Santalene synthase mutant as well as preparation method and application thereof

Abstract

The invention relates to the field of enzyme engineering, and discloses a sandalene synthase mutant, a preparation method and application thereof, the sandalene synthase mutant comprises one of a sandalene synthase mutant Q33A, a sandalene synthase mutant T34A, a sandalene synthase mutant K133A and a sandalene synthase mutant F452A, and the sandalene synthase mutant is one of the sandalene synthase mutant Q33A, the sandalene synthase mutant T34A and the sandalene synthase mutant F452A. The mutant is obtained by mutating threonine at the 34th position of wild-type santalene synthase into alanine, glutamine at the 33rd position of wild-type santalene synthase into alanine, lysine at the 133rd position of wild-type santalene synthase into alanine and phenylalanine at the 452nd position of wild-type santalene synthase into alanine. The yield of alpha-alkene synthase produced by fermentation of genetically engineered bacteria prepared by transforming the mutant into saccharomycetes is 0.35-2.24 times that of wild santalene synthase.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering, and in particular to a santalene synthase mutant, a preparation method and an application thereof. Background Art

[0002] α-Santalene is a sesquiterpenoid with significant application value. It is one of the main aroma components of natural sandalwood oil and is widely used in fragrances, medicine, and daily chemicals. In plants, α-santalene is synthesized via farnesyl pyrophosphate and santalene synthase (SanSyn). However, the catalytic products of santalene synthase also contain β-santalene, epi-β-santalene, and exo-α-bergamotene. Therefore, the yield of α-santalene produced from plant-derived raw materials is relatively low. Currently, α-santalene is primarily extracted from the heartwood and roots of Indian sandalwood via steam distillation. However, the long growth cycle and increasingly depleted resources of Indian sandalwood limit the large-scale production of α-santalene.

[0003] To increase the content of α-santalene, existing technologies have proposed using genetically engineered bacteria to synthesize sandalwood volatile oil. For example, publication number CN116694609A discloses a santalene synthase mutant, an engineered bacterium, and their uses. This technical solution optimizes the wild-type santalene synthase SanSyn gene and the wild-type santalene synthase SaSSy gene through codon optimization to obtain corresponding wild-type santalene synthase SanSyn mutants and wild-type santalene synthase SaSSy gene mutants. A combination of mutants with a similar ratio of components found in sandalwood plants is screened and transformed into Saccharomyces cerevisiae to obtain an engineered bacterium, which is then used to ferment and synthesize sandalwood volatile oil.

[0004] The present invention discovers that α-santalene can be synthesized into patchouli alcohol under the catalysis of patchouli alcohol synthase. However, the currently known α-santalene synthesis is mainly used in the synthesis of sandalwood volatile oil and has a low yield. Therefore, improving the conversion rate and yield of α-santalene synthase for the synthesis of patchouli alcohol is of great significance for the large-scale production of patchouli alcohol. Summary of the Invention

[0005] To overcome the problem of low α-santalene synthesis yield in the prior art, the present invention provides a santalene synthase mutant and its application. The mutants are obtained by subjecting the wild-type santalene synthase to site-directed mutagenesis at positions 33, 34, 133, and 452 of its amino acid sequence, SEQ ID NO. 1, to obtain santalene synthase mutants Q33A, T34A, K133A, and F452A. When these santalene synthase mutants are prepared into genetically engineered bacteria, the resulting genetically engineered bacteria synthesize α-santalene at a rate 0.35 to 2.24 times that of the wild-type santalene synthase, significantly increasing the microbial synthesis yield of α-santalene.

[0006] The specific technical solutions of the present invention are: A santalene synthase mutant, comprising one of a santalene synthase mutant Q33A, a santalene synthase mutant T34A, a santalene synthase mutant K133A, and a santalene synthase mutant F452A; the amino acid sequence of the santalene synthase mutant Q33A is shown in SEQ ID NO.3; the amino acid sequence of the santalene synthase mutant T34A is shown in SEQ ID NO.4; the amino acid sequence of the santalene synthase mutant K133A is shown in SEQ ID NO.5; and the amino acid sequence of the santalene synthase mutant F452A is shown in SEQ ID NO.6.

[0007] Preferably, the santalene synthase mutant T34A is obtained by mutating the threonine at position 34 of the wild-type santalene synthase having the amino acid sequence shown in SEQ ID NO. 2 to alanine.

[0008] Preferably, the santalene synthase mutant Q33A is obtained by mutating the 33rd glutamine of the wild-type santalene synthase having the amino acid sequence shown in SEQ ID NO. 2 to alanine.

[0009] Preferably, the santalene synthase mutant K133A is obtained by mutating lysine at position 133 of the wild-type santalene synthase with the amino acid sequence shown in SEQ ID NO 2 to alanine.

[0010] Preferably, the santalene synthase mutant F452A is obtained by mutating the phenylalanine at position 452 of the wild-type santalene synthase having the amino acid sequence shown in SEQ ID NO. 2 to alanine.

[0011] Preferably, the nucleotide sequence of the wild-type santalene synthase is shown in SEQ ID NO.1.

[0012] A method for preparing the above-mentioned santalene synthase mutant comprises the following steps: (1) The wild-type santalene synthase gene was ligated into the plasmid pESC to generate the plasmid pESC-SanSyn; (2) Design mutation primers to perform site-directed mutagenesis on the plasmid pESC-SanSyn to produce a plasmid containing the santalene synthase mutant gene.

[0013] (3) The plasmid was transformed into bacteria to express and obtain a santalene synthase mutant.

[0014] Preferably, the mutant primer includes one of Q33A-F and Q33A-R, T34A-F and T34A-R, K133A-F and K133A-R, and F452A-F and F452A-R.

[0015] Preferably, the primer sequence of Q33A-F is shown in SEQ ID NO.7.

[0016] Preferably, the primer sequence of Q33A-R is shown in SEQ ID NO.8.

[0017] Preferably, the primer sequence of T34A-F is shown in SEQ ID NO.9.

[0018] Preferably, the primer sequence of T34A-R is shown as SEQ ID NO.10.

[0019] Preferably, the primer sequence of K133A-F is shown as SEQ ID NO.11.

[0020] Preferably, the primer sequence of K133A-R is shown as SEQ ID NO.12.

[0021] Preferably, the primer sequence of F452A-F is shown as SEQ ID NO.13.

[0022] Preferably, the primer sequence of F452A-R is shown in SEQ ID NO.14.

[0023] A use of the aforementioned santalene synthase mutant in the preparation of α-santalene synthase comprises the following steps: transforming a plasmid containing the santalene synthase mutant gene into yeast to obtain a genetically engineered bacterium, inoculating the genetically engineered bacterium into a defective culture medium for cultivation, picking colonies from the defective culture medium to prepare a seed liquid, inoculating the seed liquid into a fermentation medium for fermentation and cultivation to obtain a culture liquid, and centrifuging the culture liquid to obtain the α-santalene synthase.

[0024] Preferably, n-dodecane is added 10 to 12 hours after the start of fermentation.

[0025] Compared with the existing technology, this application has the following technical effects: The santalene synthase mutant provided by the present invention is obtained by performing site-directed mutagenesis on positions 33, 34, 133 and 452 of the amino acid sequence of the wild-type santalene synthase as a template.

[0026] The santalene synthase mutant provided by the present invention significantly improves the yield of α-santalene synthesized by catalysis compared with the wild-type sandalwood synthase; the α-santalene yield of the santalene synthase mutant Q33A after culturing for 7 days reaches 182.70 mg / L, and the α-santalene yield of the santalene synthase mutant Q33A reaches 0.35 times that of the wild-type sandalwood synthase; the α-santalene yield of the santalene synthase mutant T34A after culturing for 7 days reaches 210.80 mg / L, and the α-santalene yield of the santalene synthase mutant T34A reaches 210.80 mg / L. The yield reached 2.24 times that of the wild-type sandalwood synthase; the α-santalene production of the santalene synthase mutant K133A reached 142.03 mg / L after 7 days of cultivation, and the α-santalene production of the santalene synthase mutant K133A reached 1.51 times that of the wild-type sandalwood synthase; the α-santalene production of the santalene synthase mutant F452A reached 194.88 mg / L after 7 days of cultivation, and the α-santalene production of the santalene synthase mutant F452A reached 2.07 times that of the wild-type sandalwood synthase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the three-dimensional structural model of santalene synthase.

[0028] Figure 2 This is the plasmid map of plasmid pESC-SanSyn. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the embodiments.

[0030] Example 1: The protein structure prediction tool (specific tool) was used to model the structure of santalene synthase (source). After evaluation and verification, a reliable three-dimensional structural model was obtained. The three-dimensional structural model is shown in the figure below. Figure 1 shown.

[0031] Example 2: Using PCR technology, using the plasmid pESC-SanSyn expressing a wild-type santalene synthase gene (the nucleotide sequence of the wild-type santalene synthase is shown in SEQ ID NO. 1, and the amino acid sequence of the wild-type santalene synthase is shown in SEQ ID NO. 2; the wild-type santalene synthase is derived from the plant Santalum album. The amino acid sequence of the wild-type santalene synthase was obtained by extracting mRNA from sandalwood leaves and performing reverse transcription-PCR, and was named ClTps2-1; its CNBI accession number is HQ452480.1) as a template, site-directed mutagenesis was performed at sites 33, 34, 133, and 452 of the santalene synthase to obtain santalene synthase mutants Q33A, T34A, K133A, and F452A.

[0032] The amino acid sequence of the santalene synthase mutant Q33A is shown in SEQ ID NO.3.

[0033] The amino acid sequence of the santalene synthase mutant T34A is shown in SEQ ID NO.4.

[0034] The amino acid sequence of the santalene synthase mutant K133A is shown in SEQ ID NO.5.

[0035] The amino acid sequence of the santalene synthase mutant F452A is shown in SEQ ID NO.6.

[0036] The PCR reaction system was as follows: 25 μL of PrimeSTAR Max (purchased from Takara), 1 μL each of the forward primer, reverse primer, and template DNA, and 22 μL of sterilized water.

[0037] The preparation method of the santalene synthase mutant Q33A comprises the following steps: Mutation primers Q33A-F and Q33A-R were designed. The primer sequences for Q33A-F are shown in SEQ ID NO. 7, and the primer sequence for Q33A-R is shown in SEQ ID NO. 8. PCR amplification was performed using the mutant primers and the plasmid pESC-SanSyn as a template. Ligation reactions were performed at 50°C overnight using 5 μL of OneStep Cloning enzyme and 5 μL of sample. 10 μL of the ligation product was added to competent E. coli DH5α cells, mixed thoroughly, and incubated on ice for 5 minutes. The mixed cell suspension and plasmid were then heat-shocked for 90 seconds in a 42°C water bath. After the heat shock, the cells were placed in ice water for 2 minutes, and 600 μL of LB medium was added, mixed thoroughly, and allowed to recover at 37°C for 45 minutes. The cells were then plated onto LB plates containing ampicillin resistance and cultured overnight at 37°C. Transformants from the plates were sent to Jerui Biotechnology Co., Ltd. for sequencing verification.

[0038] Agarose gel electrophoresis is used to separate the target DNA fragments. The agarose gel containing the target fragments is rapidly cut. The cut agarose gel is weighed and transferred to a clean 1.5 mL centrifuge tube. 100 μL of GDP solution is added to every 100 mg of agarose gel. The tube is then placed in a metal bath to melt the gel until completely dissolved. Once completely dissolved, the solution on the lid is centrifuged and transferred to a HiPureDNA Column. The column is placed in a collection tube and centrifuged at 12,000 rpm for 30 seconds. The filtrate in the cannula is discarded, and 300 μL of GDP buffer is added to the column. The column is allowed to stand for approximately 1 minute and then centrifuged at 12,000 rpm for 30 seconds. The filtrate is again discarded and 600 μL of DW2 buffer is added to the column. The column is centrifuged at 12,000 rpm for 30 seconds. The filtrate is again discarded and the column is washed again. It is important to dilute the DW2 buffer with anhydrous ethanol before use. Discard the filtrate, return the column to the collection tube, and centrifuge at 12,000 rpm for 3 minutes. Place the column in a clean 1.5 mL centrifuge tube, open the lid, and dry for approximately 10 minutes to remove any residual ethanol. Add 20-40 μL of preheated double-distilled water at 65°C and let stand for approximately 2 minutes. Centrifuge at 12,000 rpm for 2 minutes. Determine the concentration of the solution in the 1.5 mL centrifuge tube using a Nanodrop analyzer and store the final DNA solution in a -20°C freezer.

[0039] The preparation method of the santalene synthase mutant T34A comprises the following steps: Mutation primers T34A-F and T34A-R were designed. The primer sequences for T34A-F are shown in SEQ ID NO. 9, and the primer sequence for T34A-R is shown in SEQ ID NO. 10. PCR amplification was performed using the mutant primers and the plasmid pESC-SanSyn as a template. Ligation reactions were performed at 50°C overnight using 5 μL of One-Step Cloning enzyme and 5 μL of sample. 10 μL of the ligation product was added to competent E. coli DH5α cells, mixed thoroughly, and incubated on ice for 5 minutes. The mixed cell suspension and plasmid were then heat-shocked for 90 seconds in a 42°C water bath. After the heat shock, the cells were placed in ice water for 2 minutes, and 600 μL of LB medium was added, mixed thoroughly, and allowed to recover at 37°C for 45 minutes. The cells were then plated onto LB plates containing ampicillin resistance and cultured overnight at 37°C. Transformants from the plates were sent to Jerui Biotechnology Co., Ltd. for sequencing verification.

[0040] Agarose gel electrophoresis is used to separate the target DNA fragments. The agarose gel containing the target fragments is rapidly cut. The cut agarose gel is weighed and transferred to a clean 1.5 mL centrifuge tube. 100 μL of GDP solution is added to every 100 mg of agarose gel. The tube is then placed in a metal bath to melt the gel until completely dissolved. Once completely dissolved, the solution on the lid is centrifuged and transferred to a HiPureDNA Column. The column is placed in a collection tube and centrifuged at 12,000 rpm for 30 seconds. The filtrate in the cannula is discarded, and 300 μL of GDP buffer is added to the column. The column is allowed to stand for approximately 1 minute and then centrifuged at 12,000 rpm for 30 seconds. The filtrate is again discarded and 600 μL of DW2 buffer is added to the column. The column is centrifuged at 12,000 rpm for 30 seconds. The filtrate is again discarded and the column is washed again. It is important to dilute the DW2 buffer with anhydrous ethanol before use. Discard the filtrate, return the column to the collection tube, and centrifuge at 12,000 rpm for 3 minutes. Place the column in a clean 1.5 mL centrifuge tube, open the lid, and dry for approximately 10 minutes to remove any residual ethanol. Add 20-40 μL of preheated double-distilled water at 65°C and let stand for approximately 2 minutes. Centrifuge at 12,000 rpm for 2 minutes. Determine the concentration of the solution in the 1.5 mL centrifuge tube using a Nanodrop analyzer and store the final DNA solution in a -20°C freezer.

[0041] The preparation method of the santalene synthase mutant K133A comprises the following steps: Mutation primers K133A-F and K133A-R were designed. The primer sequences for K133A-F are shown in SEQ ID NO. 11, and the primer sequence for K133A-R is shown in SEQ ID NO. 12. PCR amplification was performed using the mutant primers and the plasmid pESC-SanSyn as a template. Ligation reactions were performed at 50°C overnight using 5 μL of One Step Cloning enzyme and 5 μL of sample. 10 μL of the ligation product was added to competent E. coli DH5α cells, mixed thoroughly, and incubated on ice for 5 minutes. The mixed cell suspension and plasmid were then heat-shocked for 90 seconds in a 42°C water bath. After the heat shock, the cells were placed in ice water for 2 minutes, and 600 μL of LB medium was added, mixed thoroughly, and allowed to recover at 37°C for 45 minutes. The cells were then plated onto LB plates containing ampicillin resistance markers and cultured overnight at 37°C. Transformants from the plates were sent to Jerui Biotechnology Co., Ltd. for sequencing verification.

[0042] Agarose gel electrophoresis is used to separate the target DNA fragments. The agarose gel containing the target fragments is rapidly cut. The cut agarose gel is weighed and transferred to a clean 1.5 mL centrifuge tube. 100 μL of GDP solution is added to every 100 mg of agarose gel. The tube is then placed in a metal bath to melt the gel until completely dissolved. Once completely dissolved, the solution on the lid is centrifuged and transferred to a HiPureDNA Column. The column is placed in a collection tube and centrifuged at 12,000 rpm for 30 seconds. The filtrate in the cannula is discarded, and 300 μL of GDP buffer is added to the column. The column is allowed to stand for approximately 1 minute and then centrifuged at 12,000 rpm for 30 seconds. The filtrate is again discarded and 600 μL of DW2 buffer is added to the column. The column is centrifuged at 12,000 rpm for 30 seconds. The filtrate is again discarded and the column is washed again. It is important to dilute the DW2 buffer with anhydrous ethanol before use. Discard the filtrate, return the column to the collection tube, and centrifuge at 12,000 rpm for 3 minutes. Place the column in a clean 1.5 mL centrifuge tube, open the lid, and dry for approximately 10 minutes to remove any residual ethanol. Add 20-40 μL of preheated double-distilled water at 65°C and let stand for approximately 2 minutes. Centrifuge at 12,000 rpm for 2 minutes. Determine the concentration of the solution in the 1.5 mL centrifuge tube using a Nanodrop analyzer and store the final DNA solution in a -20°C freezer.

[0043] The preparation method of the santalene synthase mutant F452A comprises the following steps: Mutation primers F452A-F and F452A-R were designed. The primer sequences for F452A-F are shown in SEQ ID NO. 13, and the primer sequence for F452A-R is shown in SEQ ID NO. 14. PCR amplification was performed using the mutant primers and the plasmid pESC-SanSyn as a template. Ligation reactions were performed at 50°C overnight using 5 μL of One Step Cloning enzyme and 5 μL of sample. 10 μL of the ligation product was added to competent E. coli DH5α cells, mixed thoroughly, and incubated on ice for 5 minutes. The mixed cell suspension and plasmid were then heat-shocked for 90 seconds in a 42°C water bath. After the heat shock, the cells were placed in ice water for 2 minutes, and 600 μL of LB medium was added, mixed thoroughly, and allowed to recover at 37°C for 45 minutes. The cells were then plated onto LB plates containing ampicillin resistance and cultured overnight at 37°C. Transformants from the plates were sent to Jerui Biotechnology Co., Ltd. for sequencing verification.

[0044] Agarose gel electrophoresis is used to separate the target DNA fragments. The agarose gel containing the target fragments is rapidly cut. The cut agarose gel is weighed and transferred to a clean 1.5 mL centrifuge tube. 100 μL of GDP solution is added to every 100 mg of agarose gel. The tube is then placed in a metal bath to melt the gel until completely dissolved. Once completely dissolved, the solution on the lid is centrifuged and the mixture is transferred to a HiPureDNA Column, placed in a collection tube, and centrifuged at 12,000 rpm for 30 seconds. Discard the filtrate from the cannula, add 300 μL of GDP buffer to the column, let it sit for about 1 minute, and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate again, add 600 μL of DW2 buffer to the column, and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate again and wash the column again. It is important to dilute the DW2 buffer with anhydrous ethanol before use. Discard the filtrate, return the column to the collection tube, and centrifuge at 12,000 rpm for 3 minutes. Place the column in a clean 1.5 mL centrifuge tube, open the lid, and dry for approximately 10 minutes to remove any residual ethanol. Add 20-40 μL of preheated double-distilled water at 65°C and let stand for approximately 2 minutes. Centrifuge at 12,000 rpm for 2 minutes. Determine the concentration of the solution in the 1.5 mL centrifuge tube using a Nanodrop analyzer and store the final DNA solution in a -20°C freezer.

[0045] Example 3: Plasmids containing santalene synthase mutants Q33A, T34A, K133A, F452A, and wild-type santalene synthase genes were transformed into Saccharomyces cerevisiae REL003 to prepare genetically engineered bacteria. The genetically engineered bacteria were spread onto corresponding defective solid plates and cultured at 30°C for 3 days. Colonies on the corresponding solid plates were picked and inoculated into YPD test tubes for activation at 30°C for 24 hours. The bacteria in the test tubes were then inoculated into conical flasks containing 20 mL of seed medium (YPD medium) and continued to be cultured. The initial OD600 was 0.3. After culturing for 12 to 14 hours, the cultured seed solution was inoculated into 50 mL of YPD fermentation medium and cultured for 7 days. The initial OD600 was 0.3. n-Dodecane was added 10 to 12 hours after the start of fermentation to extract α-santalene.

[0046] The formula of YPD medium is 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone.

[0047] The entire fermentation broth was transferred to a centrifuge tube and centrifuged at high speed. The supernatant was removed and dehydrated, centrifuged at high speed, and the supernatant was transferred to a gas chromatography vial for gas chromatography analysis. The gas chromatography conditions were as follows: column: HP-5 (30 m × 0.32 mm, 0.25 μm, Agilent, USA); injection port temperature: 250°C; temperature program: initial column temperature: 100°C, hold for 2 min, then increase to 210°C at a rate of 10°C / min, then to 290°C at a rate of 20°C / min, hold for 5 min. Injection method: split injection, injection volume: 1 μL, split ratio: 30:1; flame ionization detector (FID) temperature: 280°C; hydrogen flow rate: 30 mL / min; air flow rate: 300 mL / min; makeup gas flow rate (N2): 15 mL / min.

[0048] The concentration of α-santalene in the fermentation broth is shown in Table 1.

[0049] Table 1 Concentration of α-santalene in fermentation broth santalene synthase Concentration of α-santalene (mg / L) Wild-type santalene synthase 93.69 Santalene synthase mutant Q33A 182.70 Santalene synthase mutant T34A 210.80 Santalene synthase mutant K133A 142.03 Santalene synthase mutant F452A 194.88 As shown in Table 1, the concentration of α-santalene in the fermentation broth of the wild-type santalene synthase reached 93.69 mg / L after seven days of fermentation. The α-santalene production of the santalene synthase mutant Q33A reached 182.70 mg / L after seven days of cultivation, which was 0.35 times that of the wild-type sandalwood synthase. The α-santalene production of the santalene synthase mutant T34A reached 210.80 mg / L after seven days of cultivation, which was 0.35 times that of the wild-type sandalwood synthase. The production of α-santalene reached 2.24 times that of the wild-type sandalwood synthase; the production of α-santalene of the santalene synthase mutant K133A reached 142.03 mg / L after 7 days of cultivation, and the α-santalene production of the santalene synthase mutant K133A reached 1.51 times that of the wild-type sandalwood synthase; the production of α-santalene of the santalene synthase mutant F452A reached 194.88 mg / L after 7 days of cultivation, and the α-santalene production of the santalene synthase mutant F452A reached 2.07 times that of the wild-type sandalwood synthase.

[0050] The above results show that the santalene synthase mutants obtained in the present invention have higher α-santalene production than the wild-type santalene synthase. Among them, the α-santalene production of the mutant T34A is 2.24 times that of the wild-type santalene synthase. Compared with the wild-type santalene synthase, the santalene synthase mutants obtained in the present invention can significantly increase the synthesis amount of α-santalene.

[0051] Example 4: The genetically engineered bacteria of the santalene synthase mutant T34A obtained in Example 3 were cultured in a fermenter, including the following steps: a single colony was picked from YPD solid medium and inoculated into a 5 mL YPD test tube, incubated on a shaker at 30°C for approximately 24 hours. The culture was then transferred to 50 mL YPD seed medium at an initial OD600 concentration of 0.3 and incubated on a shaker at 30°C for approximately 12 hours. The cultured seed solution was then transferred to three 100 mL bottles of YPD medium at an initial OD600 concentration of 0.3. After incubation at 30°C for approximately 20 hours, the culture was prepared for inoculation into the fermenter. Prior to inoculation of the fermenter, equipment preparation was performed, including zero calibration of the dissolved oxygen electrode using saturated sodium sulfite and calibration of the pH electrode using standard solutions at pH 4 and 7, respectively. Next, the feed bottle, electrode, and vent filter were installed, and the condensate water system was connected. Once the equipment was installed, the inoculation was ignited through the inoculation port, and the dissolved oxygen level was set to 100%. The fermentation temperature was set at 30°C, and the pH was adjusted to between 5.5 and 6 using ammonia. The air flow was controlled at 1.5 vvm, and the agitation rate was set at 200 r / min. When the dissolved oxygen in the fermenter dropped to 40%, the air flow was adjusted to 2.5 vvm, and the propeller speed was automatically adjusted between 200 and 800 r / min based on the dissolved oxygen. Finally, the feed rate was set according to the sugar consumption rate, and the residual sugar concentration in the initial culture medium was controlled at around 1 g / L to ensure continuous cell growth.

[0052] Trace element solution: 15g / L EDTA, 10.2g / L ZnSO4·7H2O, 5.12g / L FeSO4·7H2O, 3.84g / L CaCl2·2H2O, 0.86g / L CoCl2·6H2O, 0.56g / L Na2MoO4·2H2O, 0.5g / L MnCl2·4H2O, and 0.5g / L CuSO4.

[0053] Vitamin solution: 25 g / L inositol, 1 g / L calcium pantothenate, 1 g / L niacin, 1 g / L thiamine hydrochloride, 1 g / L pyridoxine, 0.2 g / L p-aminobenzoic acid, 0.05 g / L biotin; filter sterilize and store at 4°C.

[0054] Culture medium in a 5-L fermenter (3 L): glucose 75 g (needs to be sterilized separately at 115°C for 20 min), Peptone 60 g, Yeast extract 30 g, 3 g each of Met / His / Leu / Ura, 1.8 g of MgSO4 (anhydrous), 36 mL of vitamin solution, and 30 mL of trace elements.

[0055] Feed 1 (2 L): glucose 700 g / L (needs to be sterilized separately at 115°C for 20 min), Peptone 20 g / L, Yeast extract 10 g / L, Met / His / Leu / Ura 1 g / L each, MgSO4 (anhydrous) 0.6 g / L, vitamin solution 12 ml / L, trace elements 10 ml / L.

[0056] Around 16 hours into fermentation, the initial glucose in the fermenter was depleted, and a high-concentration glucose feed of 700 g / L was subsequently added to the tank, keeping the residual sugar at 1 g / L. Around the 32nd hour of fermentation, the OD600 reached 47.3, at which point 10% n-dodecane was added for a two-phase fermentation. After rapid growth for 40.2 hours, the OD600 gradually stabilized, reaching a peak of 70.7 at 54.3 hours. At a fermentation time of 159.1 hours, α-santalene production reached a peak of 1557.4 mg / L.

[0057] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A santalene synthase mutant, characterized in that: The santalene synthase mutant includes one of the santalene synthase mutant Q33A, the santalene synthase mutant T34A, the santalene synthase mutant K133A and the santalene synthase mutant F452A; the amino acid sequence of the santalene synthase mutant Q33A is shown in SEQ ID NO. 3; the amino acid sequence of the santalene synthase mutant T34A is shown in SEQ ID NO. 4; the amino acid sequence of the santalene synthase mutant K133A is shown in SEQ ID NO. 5; and the amino acid sequence of the santalene synthase mutant F452A is shown in SEQ ID NO.

6.

2. The santalene synthase mutant according to claim 1, wherein The santalene synthase mutant T34A is obtained by mutating the threonine at position 34 of the wild-type santalene synthase with the amino acid sequence shown in SEQ ID NO. 2 to alanine.

3. The santalene synthase mutant according to claim 1, wherein The santalene synthase mutant Q33A is obtained by mutating the 33rd glutamine of the wild-type santalene synthase with the amino acid sequence shown in SEQ ID NO. 2 to alanine.

4. The santalene synthase mutant according to claim 1, wherein The santalene synthase mutant K133A is obtained by mutating the 133rd lysine of the wild-type santalene synthase with the amino acid sequence shown in SEQ ID NO. 2 to alanine.

5. The santalene synthase mutant according to claim 1, wherein The santalene synthase mutant F452A is obtained by mutating the phenylalanine at position 452 of the wild-type santalene synthase with the amino acid sequence shown in SEQ ID NO. 2 to alanine.

6. The santalene synthase mutant according to claim 2, 3, 4 or 5, characterized in that: The nucleotide sequence of the wild-type santalene synthase is shown in SEQ ID NO.

1.

7. A method for preparing the santalene synthase mutant according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) The wild-type santalene synthase gene was ligated into the plasmid pESC to generate the plasmid pESC-SanSyn; (2) Designing mutation primers to perform site-directed mutagenesis on the plasmid pESC-SanSyn to produce a plasmid containing the santalene synthase mutant gene; (3) The plasmid was transformed into bacteria to express the santalene synthase mutant.

8. The preparation method according to claim 7, characterized in that: The mutation primers included one of Q33A-F and Q33A-R, T34A-F and T34A-R, K133A-F and K133A-R, and F452A-F and F452A-R.

9. Use of the santalene synthase mutant according to any one of claims 1 to 6 in the preparation of α-santalene synthase, characterized in that: The following steps are involved: A plasmid containing a mutant gene of santalene synthase is transformed into yeast to obtain a genetically engineered bacterium, the genetically engineered bacterium is inoculated into a defective culture medium for cultivation, colonies in the defective culture medium are picked to prepare a seed liquid, the seed liquid is inoculated into a fermentation medium for fermentation cultivation to obtain a culture liquid, and the culture liquid is centrifuged to obtain α-santalene synthase.

10. The use according to claim 9, characterized in that: n-Dodecane was added 10-12 h after the start of fermentation.

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