High-efficiency production of novel drimane-type sesquiterpenoids using SsDMS mutants

Abstract

This invention discloses a directed evolution strategy for SsDMS, a type II sesquiterpene cyclase derived from *Streptomyces showdoensis*, based on alanine scanning, and its applications. The invention describes several key mutant sites in SsDMS capable of catalyzing the generation of novel dried sesquiterpene compounds, including substitutions at positions 208, 248, 249, 497, and 505. This invention also discloses a highly efficient *E. coli* cell factory applied to the heterologous expression of SsDMS mutants and the efficient production of novel dried sesquiterpene compounds, further yielding structurally novel dried sesquiterpene compounds. This invention achieves the efficient production of dried sesquiterpene compounds using SsDMS and its mutants. Its modification strategy has broad applicability, providing important enzymatic resources for the biosynthesis of novel terpenoids and offering more options for the discovery of drug lead compounds.

Description

Technical Field

[0001] This invention relates to genetic engineering and enzyme engineering, specifically to a type II sesquiterpene cyclase SsDMS obtained through genetic engineering that can efficiently produce novel drimane-type sesquiterpene compounds. Background Technology

[0002] Drimane-type sesquiterpenes are a class of natural products with a unique trans-decahydronaphthalene bicyclic skeleton, composed of three isoprene units. They possess a wide range of physiological activities, including antifungal, antibacterial, antiviral, and cytotoxic activities, making them significant for clinical research. Among them, drimenol exhibits antibacterial and antioxidant activities and is a synthesizer for various active compounds such as polydialdehyde and ambroxol in chemical synthesis. Its isomer, albicanol, is a cytotoxic agent with strong antioxidant and anti-heavy metal toxicity effects. However, these compounds are scarce in nature, and traditional extraction and separation methods are insufficient to support the development of subsequent functional products. Furthermore, existing chemical synthesis methods for these sesquiterpenes are cumbersome and environmentally unfriendly. Utilizing *E. coli* for efficient production of these compounds is a highly economical and effective approach.

[0003] Traditional terpene biosynthesis systems suffer from low catalytic efficiency, hindering the efficient production of novel drimane-type sesquiterpenes. Currently, a two-step artificial terpene production pathway in *E. coli* has been studied, achieving a high drimenol yield of 455.6 mg / L through strain optimization. However, this work currently only specifically and efficiently produces drimenol, failing to produce other drimane-type compounds, and lacks a systematic SsDMS modification strategy, making it difficult to selectively obtain mutants with novel catalytic functions. Previous research on the mutant AtCPS type II diterpene cyclase revealed that mutants H263A and N322A can produce hydroxylated and double-bond isomers, while mutant H263Y produces the rearranged product (-)-kolavenyldiphosphate, demonstrating that modification of the active cavity of type II terpene cyclases may generate new catalytic products.

[0004] The applicant's research found that by mutating key amino acids in the active cavity of the type II terpene cyclase SsDMS, the catalytic activity of the enzyme can be partially or completely altered, leading to the generation of structurally diverse drimane-type rearrangement products. This further enriches the diversity of SsDMS catalytic products and enables the efficient production of compounds such as albicanol, bicyclofarnesol, and drimane-8α,11-diol. This is of great significance for the drug development of drimane-type compounds with complex structures and good physiological activities. Summary of the Invention

[0005] Objectives of the Invention: This invention provides an SsDMS-based directed evolution strategy based on alanine scanning to obtain mutants capable of catalyzing the generation of novel drimane-type sesquiterpenoid compounds, thus providing more candidate compounds for subsequent drug development. Another objective of this invention is to provide an E. coli cell factory that can achieve heterologous expression of SsDMS mutants and efficient production of novel drimane-type sesquiterpenoid compounds.

[0006] Technical solution: An SsDMS enzyme mutant, wherein the amino acid sequence of the SsDMS enzyme shown in SEQ ID NO: 1 is modified such that the 208th amino acid is changed from asparagine to glycine, alanine, valine, serine, cysteine, glutamine, aspartic acid, or glutamic acid; the 248th amino acid is changed from phenylalanine to glycine, alanine, valine, leucine, methionine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, or histidine; the 249th amino acid is changed from isoleucine to alanine, methionine, phenylalanine, cysteine, or histidine; the 497th amino acid is changed from glutamine to glycine, alanine, proline, serine, threonine, cysteine, asparagine, or glutamic acid; and the 505th amino acid is changed from tyrosine to alanine, isoleucine, phenylalanine, tryptophan, or histidine.

[0007] The SsDMS enzyme is a type II terpene cyclase derived from the bacterium Streptomyces showdoensis.

[0008] The SsDMS enzyme mutant was obtained through site-directed mutagenesis.

[0009] A directed evolution method for a type II terpene cyclase, SsDMS, is used to search for active cavity substrates near the protein co-crystal structure of the SsDMS enzyme-substrate complex. Alanine was scanned within the range of amino acids, and saturation mutations were performed on novel active mutation sites.

[0010] Application of the SsDMS enzyme mutant in the biosynthesis of terpenoid compounds.

[0011] A highly efficient fusion expression vector modified with a site-directed mutation site on the type II terpene cyclase SsDMS is an expression vector that fuses the hydrolase and the SsDMS enzyme mutant; wherein the amino acid sequence of the fusion of the hydrolase and the SsDMS enzyme mutant is as shown in SEQ ID. In the amino acid sequence shown in NO:3, position 397 is changed from asparagine to glycine, alanine, valine, serine, cysteine, glutamine, aspartic acid, or glutamic acid; position 437 is changed from phenylalanine to glycine, alanine, valine, leucine, methionine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, or histidine; position 438 is changed from isoleucine to alanine, methionine, phenylalanine, cysteine, or histidine; position 686 is changed from glutamine to glycine, alanine, proline, serine, threonine, cysteine, asparagine, or glutamic acid; and position 694 is changed from tyrosine to alanine, isoleucine, phenylalanine, tryptophan, or histidine.

[0012] The highly efficient fusion expression vector is an expression vector that fuses the hydrolase and the SsDMS enzyme mutant.

[0013] Escherichia coli that efficiently produces novel drimane-type sesquiterpene compounds was introduced into E. coli cells via the aforementioned efficient fusion expression vector and an expression vector containing the amino acid sequences SEQ ID NO: 5-7 and SEQ ID NO: 9.

[0014] Escherichia coli that efficiently produces novel drimane-type sesquiterpene compounds was introduced into E. coli cells via the aforementioned efficient fusion expression vector and the expression vector with nucleic acid sequences SEQ ID NO: 8 and SEQ ID NO: 10.

[0015] The application of Escherichia coli, which efficiently produces the novel drimane-type sesquiterpenes, in the production of drimane-type sesquiterpenes.

[0016] The application described herein includes one or more compounds selected from drimenol, albicanol, bicyclofarnesol, and drimane-8α,11-diol.

[0017] The method for producing drimane-type sesquiterpenoid compounds using Escherichia coli involves fermentation medium containing 1-5% glycerol, with the bacterial culture cultured to OD0.05. 600 Induction was performed at a concentration of 1.3–1.4, with the addition of isopropyl BD-1-thiopyranogalactoside, 3-methyl-2-buten-1 alcohol, and 3-methyl-3-buten-1 alcohol during induction.

[0018] Alanine scanning mutation of SsDMS: The SsDMS described in this invention is the first type II sesquiterpene cyclase discovered in the laboratory through genome mining. It is derived from the bacterium Streptomyces showdoensis, and its amino acid sequence has the NCBI accession number A0A2P2GK84. The amino acid sequence of the encoded protein is shown in SEQ ID NO: 1 of the sequence listing, and its nucleotide sequence is shown in SEQ ID NO: 2 of the sequence listing.

[0019] This invention is based on SsDMS in the laboratory. D303E -FPP-Mg 2+ Based on the crystal structure of the complex protein, the active site was analyzed using PyMol software. Alanine scanning was performed within the radius, with a focus on aromatic residues within the β-domain. These residues may stabilize and influence water-mediated catalysis through their large side chains, and may reveal different catalytic bases that produce different catalytic products, thereby altering the catalytic activity of wild-type SsDMS. Specific implementation methods are described in Example 1.

[0020] This invention designs and constructs highly efficient expression vectors containing mutant SsDMS genes, all based on the laboratory-developed fusion expression plasmid pETDuet-SsNDH-SsDMS(R) containing a hydrolase and a cyclase. This plasmid introduces a hydrolase to achieve efficient hydrolysis of drimane-type sesquiterpene pyrophosphate products. The amino acid sequence encoding the protein is shown in SEQ ID NO: 3, and its nucleotide sequence is shown in SEQ ID NO: 4. Using site-directed mutagenesis, the target site was systematically replaced with alanine to obtain a series of SsDMS mutants. The activity and product types of the mutants were preliminarily screened and identified, revealing key mutation sites with novel catalytic activity, such as F248 and Y505. Specific implementation methods are described in Example 2. This invention also includes amino acid sequences with equivalent functions formed by replacing, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 3, and the nucleotide sequences encoding these amino acid sequences.

[0021] This invention applies a two-step artificial pathway plasmid for high-yield production of terpenoid precursors and optimized fermentation conditions to an *E. coli* cell factory, achieving heterologous expression of the SsDMS mutant and efficient production of novel drimane-type sesquiterpenoid compounds. This high-yield system for drimane-type compounds incorporates three plasmids: a high-efficiency expression vector containing the mutant SsDMS gene, and the laboratory-developed high-yield terpenoid precursor plasmids pCDFDuet-Ipk-IspA-IDI and pRSFDuet-PhoN(△). The nucleotide sequence of plasmid pCDFDuet-Ipk-IspA-IDI is shown in SEQ ID NO: 8, with the Ipk amino acid sequence shown in SEQ ID NO: 5, the IspA amino acid sequence shown in SEQ ID NO: 6, and the IDI amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence of the protein encoded by plasmid pRSFDuet-PhoN(△) is shown in SEQ ID NO: 9, and its nucleotide sequence is shown in SEQ ID NO: 10. The plasmid was introduced into a self-made electrocompetent BL21 cell using electroconversion. Positive single clones were selected and cultured, and then fermented to produce mutant products. The specific implementation method is described in Example 3.

[0022] Based on alanine scanning, this invention utilizes site-directed mutagenesis to select key sites for saturation mutagenesis, introducing all 20 amino acids at the target sites to obtain more high-yield SsDMS mutants, further optimizing enzyme activity and product specificity. These mutants were then introduced into a high-yield system for drimane-type compounds, and fermentation was performed on each mutant strain. The activity and product types of the mutants were screened and identified using TLC and HPLC analyses, yielding dominant mutants capable of catalyzing the formation of novel drimane-type sesquiterpenes, such as F248A, F248Y, and Y505W. Specific implementation methods are detailed in Examples 4 and 5.

[0023] This invention also describes the fermentation production and product validation of novel drimane-type sesquiterpenoids. A high-efficiency expression vector containing the mutant SsDMS gene, combined with a high-yield plasmid of terpene precursors, was co-transformed into *E. coli* to carry out fermentation production of the SsDMS mutant. After fermentation, the fermentation products were analyzed using TLC and HPLC. The mutant with novel catalytic activity was scaled up to 3L for fermentation. After post-processing, the fermentation products were separated and purified by silica gel column chromatography, HPLC, and other methods, and finally purified by H₂O₂. 1 -NMR, C 13 The structure was identified and confirmed by ¹HMR and database comparison methods to obtain novel drimane-type sesquiterpene compounds. The specific implementation methods are described in Examples 5 and 6.

[0024] This invention analyzes the yield of a high-yielding mutant strain containing novel activity. For the isolated and confirmed compounds, a content-related standard curve was established using HPLC. The high-yielding mutant strain was fermented according to the fermentation method of Example 3, and post-processed and analyzed by HPLC according to the method of Example 5. The analytical data were then input into the standard curves of each compound to obtain the yield of the high-yielding mutant strain. Specific implementation methods are described in Examples 7 and 8. The yield data were obtained from three parallel experiments under the same experimental conditions.

[0025] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: The SsDMS directed evolution strategy established in this invention can effectively guide the modification of other terpene cyclases and has wide applicability; the discovered key SsDMS mutants greatly expand the catalytic function of the enzymes, providing important enzymatic resources for the biosynthesis of novel terpene compounds; the constructed E. coli cell factory achieves the efficient production of novel drimane-type sesquiterpene compounds such as albicanol, bicyclofarnesol, and drimane-8α,11-diol, among which albicanol exhibits significant physiological activity; the novel drimane-type sesquiterpene compounds obtained through efficient production provide more options for the discovery of drug lead compounds. Attached Figure Description

[0026] Figure 1 For Albicanol standard curve;

[0027] Figure 2 This is the Drimenol standard curve;

[0028] Figure 3 For Bicyclofarnesol standard curve;

[0029] Figure 4 H for Albicanol 1 -NMR spectrum;

[0030] Figure 5 C for Albicanol 13 -NMR spectrum;

[0031] Figure 6 H of Bicyclofarnesol 1 -NMR spectrum;

[0032] Figure 7 C of Bicyclofarnesol 13 -NMR spectrum;

[0033] Figure 8 H for drimane-8α,11-diol1 -NMR spectrum;

[0034] Figure 9 C for drimane-8α,11-diol 13 -NMR spectrum;

[0035] Figure 10 This is the result of an alanine mutation scan. Detailed Implementation

[0036] Example 1: Design of SsDMS mutation sites

[0037] The cyclase SsDMS described in this embodiment is a type II sesquiterpene cyclase discovered in previous genome mining in the laboratory. It can catalyze the production of drimenol pyrophosphorylation products from farnesyl diphosphate (FPP). Based on existing SsDMS technology in the laboratory... D303E -FPP-Mg 2+ Based on the crystal structure of the complex protein, active sites were selected using the visualization software PyMol. Amino acids within the radius range were selected, with a focus on aromatic residues within the β-domain. A total of 19 amino acids were chosen: T129, M136, W165, L166, N208, F248, I249, M252, F255, F293, T343, F344, M354, W393, Q497, A498, PS00, and Y505.

[0038] Example 2: Construction of high-yield SsDMS mutant

[0039] Based on laboratory research, this embodiment constructs a high-yield SsDMS mutant using the hydrolase and cyclase fusion expression plasmid pETDuet-SsNDH-SsDMS(R) as a template. The SsDMS mutation site was identified using a two-step PCR method: The first PCR used the original plasmid pETDuet-SsNDH-SsDMS(R) as a template. A total of 27 bases upstream and downstream of the mutation site were selected as primers S and X. The upstream and downstream fragments of the mutation site were amplified using primers F and X (encoding the total length of the protein) and primers R and X (encoding the total length of the protein). After gel electrophoresis, the bands with the correct number of bases were selected for gel recovery to obtain the upstream and downstream fragments of the mutation site. The second PCR used an equal amount of the upstream and downstream fragments as a template. The full-length target mutant band was amplified using primers F and R (encoding the full-length protein). Linear sequences containing the designed mutation site were obtained through gel electrophoresis and gel recovery experiments. The primers are listed in Table 1. The PCR amplification experiment consisted of a 50 μL system, including 25 μL of 2×PhantaMax Buffer, 18.5 μL of ddH2O, 2.5 μL of DMSO, 1 μL of 10 mM dNTPs, 0.5 μL each of forward and reverse primers, 1 μL of template, and 1 μL of 2.5 U of high-fidelity polymerase (PhantaMax Super-Fidelity DNA Polymerase). The PCR amplification conditions were as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, annealing for 15 s (annealing temperature based on the higher Tm value of the two primers), extension at 72℃ (extension time based on fragment length 1 min / 1000 bp), for a total of 30 cycles, and a final extension at 72℃ for 10 min.

[0040] Table 1 Primer list for mutants

[0041]

[0042]

[0043] The vector pETDuet was digested using restriction endonucleases BamHI and HindIII, and then ligated to the full-length target gene containing the mutation site via homologous recombination. The recombinant product was introduced into E. coli DH5α competent cells via chemical transformation. Single colonies were picked and cultured in LB broth containing 100 μg / mL ampicillin for 12–20 h. Plasmids were extracted and sequenced. The sequencing results were compared with the unmutated original plasmid sequence using SnapGene software. Successful alignment of the mutation site and recombination ligation site indicated successful construction of a high-efficiency expression vector containing the mutation. The chemical transformation procedure was as follows: the recombinant product was added to competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 65 s, cooled on ice for 3 min, and grown on a shaker at 37°C and 230 rpm for 40 min. 100 μg / mL ampicillin was added to the bacterial culture, which was then evenly spread on LB agar and dried. The culture was then inverted and incubated at 37°C for 12 h.

[0044] Example 3: In vivo expression of high-yield plasmids of drimane-type terpenoids

[0045] In this embodiment, a modified artificial "two-step" high-yield drimenol strain was used as the chassis strain, and the in vivo expression of this type of compound was carried out by introducing a high-efficiency fusion expression plasmid containing the correct mutation site.

[0046] Plasmids containing the correct mutation sites, along with high-yield terpene precursor plasmids pCDFDuet-Ipk-IspA-IDI and pRSFDuet-PhoN(△), were co-introduced into self-made *E. coli* BL21 competent cells via electroporation. Single colonies were picked and cultured in LB broth containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 50 μg / mL streptomycin sulfate for approximately 10 h. Then, 250 μL of the bacterial culture was added to 50 mL of LB broth containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 50 μg / mL streptomycin sulfate, and 2% glycerol in a clean bench and cultured at 37°C and 230 rpm for 2-4 h until OD (digestive activity) was reached. 600 =1.3~1.4, cool the bacterial culture in an ice bath for 20 min, add 0.25 mM IpTG (isopropyl BD-1-thiopyranogalactopyranoside) and 10 mM DMAA:ISO = 1:2 (3-methyl-2-buten-1 alcohol: 3-methyl-3-buten-1 alcohol = 1:2) mixture to a clean bench, and culture at 18℃ and 200 rpm for 72 h to complete the in vivo expression of high-yield plasmids of drimane-type terpenoid compounds.

[0047] Preparation of E. coli BL21 electrotransformation competent cells: A small amount of E. coli BL21 bacterial culture stored at -80℃ was streaked onto LB agar solid medium and cultured at 37℃ for 12 hours. Single colonies were then picked and transferred to 5 mL of LB liquid medium and cultured at 37℃ and 230 rpm for 12-18 hours. Then, 5 mL of the bacterial culture was added to 1 L of LB liquid medium in a clean bench and cultured at 37℃ and 230 rpm for 1-3 hours until OD (digestive activity) was reached. 600 =0.5~0.6. Pour the bacterial culture into 10 pre-chilled 50mL centrifuge tubes, then quickly place them in an ice-water mixture for quenching. After cooling, centrifuge at 4000rpm for 10min in a low-temperature refrigerated centrifuge (4℃). Discard the supernatant, and repeat the above operation to collect the bacterial cells. Gently resuspend the bacterial cells in each tube with a small amount of pre-chilled sterile distilled water. Combine the bacterial cultures and add distilled water to 40mL. Centrifuge at 4000rpm for 10min in a low-temperature refrigerated centrifuge (4℃). Discard the supernatant, and repeat the above operation once. Then repeat the above steps twice with pre-chilled sterile 10% glycerol, centrifuge at 4000rpm for 10min in a low-temperature refrigerated centrifuge (4℃). After the last centrifuge, discard all the supernatant and add 4mL of pre-chilled sterile 10% glycerol to gently resuspend the cells at the bottom of the centrifuge tube. After resuspending, aliquot the bacterial culture into 1.5mL centrifuge tubes and immediately place them in liquid nitrogen for flash freezing. Store at -80℃ or use immediately for electroporation.

[0048] The specific experimental steps for electroporation are as follows: Thaw the electroporation competent cells on ice for 3 minutes, add an appropriate amount of plasmid to be transformed (approximately 500 ng of each plasmid in the three-plasmid transformation system), incubate on ice for 5 minutes, transfer to a pre-cooled electroporation cuvette and let stand for 10 minutes. Set the electroporation instrument conditions to U = 1500 V, R = 400 Ω, C = 25 μF, pre-charge and discharge, and then perform electroporation. Quickly add 700 μL of preheated LB agar at 37°C, and incubate at 230 rpm for 1 hour in a shaker at 37°C. Add 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 50 μg / mL streptomycin sulfate (relative to a solid LB agar plate), mix well, spread evenly on a solid LB agar plate, dry, and invert incubate at 37°C for 12 hours. The single colonies on the solid LB agar plate are E. coli BL21 cells that have been successfully transformed with plasmids.

[0049] Example 4: Construction of SsDMS site-directed saturation high-yield mutant

[0050] SsDMS site-directed saturation mutagenesis involves mutating the target site with 19 amino acids other than the original amino acid, following the experimental method in Example 2. The 20 amino acids involved in the saturation mutagenesis are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), tryptophan (W), serine (S), tyrosine (Y), cysteine ​​(C), phenylalanine (F), asparagine (N), glutamine (G), threonine (T), aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), and histidine (H). The primers for the five rounds of saturation mutagenesis sites in this example are shown in Table 2.

[0051] Table 2 Primers for saturated mutants

[0052]

[0053]

[0054]

[0055]

[0056] Example 5: Screening for novel catalytically active mutants

[0057] After constructing the high-yield mutant and expressing the high-yield plasmid of the mutant in vivo, the successfully fermented bacterial broth was transferred to 50 mL centrifuge tubes and centrifuged at 4000 rpm for 20 min in a refrigerated centrifuge (4℃). The supernatant was discarded, and the bacterial pellet was mixed with 3 mL of acetone solution by vortexing. The mixture was then sonicated for 4 h in an ultrasonic cleaner at a frequency of 40 kHz and a power of 200 W. After sonication, the mixture was centrifuged at 4000 rpm for 20 min in a refrigerated centrifuge (4℃). The supernatant was transferred to 5 mL centrifuge tubes and centrifuged at 12000 rpm for 10 min in an ultracentrifuge. An appropriate amount of the supernatant was filtered through a 0.22 μm nylon filter into a liquid chromatography vial for subsequent HPLC analysis. The remaining supernatant was analyzed by TLC. HPLC analysis was performed using an Agilent 1260 Infinity system equipped with a YMC-Pack ODS-A column (150 × 4.6 mm, 5 μm) at a flow rate of 0.8 mL / min. The analytical conditions are shown in Table 3. The analytical results are shown in [Table 3]. Figure 10 Silica gel GF254 plates were used. After spotting, the samples were developed using petroleum ether:ethyl acetate = 5:1 as the developing solvent and 10% sulfuric acid-ethanol solution as the colorimetric reagent. Wild-type cells were used as a control for each batch of samples. Mutant fermentation samples with new spots or peaks compared to the wild type were repeated. After confirming the results, the new compounds were enriched, separated, and identified.

[0058] Table 3. HPLC analysis methods for fermentation products

[0059]

[0060] Example 6: Enrichment, separation and identification of drimane-type terpenoids

[0061] Based on the experimental results of Example 5, this embodiment scales up the fermentation of a novel active mutant strain to 3L (60 50mL bottles) using the fermentation method of Example 3. After fermentation, the bacterial cultures were combined and poured into 500mL white centrifuge bottles, balanced, and centrifuged at 4000rpm for 25min in a low-temperature refrigerated centrifuge (4℃). The supernatant was discarded, and the above steps were repeated until all bacterial precipitates were collected. The bacterial cultures were combined in white centrifuge bottles, 200mL of acetone was added, and the mixture was ultrasonically cleaned for 2h at a frequency of 40kHz and a power of 200W. After ultrasonication, ultrafiltration was performed using a Buchner funnel to separate the supernatant and precipitate. The precipitate was then treated with 200mL of acetone twice more. The supernatants from the three extractions were combined, and the acetone was evaporated in a rotary evaporator. The remaining components were extracted three times with ethyl acetate. The upper solutions from the three extractions were combined, an appropriate amount of saturated sodium sulfate solid was added, the water was absorbed, and the ethyl acetate was evaporated in a rotary evaporator to obtain 3L of fermentation extract.

[0062] Take 2-3 times the amount of 200-300 mesh silica gel powder and mix it with the fermentation extract. Separate the fermentation products using silica gel column chromatography. For F248A and Y505W fermentation samples (whose fermentation products were identified as albicanol and bicyclofarnesol, respectively), the mobile phase ratio was petroleum ether: ethyl acetate = 15:1, and the colorimetric reagent was 10% sulfuric acid-ethanol solution. After the new target spot appears during elution, it usually mixes with Drimenol or FOH. Combine the samples containing the new spot after column chromatography, and evaporate the mobile phase to dryness in a rotary evaporator to obtain the crude product. The crude product was dissolved in an appropriate amount of chromatographic methanol, centrifuged at 12,000 rpm for 10 min, and filtered through a 0.22 μm nylon membrane into a liquid chromatography vial. Subsequent preparation of new compounds was performed using an Agilent 1260 Infinity analytical HPLC system equipped with an Agilent Poroshell 120 EC-C18 column. For crude F248A, a semi-preparative separation was performed using a flow rate of 1 mL / min and isocratic elution with 70% acetonitrile. For crude Y505W, a semi-preparative separation was performed using a flow rate of 0.8 mL / min and isocratic elution with 70% acetonitrile. For the F248N fermentation sample (identified as drimane-8α,11-diol), the mobile phase ratio was petroleum ether:ethyl acetate = 2:1, and the colorimetric reagent was 10% sulfuric acid-ethanol solution. After eluting the target new spot, samples containing the new spot were combined, and the mobile phase was evaporated to dryness in a rotary evaporator to obtain the separated product. After the compounds were separated, the liquid was evaporated to dryness in a rotary evaporator. An appropriate amount of the evaporated sample was dissolved in deuterated chloroform and transferred to an NMR tube for NMR analysis. The separated products were identified by data comparison.

[0063] like Figure 4-9 As shown, modifying the SsDMS mutation site can generate other drimane-type compounds besides drimenol, such as albicanol, bicyclofamesol, and drimane-8α,11-diol, which greatly expands the catalytic function of SsDMS and provides more candidate compounds for subsequent drug development. Furthermore, albicanol has been shown to have cytotoxicity and has certain application value.

[0064] Example 7: Establishment of a standard curve for drimane-type terpenoids

[0065] This embodiment establishes a standard curve for the compounds with UV absorption obtained in Example 6. 4 mg of a drimane-type terpene compound standard was accurately weighed and added to 1 mL of methanol solution to prepare a 4 mg / mL standard stock solution. This stock solution was then diluted with methanol to seven different concentration gradients: 4 mg / mL, 2 mg / mL, 1.5 mg / mL, 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. These samples were analyzed by HPLC using the same method as shown in Table 3 of Example 5, with an injection volume of 5 μL each time. Based on the different peak areas at the retention time corresponding to the 210 nm absorption wavelength of the drimane-type terpene compound samples at different concentration gradients, a standard curve was established. Figure 1-3 The function is obtained by fitting the data.

[0066] Example 8: Yield analysis of a novel catalytically active mutant

[0067] This embodiment, based on the screening results of Example 5, performs a unified yield analysis on mutants with novel catalytic activity. The mutant bacterial culture, frozen at -80℃, was thawed on ice. 10 μL of the culture was added to LB liquid containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 50 μg / mL streptomycin sulfate. The culture was then incubated at 37℃ and 230 rpm for approximately 10 h on a shaker. The mutant bacterial culture was then re-fermented according to the fermentation method of Example 3. After fermentation, the fermentation products were analyzed by HPLC according to the product processing method of Example 5. The HPLC analysis results were integrated, and the peak area corresponding to the retention time of the new compound was recorded. The peak area was then converted into the fermentation yield of the mutant. The fermentation yields of the mutants involved in this embodiment are shown in Table 4.

[0068] In this example, the inventors discovered that the mutant strain F248Y can completely alter the catalytic function of the wild-type enzyme, producing the compound albicanol completely, which fully demonstrates the practicality of the targeted modification strategy of the present invention.

[0069] Table 4. Mutant Yield

[0070]

[0071]

[0072] SEQ ID NO:1

[0073] MNASPTPTATTTTEPATAVVRCRTRLARRVVAAVGPDGLLPAPCESRVLESALALALLTEERAEADATARLTAYLRTTLRTAPPDPFQCAVARAVLGGAGERGERVGDEGDMDAGTALDAGLDGFDHFTAGRKRLMFRTVLAALGATGFPAVPWEAYDTRPQQSWLHMEMKALKVLAAHGTGHPDVVRDEDWRALLPALEPGPAWECNNLAQLLALLALRHSPRHRPALGDVLKHVAGRLRPDGGMPFIDGMTVFTTAAAGLALSLLPAPPACVTPMADALALRRNPDGGYGFHSGVAQSDVDDTCYVLEFLRRAAPDRHRTAVAEAEGYLLALRNPDGGFPTFARGTSSEIAMTAAAASALAHDPDRREEVDEAVRYVVRHQRPDGTFERSWSRNATNAVFRAVLALTGVAAHGEERRSRARAAERALAHLAATQNGDGGWGHAEAEPSDPISTAYAVIALARGPRARPGGPLDRALAYLVERQHPDGGYRSRPDQAGPRPLLYDVPALADVFVLLALAHATATPDPEGCSR*

[0074] SEQ ID NO:2

[0075]

[0076] SEQ ID NO:3

[0077] VTAHETVDYVDADDRRVTRGPRGGAAARGLYYRVAATVCADAAGRILVYRRSARAAVYPAHHDILIGGCPRAGEGYAEAAARELREELGIRAAPRPVLHEPRPSPVGRCWLAVHVAVVEAPPDVDPREIADHFFAPPQDLLDHPPAPFVPEGRRILARLLAHGLLPPLTPSRPSPTTRPTGSGGSGGSGMNASPTPTATTTTEPATAVVRCRTRLARRVVAAVGPDGLLPAPCESRVLESALALALLTEERAEADATARLTAYLRTTLRTAPPDPFQCAVARAVLGGAGERGERVGDEGDMDAGTALDAGLDGFDHFTAGRKRLMFRTVLAALGATGFPAVPWEAYDTRPQQSWLHMEMKALKVLAAHGTGHPDVVRDEDWRALLPALEPGPAWECNNLAQLLALLALRHSPRHRPALGDVLKHVAGRLRPDGGMPFIDGMTVFTTAAAGLALSLLPAPPACVTPMADALALRRNPDGGYGFHSGVAQSDVDDTCYVLEFLRRAAPDRHRTAVAEAEGYLLALRNPDGGFPTFARGTSSEIAMTAAAASALAHDPDRREEVDEAVRYVVRHQRPDGTFERSWSRNATNAVFRAVLALTGVAAHGEERRSRARAAERALAHLAATQNGDGGWGHAEAEPSDPISTAYAVIALARGPRARPGGPLDRALAYLVERQHPDGGYRSRPDQAGPRPLLYDVPALADVFVLLALAHATATPDPEGCSR*

[0078] SEQ ID NO:4

[0079]

[0080] SEQ ID NO:5

[0081] MDPFTMMILKIGGSVITDKSAYRTARTYAIRSIVKVLSGIEDLVCVVHGGGSFGHIKAMEFGLPGPKNPRSSIGYSIVHRDMENLDLMVIDAMIEMGMRPISVPISALRYDGRFDYTPLIRYIDAGFVPVSYGDVYIKDEHSYGIYSGDDIMADMAELLKPDVAVFLTDVDGIYSKDPKRNPDAVLLRDIDTNITFDRVQNDVTGGIGKKFESMVKMKSSVKNGVYLINGNHPERIGDIGKESFIGTVIR*

[0082] SEQ ID NO:6

[0083] MDFPQQLEACVKQANQALSRFIAPLPFQNTPVVETMQYGALLGGKRLRPFLVYATGHMFGVSTNTLDAPAAAVECIHAYSLIHDDLPAMDDDDLRRGLPTCHVKFGEANAILAGDALQTLAFSILSDADMPEVSDRDRISMISELASASGIAGMCGGQALDLDAEGKHVPLDALERIHRHKTGALIRAAVRLGALSAGDKGRRALPVLDKYAESIGLAFQVQDDILDVVGDTATLGKRQGADQQLGKSTYPALLGLEQARKKARDLIDDARQSLKQLAEQSLDTSALEALADYIIQRNK*

[0084] SEQ ID NO:7

[0085] MQTEHVILLNAQGVPTGTLEKYAAHTADTRLHLAFSSWLFNAKGQLLVTRRALSKKAWPGVWTNSVCGHPQLGESNEDAVIRRCRYELGVEITPPESIYPDFRYRATDPSGIVENEVCPVFAARTTSALQINDDEVMDYQWCDLADVLHGIDATPWAFSPWMVMQATNREARKRLSAFTQLK*

[0086] SEQ ID NO:8

[0087]

[0088] SEQ ID NO:9

[0089] MKRQLFTLSIVGVFSLNTFASIPPGNDVTTKPDLYYLTNNDNAIDSLALLPPPPQIGSIAFLNDQAMYEKGRLLRNTERGKLAAEDANLSSGGVANVFSAAFGSPITAKDSPELHKLLTNMIRDAGDLATRSAKEYYMRIRPFAFYGVSTCNTKEQDKLSKNGSYPSGHTSIGWATALVLSEINPARQDTILKRGYELGDSRVICGYHWQSDVDAARIVGSAIVATLHSNPVFQAQLQKAKDEFANNQKK*

[0090] SEQ ID NO:10

[0091] atgaagcgccagctgtttaccctgagcattgtgggtgtttttagtctgaatacctttgccagtattccgccgggtaatgatgttaccaccaaaccggatctgtattatctgaccaatgataatgcaattgacagcctggccctgctgccgccgccgcctcagattggtagcattgcatttctgaatgatcaggcaatgtatgaaaaaggtcgcctgctgcgtaataccgaacgtggtaaactggccgccgaagatgcaaatctgagcagtggcggtgttgcaaatgtgtttagcgccgcctttggtagcccgattaccgccaaagatagcccggaactgcataaactgctgaccaatatgattcgcgatgcaggcgatctggcaacccgcagcgcaaaagaatattatatgcgtattcgtccgtttgccttttatggtgttagtacctgtaataccaaagaacaggataagctgagcaagaatggcagttatccgagtggccataccagcattggttgggccaccgcactggttctgagtgaaattaatccggcccgccaggataccattctgaaacgtggctatgaactgggtgacagtcgtgtgatttgcggttatcattggcagagtgatgtggatgcagcacgtattgtgggtagtgccattgtggcaaccctgcatagcaatccggtgtttcaggcccagctgcagaaagcaaaagatgaatttgccaataatcagaaaaagtaa

[0092] The * at the end of the amino acid sequence represents the stop codon.

Claims

1. A mutant of the SsDMS enzyme characterized in that, The SsDMS enzyme with the amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid at position 248 is changed from phenylalanine to alanine, valine, leucine, methionine, tryptophan, serine, tyrosine or asparagine.

2. The mutant SsDMS enzyme of claim 1, wherein, The SsDMS enzyme is a type II terpene cyclase derived from the bacterium Streptomyces showdoensis.

3. The mutant SsDMS enzyme of claim 1, wherein, The SsDMS enzyme mutant is obtained by a site-directed mutagenesis method.

4. Use of the SsDMS enzyme mutant of claim 1 in biosynthesis of terpenoids, wherein the mutant can biosynthesize both drimenol and albicanol, the mutant with the amino acid at position 248 of SEQ ID NO: 1 changed to tryptophan can further biosynthesize bicyclofarnesol, and the mutant with the amino acid at position 248 of SEQ ID NO: 1 changed to alanine or asparagine can further biosynthesize drimane-8a, 11-diol.

5. A high efficient fusion expression vector containing SsDMS enzyme mutant, characterized in that, It is an expression vector fusing a hydrolytic enzyme and the SsDMS enzyme mutant of claim 1, wherein the amino acid sequence of the fusion of the hydrolytic enzyme and the SsDMS enzyme mutant of claim 1 is the amino acid sequence shown in SEQ ID NO: 3, wherein the amino acid at position 437 is changed from phenylalanine to alanine, valine, leucine, methionine, tryptophan, serine, tyrosine or asparagine.

6. An Escherichia coli for efficient production of a drimane-type sesquiterpene compound, characterized by, The efficient fusion expression vector of claim 5 is introduced into E. coli.

7. Use of the E. coli of claim 6 in production of drimane-type sesquiterpenoids, wherein the E. coli can biosynthesize both drimenol and albicanol, the E. coli containing the expression vector with the amino acid at position 437 of SEQ ID NO: 3 changed to tryptophan can further biosynthesize bicyclofarnesol, and the E. coli containing the expression vector with the amino acid at position 437 of SEQ ID NO: 3 changed to alanine or asparagine can further biosynthesize drimane-8a, 11-diol.

8. The method of producing a drimane-type sesquiterpene compound by Escherichia coli according to claim 6, characterized by, The fermentation medium contains 1-5% glycerol and the culture is grown to OD 600 = 1.3-1.4, induction with isopropyl β-D-1-thiogalactopyranoside and 3-methyl-2-buten-1-ol, 3-methyl-3-buten-1-ol.

Images