The ReceptorGrid Generation module in Schrödinger was used to set the most appropriate Enclosing box to perfectly encapsulate the ligand molecule in the protein crystal, and on this basis, the active site of β-himacefedrine protein was obtained. The processed ligand compound FPP was molecularly docked with the active site of the target protein (using the highest precision XP docking). The docking results are shown as follows. Figure 1As shown, the results show that FPP penetrates deep into the active pocket of β-himaquinone cedrene protein, and the protein residues PHE554, TYR555, etc. form hydrophobic forces on FPP. The ligand forms a salt bridge and a hydrogen bond with residue LYS491, three salt bridges and a hydrogen bond with residue ARG475, and a metal coordination bond and two salt bridges with magnesium ions. In addition, there are four polar amino acids in the active pocket of β-himaquinone cedrene protein, including THR325, THR329, SER433, SER434 ( Figure 1 A amino acids are labeled in light blue).

[0030] Example 2 Construction of pSP-GM1-PoTPS1 Mutants Recombinant yeast plasmid The positions of the four polar amino acids in the active pocket of β-himacadrene protein were analyzed. SER434 was found to be closest to the substrate, so a saturation mutation was performed on this site. Based on the β-himacadrene gene sequence (shown in SEQ ID NO. 2), primers were designed to amplify the target gene. The primer sequences are as follows:

[0031] Primer pSP-GM1-PoTPS1 Mutants -F: TGGTAAGTCTNNKGGCTTCGGTCCATTTTTTTTTGCAC; SEQ ID NO.3.

[0032] Primer pSP-GM1-PoTPS1 Mutants -R: CCGAAGCCNNNKAGACTTACCAGCAATCTTAATATACT; SEQID NO.4.

[0033] Using the plasmid pSP-GM1-URA-PoTPS1, which contains the gene encoding the sesquiterpene synthase PoTPS1 from a published patent (Patent Publication No. CN118853643A), as a template, PCR amplification was performed using two degenerate primers, SEQ ID NO. 3 and SEQ ID NO. 4, to perform saturation mutagenesis at the SER434 site of PoTPS1. The specific steps were as follows: 25 μL of 2× PrimerStar Mix, 20 μL of ddH2O, and primers pSP-GM1-PoTPS1 were added to the 50 μL reaction system. Mutants -F 2 μL, primer pSP-GM1-PoTPS1 Mutants-R 2 μL, template 1 μL. Reaction conditions were 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 60°C annealing for 15 s, and 72°C extension for 2 min, for a total of 35 cycles; and 72°C extension for an additional 10 min. The final reaction was stored at 4°C. After PCR, the product was electrophoresed on a 1% agarose gel. After gel excision, the target fragment was recovered. The mutant plasmid fragment obtained from gel recovery was self-ligated using the Gibson homologous recombination method: 5 μL of the target mutant plasmid fragment and 5 μL of 2× Gibson Assemble Mix were ligated at 50°C for 15 min.

[0034] The transformation was performed using the E. coli chemical transformation method, and the steps were as follows: (1) Thaw the Trans 10 competent cells on ice, add 10 μL of the ligation product, and incubate on ice for 30 minutes.

[0035] (2) Heat shock at 42°C for 30 seconds, followed by immediate ice bath for 2 minutes.

[0036] (3) Add 500 μL of antibiotic-free LB medium that has been pre-bathed on ice and shake at 37°C and 180 rpm for 1 h.

[0037] (4) After recovery, centrifuge at 6000 rpm for 2 min, remove the supernatant, retain 100 μL, resuspend the bacteria, and spread all on the LB plate containing Amp resistance.

[0038] (5) Place in a 37°C incubator and culture overnight for approximately 12 to 16 hours.

[0039] From the plate, select all uniformly morphological colonies from the solid medium and transfer them to liquid LB medium containing Amp resistance. Culture the culture at 37°C, shaking at 180 rpm for approximately 12 hours. Take 1 mL of the bacterial culture and verify it through first-generation sequencing. Select 19 mutants of SER434 with correct sequencing for subsequent fermentation.

[0040] The 19 mutants of SER434, whose amino acid sequence is SEQ ID NO.1, were obtained by the following mutations: S434A, S434C, S434D, S434E, S434F, S434G, S434H, S434I, S434K, S434L, S434M, S434N, S434P, S434Q, S434R, S434T, S434V, S434W, S434Y.

[0041] The nucleotide sequences of the 19 mutants of SER434 are as follows: 1300-1302 bp "tct" of SEQ ID NO.2 is replaced by: gca, tgc, gac, gag, ttt, gga, cac, att, aaa, ctg, atg, aat, cct, cag, aga, act, gtt, tgg, tac, respectively.

[0042] Example 3 Construction of YCP-00 / pSP-GM1-PoTPS1 (Mutants) recombinant yeast strain Transformation was performed using the Saccharomyces cerevisiae electroporation method, as follows: (1) Inoculate 5 mL of sterile YPD medium with Saccharomyces cerevisiae CEN.PK113-5D and culture overnight at 30°C and 200 rpm.

[0043] (2) Inoculate the pre-cultured cells into a 30 mL conical flask of YPD medium, with an initial OD 600 = 0.3, cultured at 30 °C for about 9 h until OD 600 =1.2~1.6 is sufficient.

[0044] (3) When the OD reaches the set value, pour the bacterial solution into a 50 mL sterile centrifuge tube and centrifuge at 3000 rpm at 4°C for 3 min.

[0045] (4) Wash once with 20 mL of pre-cooled sterile water and discard the supernatant.

[0046] (5) Wash once with 20 mL of pre-cooled sorbitol and discard the supernatant.

[0047] (6) Resuspend the cells in 16 mL of 1 M sorbitol, add 2 mL of 10×TE and 2 mL of 10×LiAc, and resuspend at 30°C, 200 rpm for 30 min.

[0048] (7) Add 200 μL of 1M DTT and continue recovery for 15 min.

[0049] (8) After recovery, collect the bacterial cells by centrifugation at 3000 rpm for 3 min and wash them twice with 20 mL of sorbitol (wash out the DTT, otherwise it will affect the subsequent transformation efficiency).

[0050] (9) After centrifugation, remove as much liquid as possible and resuspend the yeast cells in 200 μL of 1 M sorbitol. Then, aliquot 50 μL into sterile EP tubes and place on ice until ready for use.

[0051] (10) Gently add 1 μL of the recombinant plasmid constructed above to the prepared competent medium and transfer the liquid to a pre-cooled electroporation cuvette (0.2 cm gap).

[0052] (11) Place the electroporation cup in the electroporator tank, select program Sc2, and perform electroporation at 1.5 KV. Immediately after electroporation, add 1 mL of 1 M sorbitol to the electroporation cup. After the liquid is mixed, transfer the cells from the electroporation cup to 6 mL of a mixture (3 mL YPD + 3 mL 1 M sorbitol) and resuscitate at 30 °C and 200 rpm for 1 h.

[0053] (12) After recovery, centrifuge at 3000 rpm at 4°C, discard the supernatant, resuspend in 1 mL of sterile water, and spread 100 μL on SC-Ura plates (amino-free yeast nitrogen base (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), and culture in a 30°C incubator for 48 to 72 h.

[0054] Amino acid mixture composition: 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, p-aminobenzoic acid 0.2g, phenylalanine 2.0g, proline 2.0g, serine 2.0g, threonine 2.0g, tryptophan 2.0g, tyrosine 2.0g, valine 2.0g.

[0055] Take out the cultured plate and randomly select single colonies with uniform morphology for colony PCR. The products are tested by 1% agarose gel electrophoresis to check whether the band size is correct. Figure 2 As shown, the results showed that the selected transformant had a single band at about 1800 bp, proving that the transformant was a positive clone.

[0056] Example 4 Induced fermentation (1) Inoculate the verified positive clones into 5 mL of SC-Ura liquid medium and culture overnight to obtain seed solution.

[0057] (2) OD 600 The 1.6% seed solution was inoculated into 50 mL SC-Ura medium at a ratio of 1:50 and cultured at 30°C and 200 rpm for 24 h.

[0058] (3) 24 h after inoculation, 20% organic phase n-nonane was added to the fermentation broth, and then the culture was continued under the above conditions for 96 h.

[0059] (4) After the fermentation is completed, 200 μL of the organic phase of n-nonane in the upper layer of the fermentation liquid is collected for GC-MS analysis.

[0060] Example 5 GC-MS detection of fermentation and catalytic products (1) GC-MS detection method Chromatographic 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, maintain for 5 min, then increase to 300 ℃ at 30 ℃ / min, maintain for 5 min.

[0061] (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 sample and mix with 15 μL of 50 mg / L tetradecane to achieve a final tetradecane concentration of 5 mg / L. Calculate the sample yield based on the peak area ratio of the product to the standard.

[0062] Test results such as Figure 3 As shown, YCP-00 / pSP-GM1-PoTPS1 Mutant GC-MS images of recombinant yeast strain products; A is the GC spectrum of the fermentation product of the wild-type strain YCP-00 / pSP-GM1-PoTPS1, which produces 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 the mutant strain YCP-00 / pSP-GM1-PoTPS1 S434D / E / G GC spectrum of fermentation products. The strain is inactivated and no product is produced. C is the mutant strain YCP-00 / pSP-GM1-PoTPS1 S434A / C / F / H GC profile of fermentation products. Product specificity remains unchanged, but the yield is greatly reduced. D is the mutant strain YCP-00 / pSP-GM1-PoTPS1 S434I / L / N The GC spectrum of the fermentation product shows that the product specificity has changed. In addition to the original two sesquiterpenes, two new sesquiterpenes were generated: β-Farnesene with a retention time of 7.23 min and α-Farnesene with a retention time of 7.94 min. E is the mutant strain YCP-00 / pSP-GM1-PoTPS1 S434K / M / P / Q / R / W / YGC spectrum of fermentation products. Product specificity changed, only α-Farnesene and β-Farnesene were produced. F is the mutant strain YCP-00 / pSP-GM1-PoTPS1. S434T The GC profile of the fermentation product showed a change in product specificity. In addition to the original two sesquiterpenes, six new sesquiterpenes were generated, namely β-Farnesene with a retention time of 7.23 min, α-Farnesene with a retention time of 7.94 min, α-Cedrene with a retention time of 6.92 min, β-Cedrene with a retention time of 7.11 min, β-Curcumene with a retention time of 8.02 min, and Acoradiene with a retention time of 7.56 min. G is the mutant strain YCP-00 / pSP-GM1-PoTPS1 S434V The GC spectrum of the fermentation product shows that the product specificity has changed, and only one sesquiterpenoid, cedrol, is produced. The yield is greatly increased from 0.56 mg / L of the wild type to 0.56 mg / L of the mutant. S434V of 7.78mg / L.

[0063] Sesquiterpene synthase PoTPS1 mutant PoTPS1 S434V The amino acid sequence is shown in SEQ ID NO.5.

[0064] MSNLMGDHISSLSSIPSNAFNQWDDAFIQSMETPYGEPEYRERAETLAKEIKIFLKDMQSGGGDGDLIERLEIVDALQCLGIDRYFQAEIKAALDYVYNCWDESVGIG LGSQSATKDLNATALALRVFRLNRYDVSADTLKYFKDNNGRFVLCGDNKDNNDEDNSKEEKVMRSMLNLLRLSSLAFPAEIVMEEAKAFSSRYLKELLGKSGDTSKKS FLKEVEYALIYEWPRTFIRWEARNFIEIYELDNERLKDKRILELAKLDFNILQFHYKLEMKNLSSWWVESEISKLIATRERSIEYLLWAISSMDELEHSSSRIALAKI TSLITILDDIFDDYATFEQLKCIRDAIFKGWDVSIIENIPNNWKRCMEFVFKTIHQLTIDATDYQGRDMMPFVSKAWEDYVEACFEQARWKLKGYFPTYNEYIKIAGKSV SEQ ID NO.5.

[0065] Sesquiterpene synthase PoTPS1 mutant PoTPS1 S434V The nucleotide sequence is shown in SEQ ID NO.6.

[0066] gtt ggcttcggtccattttttttgcactctgctattctggcttctccaaatttgagggacgatgacattcaaaagatttacttggacaagtctaggttctaccagttgatgagag tcgctatgagattgattgatgacattcacgactttgaggaggaaaggttgcacggtaaaatggcttctgctatttcttgttatatggccgaccacccaaattgttctgagaaa gaagctatgaaccacattattgagttgaacaacgaggtcctgaaggagttgactagggaatttttgaagccctctatgattttccatgagtgggagaaaattttcgtcaactc tactaggggtgtccagttcttttatgttcatggtgatggtttcacttacactcacaaagaaattaagcatcagattttgaagattattgtcgacccaattaaggtctga; SEQ ID NO.6.

[0067] Example 6 Preparation of Pure Cedarol The fermentation broth and organic phase mixture from Example 4 was poured into a centrifuge tube and centrifuged at 4000 rpm to separate the fermentation broth and the organic n-nonane phase. The upper organic phase was removed by rotary evaporation at 50°C to remove the Cedrol-containing sesquiterpene product mixture (a pale yellow liquid) for later use.

[0068] The separated sesquiterpene product (mixture) is purified by first preparing a silica gel column, pre-treating the silica gel column with silver nitrate, and then loading the silver ion silica gel into the column. First, pass n-nonane through the column to flatten the column and expel the gas. Then, add the sesquiterpene oily liquid to the column and elute with n-nonane to separate the mixed sesquiterpene compounds. The liquid flowing down is collected in a 1.5mL centrifuge tube at the outlet of the column, and a sample is taken to test the product purity using GC-MS. The GC chromatogram of the pure product is as follows: Figure 4 As shown, the purity is greater than 99%.

[0069] Example 7 Structural Characterization of Cedarol (1) Nuclear magnetic resonance imaging NMR data were recorded on a 600 MHz NMR spectrometer using CDCl3 as solvent. Chemical shifts were referenced to tetramethylsilane (TMS) and reported in ppm. Proton chemical shifts ( 1H NMR) refers to the proton resonance of CHCl3, carbon chemical shift ( 13 C NMR) is referenced to the carbon resonances of CDCl3. Data include chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant (Hz), and integral value. Results are as follows Figure 5 shown.

[0070] (2) Infrared spectroscopy characterization Fourier transform infrared spectroscopy (FTIR) was used to analyze the absorption and reflectance spectra of the target sesquiterpenes using a Nicolet 6700 spectrometer. The chemical composition and structure of the purified samples were directly determined. Figure 6 shown.

[0071] Example 8 Determination of the toxicity of cedarol on human liver cancer cells HepG2 and SK-Hep-1 CCK8 assay was used to detect the toxicity of samples to experimental cells and IC 50 The values and cedarol concentration groups are shown in Table 1.

[0072] Table 1

[0073] The experimental steps are as follows: (1) Cells (HepG2 and SK-Hep-1) in the logarithmic growth phase were digested with trypsin, and then the cells were seeded into 96-well plates and cultured in a 37°C incubator.

[0074] (2) After the cells adhered, the culture medium was replaced with MEM (containing NEAA) containing 10% FBS, and the cells were starved for 12 h.

[0075] (3) The culture medium was replaced with cedrol at different concentrations (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM) diluted in the above culture medium and cultured for 72 h.

[0076] (4) After 72 h of culture, add 10 μL of CCK-8 solution to the wells of each experimental group in turn, and then incubate them in a 37 °C incubator for no less than 1 h and no more than 4 h.

[0077] (5) At a wavelength of 450 nm, the absorbance was measured using an enzyme-labeled instrument, and the effect of the drug on cell viability was calculated using the following formula: Proliferation rate % = (experimental group - blank) / (control group - blank) × 100% Blank: absorbance value of culture medium; Experimental group: absorbance value of cells after intervention with different concentrations of cedarol (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM); Control group: absorbance value of cells after intervention with 0 μM cedarol.

[0078] The experimental results are as follows Figure 7 As shown in the figure, the results showed that with the increase of drug concentration, the survival activity of the two human liver cancer cells became lower and lower, indicating that the cells were drug-dependent, and the IC values of the two liver cancer cells were determined. 50 values, which were 20.19 and 23.86 μM ( Figure 7 A). Select IC at the same time 50 The cell activity test was conducted at a concentration of one-fourth of that of cedrol. It was found that the cell proliferation treated with cedrol was significantly inhibited in a concentration- and time-dependent manner, and the activity of two human liver cancer cells was reduced ( Figure 7 B).

[0079] Example 9 The cell apoptosis rate was detected by TUNEL staining. The experimental groups are shown in Table 2.

[0080] Table 2

[0081] After pre-treatment, the cells and tissue sections of each experimental group were washed once with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed again with PBS, and then incubated at room temperature for 5 minutes in a PBS solution containing 0.3% TritonX-100. Then, they were washed three times with PBS, and 50 μl of TUNEL detection solution (TdT enzyme and fluorescent labeling solution were prepared at a ratio of 1:9, Beyotime (Biyuntian) - One-step TUNEL Cell Apoptosis Detection Kit - C1088) was added and incubated at 37 ° C in the dark for 1 hour. After washing again with PBS three times, the sections were sealed with anti-fluorescence quenching sealing solution and observed under a fluorescence microscope and photographed. The experimental results are shown in Figure 2. Figure 8 As shown in the results, the number of apoptotic cells in the two liver cancer cells added with cedarwood increased significantly, indicating that cedarwood promoted cell apoptosis ( Figure 8 A). Cedarol increased the apoptosis rate of HepG-2 cells from 3.18% to 15.68%; and the apoptosis rate of SK-Hep-1 cells from 2.61% to 17.08% ( Figure 8 B).

[0082] Example 10 Western blot detection of the expression of key apoptosis proteins in cells The cells in each group in Table 2 were subjected to the following experimental procedures: (1) Total protein extraction Total protein extraction of cells: 1×10 6 Cells were seeded into each well of a 6-well plate. After the cells adhered to the wall, the cells were treated according to their grouping. After the experimental treatment, the 6-well plate was removed, the cells were collected, and the cell samples were washed twice with pre-chilled PBS. 0.5 mL of RIPA lysis buffer supplemented with a final concentration of 1 mM PMSF was added to each well. After thorough lysis, the cells were centrifuged at 12,000 g for 5 minutes at 4°C. The supernatant was immediately aspirated into a pre-chilled EP tube, which was the extracted cellular protein and stored at -80°C until further use.

[0083] (2) Protein quantification by BCA method Prepare the appropriate volume of BCA working solution based on the number of samples: 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1) and mix thoroughly. Completely dissolve the protein standard and dilute 10 μL of the protein standard to 100 μL with PBS for a final concentration of 0.5 mg / mL. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the standard wells of a 96-well plate, adding the solution used to dilute the standard to 20 μL. Add the appropriate volume of sample to the sample wells of the 96-well plate. If the sample volume is less than 20 μL, add the standard diluent 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 calculate the protein concentration of the sample in mg / mL using the formula (Figure 5). Finally, add 5× Loading Buffer (to a final concentration of 1×) and boil in water for 10 min. The sample is ready and can be stored at -20°C.

[0084] (3) Gel electrophoresis and membrane transfer, blocking and antibody incubation Prepare a 12% separating gel and a 5% stacking gel and cast an SDS-PAGE gel. After adding an appropriate amount of pre-chilled 1× electrophoresis buffer, add the previously extracted total cell protein extract and a prestained protein marker to the lanes. Run electrophoresis at 80 V for approximately 30 minutes. Once the sample has entered the separating gel, adjust the voltage to 120 V and continue electrophoresis. End electrophoresis when the target band reaches the appropriate position (referenced by the position of the prestained protein marker). Cut the PVDF membrane according to the size of the gel and activate it in methanol for 1 minute. Then, soak it in transfer buffer. Also soak the filter paper in transfer buffer for 15 minutes. Prepare the transfer "sandwich" according to the principle of filter paper ≥ PVDF membrane ≥ gel ≥ filter paper (i.e., gel on the negative side and membrane on the positive side). Ensure that no bubbles are present before starting the constant voltage transfer. After transfer, wash with TBST for 2 minutes. Block the membrane with rapid blocking solution on a shaker at room temperature for 15 minutes. Incubate with primary antibody overnight at 4°C. Wash the membrane three times with TBST (15 minutes each). Then, add secondary antibody and incubate at room temperature for 2 hours. Wash the membrane three times with TBST (15 minutes each).

[0085] (4) Chemiluminescence, development, and fixing Equal volumes of chemiluminescent reagents A and B (China); ECL luminescent solution AB (P2100) (NCMBiotech, China) were mixed, and the PVDF membrane was placed protein-side down in full contact with the mixture. After 3 minutes, the PVDF membrane was detected using a JP-K6000 chemiluminescence imager. The optical density values of protein expression were analyzed using Image J software, and the relative protein expression was calculated as the grayscale value of the target protein / the grayscale value of the internal reference protein.

[0086] Antibody dilution factors are shown in Table 3.

[0087] Table 3

[0088] The experimental results are as follows Figure 9 As shown, the results showed that the protein expression levels of the three apoptosis proteins were significantly upregulated in both HepG2 and SK-Hep-1 liver cancer cells, indicating that cedrol promoted the apoptosis of liver cancer cells.

[0089] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A β-himacladusin synthase mutant, characterized in that: It is S434V; The amino acid sequence of the β-himacadrene synthase mutant is shown in SEQ ID NO.

5.

2. The biomaterial related to the β-himacadrene synthase mutant according to claim 1, characterized in that: At least one of the following (1)-(4): (1) a nucleic acid molecule encoding a mutant of β-himacladus synthase; (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 nucleic acid molecule 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. Use of the β-himacadrene synthase mutant according to claim 1 or the related biomaterial according to claim 2 in increasing cedrol production.

4. Use of the β-himacadrene synthase mutant according to claim 1 or the related biomaterial according to claim 2 in the production of cedarol.

5. The use according to claim 3 or 4, characterized in that The application of the cedrol in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that The tumor is liver cancer.

Images