Efficient production of novel rimane type sesquiterpene compound by using SsDMS mutant

Abstract

The invention discloses a directed evolution strategy of type II sesquiterpene cyclase (SsDMS) derived from Streptomyces shedoensis based on alanine scanning, and an application of the type II sesquiterpene cyclase (SsDMS) derived from Streptomyces shedoensis based on alanine scanning. The invention relates to a plurality of SsDMS key mutant sites capable of catalytically generating a novel drive type sesquiterpenoid compound, which comprise substitution of sites 208, 248, 249, 497 and 505, and the SsDMS key mutant sites comprise substitution of sites 208, 248, 249, 497 and 505. The invention discloses an efficient escherichia coli cell factory applied to heterologous expression of an SsDMS mutant and efficient production of a novel drive type sesquiterpene compound, and further obtains the drive type sesquiterpene compound with a novel structure. According to the present invention, the efficient production of the drive type sesquiterpene compound by using the SsDMS and the mutant thereof is achieved, the modification strategy has wide applicability, the important enzymology resource is provided for the biosynthesis of the novel terpene compound, and the more choices are provided for the discovery of the drug lead compound.

Description

Technical Field

[0001] The invention relates to genetic engineering and enzyme engineering, and in particular to a type II sesquiterpene cyclase SsDMS obtained by genetic engineering and capable of efficiently producing novel drimane type sesquiterpene compounds. Background Art

[0002] Drimane-type sesquiterpenoid compounds are a class of natural products with a unique trans-decahydronaphthalene bicyclic skeleton, composed of three isoprene units, and have rich physiological activities such as antifungal, antibacterial, antiviral, and cytotoxic activities, which are of great significance for clinical research. Among them, drimenol has antibacterial and antioxidant activities, and is a synthon of various active compounds such as polygoni aldehyde and ambroxan in chemical synthesis. Its isomer albicanol is a cytotoxic agent with strong antioxidant and heavy metal toxicity effects. However, the content of such compounds in nature is relatively small, and it is difficult to support the subsequent functional product development by traditional raw material extraction and separation; in terms of chemical synthesis, the existing chemical synthesis of such sesquiterpenoid compounds has problems such as cumbersome steps and unfriendly environment. Using Escherichia coli to efficiently produce such compounds is a very economical and effective method.

[0003] The catalytic efficiency of traditional terpene biosynthesis systems is low, making it difficult to achieve efficient production of novel drimane-type sesquiterpenoid compounds. Currently, there has been research on the production of drimane-type compounds in Escherichia coli using an artificial "two-step" terpene production pathway to produce high-yield drimenol in Escherichia coli, which achieved a fermentation yield of 455.6 mg / L by optimizing the strain. However, this work can only produce drimenol specifically and efficiently, and has not completed the production of other drimane-type compounds. In addition, there is a lack of a systematic SsDMS transformation strategy, making it difficult to obtain mutants with novel catalytic functions. Previous researchers have found that the mutants H263A and N322A of the enzyme AtCPS can produce hydroxylation and double bond isomerization products, and the mutant H263Y can produce the rearrangement product (-)-kolavenyldiphosphate, proving that the transformation of the active cavity of type II terpene cyclase may produce new catalytic products.

[0004] The applicant's research found that by conducting mutation experiments on the key amino acids in the active cavity of type II terpene cyclase SsDMS, the catalytic activity of the enzyme can be partially or completely changed, so that it can generate drimane-type rearrangement products with diverse structures, further enriching the diversity of the catalytic products of SsDMS, and achieving efficient production of compounds such as albicanol, bicyclofarnesol, drimane-8α, 11-diol, etc., which is of great significance for the drug development of drimane-type compounds with complex structures and good physiological activity. Summary of the invention

[0005] Purpose of the invention: The present invention provides an SsDMS directed evolution strategy based on alanine scanning to obtain mutants that can catalyze the production of novel drimane-type sesquiterpene compounds, providing more candidate compounds for subsequent drug development; another object of the present invention is to provide an Escherichia coli cell factory that can achieve heterologous expression of SsDMS mutants and efficient production of novel drimane-type sesquiterpene compounds.

[0006] Technical solution: A SsDMS enzyme mutant, wherein the amino acid sequence of the SsDMS enzyme shown in SEQ ID NO: 1 is 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 is obtained by site-directed mutagenesis.

[0009] A directed evolution method for type II terpene cyclase SsDMS enzymes, searching for the active cavity substrate vicinity through the protein co-crystal structure of the SsDMS enzyme-substrate complex Alanine scanning was performed on the amino acids within the range, and saturation mutagenesis was performed on the mutation sites with novel activities.

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

[0011] A highly efficient fusion expression vector for modifying the site-directed mutation site of type II terpene cyclase SsDMS, which is an expression vector that fuses a hydrolase and the SsDMS enzyme mutant; wherein the amino acid sequence of the hydrolase and the SsDMS enzyme mutant is a sequence of 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 high-efficiency fusion expression vector is an expression vector fused with the hydrolase and the SsDMS enzyme mutant.

[0013] The invention discloses an Escherichia coli for efficiently producing novel drimane-type sesquiterpene compounds, wherein the Escherichia coli cells are simultaneously introduced with the efficient fusion expression vector and an expression vector containing the amino acid sequences of SEQ ID NO: 5-7 and SEQ ID NO: 9.

[0014] Escherichia coli for efficiently producing novel drimane-type sesquiterpene compounds, and Escherichia coli cells into which the efficient fusion expression vector and the expression vector having the nucleic acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10 are simultaneously introduced.

[0015] The novel Escherichia coli for efficiently producing drimane-type sesquiterpene compounds is used in producing drimane-type sesquiterpene compounds.

[0016] In the application, the drimane-type sesquiterpene compound includes one or more compounds selected from the group consisting of drimenol, albicanol, bicyclofarnesol, and drimane-8α, 11-diol.

[0017] The method for producing drimane-type sesquiterpene compounds by Escherichia coli comprises fermentation medium containing 1-5% glycerol, and the bacterial liquid is cultured to OD 600 =1.3-1.4, and during induction, isopropyl BD-1-thiogalactopyranoside, 3-methyl-2-butene-1-ol and 3-methyl-3-butene-1-ol were added.

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

[0019] The present invention is in the laboratory SsDMS D303E -FPP-Mg 2+ Based on the crystal structure of the complex protein, the active site was identified by PyMol software. Alanine scanning was performed within the radius, focusing on aromatic residues in the β domain, which may stabilize and affect water-mediated catalysis through their bulky side chains and may reveal that different catalytic bases produce different catalytic products, thereby achieving the purpose of changing the catalytic activity of wild-type SsDMS. The specific implementation method is shown in Example 1.

[0020] The efficient expression vectors designed and constructed by the present invention containing mutant SsDMS genes are all based on the laboratory hydrolase and cyclase fusion expression plasmid pETDuet-SsNDH-SsDMS (R), which introduces hydrolase to achieve efficient hydrolysis of drimane-type sesquiterpene pyrophosphate products. The amino acid sequence of the encoded protein is shown in the sequence table SEQ ID NO: 3, and its nucleotide sequence is shown in the sequence table SEQ ID NO: 4. Using the method of site-directed mutagenesis, the target site is systematically replaced with alanine, a series of SsDMS mutants are obtained, and the activities and product types of the mutants are preliminarily screened and identified, and key mutation sites with novel catalytic activity are found, such as F248, Y505, etc. The specific implementation method is shown in Example 2. The present 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 the sequence table SEQ ID NO: 3, and nucleotide sequences encoding these amino acid sequences.

[0021] The present invention applies the artificial "two-step method" terpene precursor high-yield pathway plasmid and fermentation optimization conditions to the Escherichia coli cell factory to achieve heterologous expression of SsDMS mutants and efficient production of novel drimane-type sesquiterpene compounds. The drimane-type compound high-yield system introduces three plasmids, namely, a high-efficiency expression vector containing a mutant SsDMS gene and laboratory terpene precursor high-yield plasmids pCDFDuet-Ipk-IspA-IDI and pRSFDuet-PhoN (△). The nucleotide sequence of plasmid pCDFDuet-Ipk-IspA-IDI is shown in the sequence listing SEQ ID NO: 8, wherein the Ipk amino acid sequence is shown in SEQ ID NO: 5, the IspA amino acid sequence is shown in SEQ ID NO: 6, and the IDI amino acid sequence is shown in SEQ ID NO: 7; the amino acid sequence of the protein encoded by plasmid pRSFDuet-PhoN (△) is shown in the sequence listing SEQ ID NO: 9, and its nucleotide sequence is shown in the sequence listing SEQ ID NO: 10. The plasmid was introduced into the self-made electrocompetent BL21 by electroporation, and the positive single clone was selected for culture, and then fermented to produce the mutant product. The specific implementation method is shown in Example 3.

[0022] The present invention uses a site-directed mutagenesis method on the basis of alanine scanning to select key sites for saturation mutation, that is, to introduce all 20 amino acids at the target sites, obtain more SsDMS high-yield mutants, and further optimize enzyme activity and product specificity. Introduce it into a drimane-type compound high-yield system, ferment each mutant strain, screen and identify the activity and product type of the mutant by TLC and HPLC and other analytical methods, and obtain advantageous mutants that can catalyze the generation of novel drimane-type sesquiterpene compounds, such as F248A, F248Y, Y505W, etc. The specific implementation method is shown in Examples 4 and 5.

[0023] The present invention also performs fermentation production and product confirmation on novel drimane-type sesquiterpene compounds. The efficient expression vector containing the mutant SsDMS gene is combined with the terpene precursor high-yield plasmid to be co-transformed into Escherichia coli to carry out fermentation production of the SsDMS mutant. After the fermentation is completed, the fermentation product is analyzed by TLC and HPLC. After the mutant with novel catalytic activity is fermented by 3L, the fermentation product is separated and purified by silica gel column chromatography, HPLC and other methods after post-treatment, and finally purified by H 1 -NMR, C 13 -HMR and database comparison methods were used to identify and confirm the structure, and a novel drimane-type sesquiterpene compound was obtained. The specific implementation method is shown in Examples 5 and 6.

[0024] The present invention conducts yield analysis on a high-yield mutant strain containing novel activity. A content-related standard curve is established by HPLC for the isolated and confirmed compounds, the high-yield mutant strain is fermented according to the fermentation method of Example 3, and post-processed and HPLC analyzed according to the method of Example 5, and the analysis data is brought into the standard curve of each compound to obtain the yield of the high-yield mutant strain. The specific implementation method is shown in Examples 7 and 8. The yield data are obtained from three parallel experiments under the same experimental conditions.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the SsDMS directed evolution strategy established by the present invention can effectively guide the transformation of other terpene cyclases and has wide applicability; the discovered SsDMS key mutants greatly expand the catalytic function of the enzyme and provide important enzymatic resources for the biosynthesis of novel terpene compounds; the constructed Escherichia coli cell factory realizes the efficient production of novel drimane-type sesquiterpene compounds such as albicanol, bicyclofarnesol, drimane-8α, 11-diol, among which albicanol has significant physiological activity; the novel drimane-type sesquiterpene compounds obtained by efficient production provide more choices for the discovery of drug lead compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the Albicanol standard curve;

[0027] Figure 2 is the Drimenol standard curve;

[0028] Figure 3 is the Bicyclofarnesol standard curve;

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

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

[0031] Figure 6 H for Bicyclofarnesol 1 -NMR spectra;

[0032] Figure 7 C for Bicyclofarnesol 13 -NMR spectra;

[0033] Figure 8 drimane-8α, 11-diol H1 -NMR spectra;

[0034] Fig. 9 C of drimane-8α,11-diol 13 -NMR spectra;

[0035] Fig.10 The results of alanine scanning mutagenesis. DETAILED DESCRIPTION

[0036] Example 1: Design of SsDMS mutation sites

[0037] The cyclase SsDMS described in this example is a type II sesquiterpene cyclase discovered by early genome mining in the laboratory, which can catalyze farnesyl diphosphate (FPP) to generate drimenol pyrophosphorylation products. D303E -FPP-Mg 2+ Based on the crystal structure of the complex protein, the active site was selected in the visualization software PyMol. The amino acids within the radius focus on the aromatic residues in the β domain. A total of 19 amino acids were selected, namely T129, M136, W165, L166, N208, F248, I249, M252, F255, F293, T343, F344, M354, W393, Q497, A498, PS00, and Y505.

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

[0039] Based on laboratory research, this embodiment uses the hydrolase and cyclase fusion expression plasmid pETDuet-SsNDH-SsDMS (R) as a template to construct a SsDMS high-yield mutant. The realization of the SsDMS mutation site adopts a two-step PCR method: the first PCR uses the original plasmid pETDuet-SsNDH-SsDMS (R) as a template, firstly selects 27 bases upstream and downstream as the mutation site primers S and X with the mutation site as the center, and amplifies the upstream and downstream fragments of the mutation site with the F-end primer of the total length of the coding protein and the primer X, and the R-end primer of the total length of the coding protein and the primer X. After gel electrophoresis, the band with the correct number of bases is selected for gel recovery to obtain the upstream and downstream fragments of the mutation site; the second PCR uses the mixture of substances such as upstream and downstream fragments as a template, and uses the full-length coding protein primers F and R to amplify the full-length target mutation band, and obtains the linear target gene containing the designed mutation site through gel electrophoresis experiments and gel recovery experiments. The above primers are shown in Table 1. The PCR amplification experimental system was a 50 μL system, including 2×Phanta Max Buffer 25 μL, ddH2O 18.5 μL, DMSO 2.5 μL, 10 mM dNTPs 1 μL, 0.5 μL each of forward and reverse primers, 1 μL of template, and 2.5 U 1 μL of high-fidelity polymerase (Phanta Max Super-Fidelity DNA Polymerase); the PCR amplification experimental conditions were 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, annealing for 15 s (annealing temperature was calculated based on the higher Tm value of the two primers), 72°C extension (extension time was calculated based on the fragment length 1 min / 1000 bp), a total of 30 cycles, and finally 72°C extension for 10 min.

[0040] Table 1 Mutant primer list

[0041]

[0042]

[0043] The vector pETDuet was cut open using restriction endonucleases BamHI and HindIII, and connected to the full-length target gene containing the mutation site by homologous recombination. The recombinant product was introduced into E.coli DH5α competent cells by chemical transformation, and the monoclonal colony was picked and cultured in LB liquid medium containing 100μg / mL ampicillin for 12-20h, and the plasmid was extracted and sequenced. The sequencing results were compared with the original plasmid sequence without mutation using Snap Gene software. If the mutation site and the recombination connection site were successfully aligned, the efficient expression vector containing the mutation was successfully constructed. Chemical transformation experimental process: The recombinant product was added to the competent cells, left on ice for 30min, heat-shocked in a 42℃ water bath for 65s, cooled on ice for 3min, grown at 37℃ and 230rpm shaking for 40min, 100μg / mL ampicillin was added to the bacterial solution, evenly spread on LB agar solid medium and blown dry, and inverted in a 37℃ incubator for 12h.

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

[0045] In this example, the artificial "two-step method" drimenol high-yielding strain was modified as the base strain, and the in vivo expression of the compound was performed by introducing a high-efficiency fusion expression plasmid containing the correct mutation site.

[0046] The plasmid containing the correct mutation site and the high-yield plasmids pCDFDuet-Ipk-IspA-IDI and pRSFDuet-PhoN (△) of terpenoid precursors were co-introduced into the homemade E. coli BL21 competent cells by electroporation, and the single clone bacterial solution was picked to LB liquid medium containing 100μg / mL ampicillin, 50μg / mL kanamycin and 50μg / mL streptomycin sulfate for about 10 hours. Take out 250μL of bacterial solution in the clean bench and add it to 50mL LB liquid medium containing 100μg / mL ampicillin, 50μg / mL kanamycin, 50μg / mL streptomycin sulfate and 2% glycerol, and culture at 37°C, 230rpm shaking for 2-4 hours until OD 600 =1.3~1.4, cool the bacterial solution in an ice bath for 20 min, add 0.25 mM IpTG (isopropyl BD-1-thiogalactopyranoside) and 10 mM DMAA:ISO=1:2 (3-methyl-2-butene-1ol:3-methyl-3-butene-1ol=1:2) mixture in a clean bench, and culture at 18°C ​​and 200 rpm in a shaking incubator for 72 h to complete the in vivo expression of the high-yield plasmid of drimane-type terpenoid compounds.

[0047] Preparation of E. coli BL21 electroporation competent cells: Take a small amount of E. coli BL21 culture medium frozen in a -80℃ refrigerator and streak it on LB agar solid medium. After culturing in a 37℃ incubator for 12 hours, pick a single clone and transfer it to 5mL LB liquid medium. Cultivate it in a shaker at 37℃ and 230rpm for 12-18 hours. Add 5mL of the culture medium to 1L LB liquid medium in a clean bench and culture it in a shaker at 37℃ and 230rpm for 1-3 hours until the OD reaches 0. 600 =0.5~0.6. Pour the bacterial solution into 10 pre-cooled 50mL centrifuge tubes, and then quickly place them in an ice-water mixture for sudden cooling. After cooling, centrifuge them at 4000rpm for 10min in a low-temperature refrigerated centrifuge (4°C), discard the supernatant, and repeat the above operation with the remaining bacterial solution to collect the bacteria; gently resuspend the bacteria in each tube with a small amount of pre-cooled sterile distilled water, add distilled water to 40mL after combining the bacterial solution, centrifuge them at 4000rpm for 10min in a low-temperature refrigerated centrifuge (4°C), discard the supernatant, and repeat the above operation once; repeat the above steps twice with pre-cooled sterile 10% glycerol, centrifuge them at 4000rpm for 10min in a low-temperature refrigerated centrifuge (4°C), pour out the supernatant for the last time, add 4mL of pre-cooled sterile 10% glycerol, and gently resuspend the cells at the bottom of the centrifuge tube; after resuspension, divide the bacterial solution into 1.5mL centrifuge tubes, immediately put them into liquid nitrogen for quick freezing, store them in a -80°C refrigerator or use them for electrotransformation immediately.

[0048] The specific experimental steps of electroporation are as follows: thaw the electroporation competent cell on ice for 3 min, add an appropriate amount of plasmid to be transferred (the amount of each plasmid added in the three-plasmid transfer system is about 500 ng), place on ice for 5 min, transfer to a pre-cooled electroporation cup and let it stand for 10 min, set the electroporation instrument conditions to U=1500V, R=400Ω, C=25μF, perform electroporation after pre-charging and discharging, quickly add 700μL of LB preheated to 37°C, culture at 230rpm in a 37°C shaker for 1 h, add 100μg / mL ampicillin, 50μg / mL kanamycin and 50μg / mL streptomycin sulfate (relative to agar solid LB plate), mix well and spread on an agar solid LB plate, blow dry and then invert in a 37°C incubator for 12 h. The monoclonal colony on the agar solid LB plate is the E. coli BL21 cell with successful plasmid transfer.

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

[0050] SsDMS site-directed saturation mutagenesis is to mutate the target site to 19 amino acids other than the original amino acid of the site according to the experimental method of Example 2. The 20 amino acids involved in the saturation mutation are glycine G, alanine A, valine V, leucine L, isoleucine I, methionine (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 mutation sites in this example are shown in Table 2.

[0051] Table 2 Saturation mutant primers 2

[0052]

[0053]

[0054]

[0055]

[0056] Example 5: Screening of mutants with novel catalytic activity

[0057] After the construction of high-yield mutants and the in vivo expression of mutant high-yield plasmids, the successfully fermented bacterial broth was transferred to 50 mL centrifuge tubes, centrifuged at 4000 rpm for 20 min in a low-temperature refrigerated centrifuge (4°C), the supernatant was discarded, 3 mL of acetone solution was added to the bacterial precipitate, vortexed and mixed, and ultrasonicated in an ultrasonic cleaner for 4 h, with an ultrasonic frequency of 40 kHz and a power of 200 W. After the ultrasonication, the supernatant was transferred to a 5 mL centrifuge tube and centrifuged at 12000 rpm for 10 min in an ultracentrifuge. An appropriate amount of supernatant was filtered through a 0.22 μm nylon filter membrane into a liquid phase vial for subsequent HPLC analysis, and the remaining supernatant was subjected to TLC analysis. HPLC analysis was performed using an Agilent 1260 Infinity equipped with a YMC-Pack ODS-A column (150×4.6 mm, 5 μm) at a flow rate of 0.8 mL / min. The analysis conditions are described in Table 3, and the analysis results are shown in Table 3. Fig.10 ; Silica gel GF254 silica gel plate was used, and petroleum ether: ethyl acetate = 5:1 was used as the developing agent after spotting, and 10% sulfuric acid-ethanol solution was used as the color developer. Each batch of samples was compared with the wild-type bacteria. If the mutant fermentation sample had new spots or new peaks compared with the wild type, the experiment was repeated. After the results were determined, the new compounds were enriched, separated and identified.

[0058] Table 3 HPLC analysis method of fermentation products

[0059]

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

[0061] Based on the experimental results of Example 5, this example uses the fermentation method of Example 3 to amplify the fermentation of a mutant strain with novel activity by 3L, i.e., 50mL*60 bottles. After the fermentation is completed, the bacterial solution is combined and poured into a 500mL white centrifuge bottle, and after balancing, it is centrifuged at 4000rpm for 25min in a low-temperature refrigerated centrifuge (4°C), and the supernatant is discarded. The above steps are repeated until all bacterial precipitations are collected. The bacterial cells are combined in a white centrifuge bottle, 200mL of acetone is added, and ultrasonic cleaning is performed for 2h in an ultrasonic cleaning instrument, with an ultrasonic frequency of 40kHz and a power of 200W. After the ultrasonic cleaning is completed, ultrafiltration is performed using a Buchner funnel to separate the supernatant and the precipitate. The precipitate continues to add 200mL of acetone and repeats the previous operation twice. The supernatant liquids obtained by combining three times are evaporated in a rotary evaporator to dry the acetone, and the remaining components are extracted three times with ethyl acetate. The upper layer solution obtained by combining three extractions is added with an appropriate amount of saturated sodium sulfate solid, and the water is dried and evaporated in a rotary evaporator to dry the ethyl acetate to obtain a fermentation extract of 3L bacterial cells.

[0062] Take 200-300 mesh silica gel powder of 2-3 times the sample volume to mix the fermentation extract, and use silica gel column chromatography to separate the fermentation products. For the fermentation samples of F248A and Y505W (the fermentation products were identified as albicanol and bicyclofarnesol, respectively), the mobile phase ratio was petroleum ether: ethyl acetate = 15:1, and the color developer was 10% sulfuric acid-ethanol solution. After the elution target new spot appears, it is usually mixed with Drimenol or FOH. The samples containing the new spot after passing through the column are combined, and the mobile phase is dried in a rotary evaporator to obtain a crude product. The crude product was dissolved with an appropriate amount of chromatographic methanol, centrifuged at 12000rpm for 10min in a high-speed centrifuge, filtered through a 0.22μm nylon membrane into a liquid phase vial, and then the new compound was prepared using an Agilent 1260Infinity analytical liquid phase, equipped with an Agilent Poroshell 120 EC-C18 column. The crude product of F248A was semi-preparatively separated using a flow rate of 1mL / min and 70% acetonitrile isocratic elution method, and the crude product of Y505W was semi-preparatively separated using a flow rate of 0.8mL / min and 70% acetonitrile isocratic elution method. For the fermentation sample of F248N (the fermentation product was identified as drimane-8α, 11-diol), the separation mobile phase ratio was petroleum ether: ethyl acetate = 2:1, and the color developer was 10% sulfuric acid-ethanol solution. After eluting the target new point, the samples containing the new point were combined, and the mobile phase was spun dry in a rotary evaporator to obtain the separated product. After the separation of the compounds, the liquid was dried in a rotary evaporator, and an appropriate amount of the dried sample was dissolved in deuterated chloroform and transferred to a nuclear magnetic resonance tube for NMR analysis. The separated product was identified by data comparison.

[0063] like Figure 4-9 As shown, modification of the mutation sites of SsDMS 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. In addition, albicanol has been proven to be cytotoxic and has certain application value.

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

[0065] In this example, a standard curve is established for the compound with ultraviolet absorption obtained in Example 6. Accurately weigh 4 mg of drimane-type terpenoid compound standard, add 1 mL of methanol solution, prepare a standard mother solution with a concentration of 4 mg / mL, dilute with methanol to seven samples with different concentration gradients of 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, respectively, and perform HPLC analysis respectively. The analysis method is the same as shown in Table 3 in Example 5, and the injection volume is 5 μL each time. According to the different peak areas at the corresponding retention time at the absorption wavelength of 210 nm of the drimane-type terpenoid compound samples with different concentration gradients, a standard curve is established ( Figure 1-3 ), and the function is obtained after fitting.

[0066] Example 8: Yield analysis of mutants with novel catalytic activity

[0067] Based on the screening results of Example 5, this example performs a unified yield analysis on mutants with novel catalytic activity. The mutant bacterial solution frozen in a -80°C refrigerator was placed on ice to thaw, 10 μL of the bacterial solution was added to LB liquid culture medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin and 50 μg / mL streptomycin sulfate, and cultured at 230 rpm in a 37°C shaker for about 10 hours, and the mutant bacterial solution was re-fermented according to the fermentation method of Example 3. After the fermentation was completed, the fermentation product was analyzed by HPLC according to the product processing method of Example 5. The HPLC analysis results were integrated, and the peak areas of the new compounds corresponding to the retention time were recorded respectively, and the peak areas were converted into the fermentation yield of the mutant. The fermentation yield of the mutants involved in this example is shown in Table 4.

[0068] In this example, the inventors found that the mutant F248Y strain can completely change the catalytic function of the wild-type enzyme and completely produce the compound albicanol, which fully demonstrates the practicality of the directed transformation 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 SsDMS enzyme mutant, characterized in that: The SsDMS enzyme with an amino acid sequence as shown in SEQ ID NO: 1, wherein 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.

2. The SsDMS enzyme mutant according to claim 1, characterized in that The SsDMS enzyme is a type II terpene cyclase derived from the bacterium Streptomyces showdoensis.

3. The SsDMS enzyme mutant according to claim 1, characterized in that The SsDMS enzyme mutant is obtained by site-directed mutagenesis.

4. A directed evolution method for type II terpene cyclase SsDMS enzyme, characterized in that: Searching for the active cavity near the substrate through the protein co-crystal structure of the SsDMS enzyme-substrate complex Alanine scanning was performed on the amino acids within the range, and saturation mutagenesis was performed on the mutation sites with novel activities.

5. Use of the SsDMS enzyme mutant according to claim 1 in the biosynthesis of terpenoid compounds.

6. A highly efficient fusion expression vector for modifying the site-directed mutation site of type II terpene cyclase SsDMS, characterized in that: The invention relates to an expression vector for fusing a hydrolase and a mutant of the SsDMS enzyme according to claim 1; wherein the amino acid sequence to which the hydrolase and the mutant of the SsDMS enzyme according to claim 1 are fused is that the 397th position of the amino acid sequence shown in SEQ ID NO: 3 is changed from asparagine to glycine, alanine, valine, serine, cysteine, glutamine, aspartic acid or glutamic acid, the 437th position is changed from phenylalanine to glycine, alanine, valine, leucine, methionine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine or histidine, the 438th position is changed from isoleucine to alanine, methionine, phenylalanine, cysteine ​​or histidine, the 686th position is changed from glutamine to glycine, alanine, proline, serine, threonine, cysteine, asparagine or glutamic acid, and the 694th position is changed from tyrosine to alanine, isoleucine, phenylalanine, tryptophan or histidine.

7. A novel Escherichia coli for efficiently producing drimane-type sesquiterpene compounds, characterized in that: The highly efficient fusion expression vector of claim 6 and the expression vector containing the amino acid sequences of SEQ ID NO: 5-7 and SEQ ID NO: 9 are simultaneously introduced into the Escherichia coli cells.

8. Use of the novel Escherichia coli for efficiently producing drimane-type sesquiterpene compounds according to claim 7 in producing drimane-type sesquiterpene compounds.

9. The use according to claim 8, characterized in that: The drimane-type sesquiterpene compounds include one or more compounds selected from the group consisting of drimenol, albicanol, bicyclofarnesol, and drimane-8α,11-diol.

10. A method for producing drimane-type sesquiterpene compounds by using Escherichia coli according to claim 7, characterized in that: The fermentation medium contains 1-5% glycerol, and the bacterial solution is cultured to OD 600 =1.3-1.4, and during induction, isopropyl β-D-1-thiogalactopyranoside, 3-methyl-2-butene-1-ol and 3-methyl-3-butene-1-ol were added.

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