Fermentation method of strain for synthesizing sclareol
By constructing an engineered strain and optimizing the fermentation process, the problems of low equipment utilization and high dissolved oxygen content in the existing technology were solved, and efficient and low-cost sclareol production was achieved. The utilization rate and dissolved oxygen content of the fermentation tank were improved, the operation process was simplified, and the yield and synthesis efficiency of sclareol were increased.
Patent Information
- Application Number
- CN202510766342.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-10
- Publication Date
- 2025-10-03
AI Technical Summary
The existing fermentation method for preparing sclareol has the disadvantages of low equipment utilization, high dissolved oxygen requirements and complex operation, which affects bacterial growth and limits fermentation yield.
An engineered strain was constructed, the intracellular metabolic pathway was optimized, key enzyme genes were overexpressed, and cigar tobacco buds were used as the only raw material. Efficient sclareol synthesis was achieved through batch fed high-density fermentation combined with low-speed stirring and moderate dissolved oxygen control.
The utilization rate and dissolved oxygen content of the fermentation tank are improved, the operation is simplified, the cost is reduced, the yield and synthesis efficiency of sclareol are increased, and green and efficient production is achieved.
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Figure CN120738232A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a strain fermentation method, in particular to a strain fermentation method for synthesizing sclareol. Background Art
[0002] Sclareol is a key raw material for the synthesis of the high-grade ambergris flavoring agent ambroxan and the tobacco flavoring agent sclareolide. Traditional methods for extracting sclareol from sclareol are significantly limited by raw materials. A currently under-researched approach is to produce sclareol via fermentation using gene-edited recombinant Saccharomyces cerevisiae, which addresses this raw material limitation. To improve the yield of sclareol fermentation and enable industrial application, current research is focused on achieving high-density fermentation using fed-batch feeding. Initially, fermentation occurs slowly. After a 12-14 hour lag phase, the cells enter the logarithmic growth phase. At this point, batch-feeding of culture medium overcomes the growth inhibition caused by high substrate concentrations, enabling high-density fermentation. Because high-density fermentation involves batch-feeding of culture medium, eliminating effluents, the initial fermentation tank fill factor is very low. Furthermore, the fermentation requires a long lag phase, during which no feed is added, maintaining a low fill factor. Batch feeding is initiated after the logarithmic growth phase, gradually increasing the fill factor.
[0003] Therefore, this method has the disadvantage of low equipment utilization, which limits fermentation yield. In addition, high-density fermentation requires high dissolved oxygen levels. In process control, dissolved oxygen levels can be increased by increasing the agitator shaft speed and increasing the fermentation tank pressure. However, the increased speed increases the shear force on the fermentation liquid and the increased pressure, which can affect bacterial growth. In view of this, the present invention has designed a strain fermentation method for synthesizing sclareol. Summary of the Invention
[0004] The main purpose of the present disclosure is to provide a strain fermentation method for synthesizing sclareol, so as to effectively solve the problems raised by the inventors in the above background technology.
[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:
[0006] A strain fermentation method for synthesizing sclareol comprises the following steps:
[0007] Step 1: constructing a strain, constructing a biosynthetic pathway for sclareol in a host strain, and optimizing the intracellular mevalonate metabolic pathway and the central metabolic pathway, thereby obtaining an engineered bacterial strain A; the host strain is Pichia pastoris; or, overexpressing or knocking out metabolic regulatory factors in the above-obtained engineered bacterial strain A, thereby obtaining an engineered bacterial strain B; or, using a cell compartmentalization strategy in the above-obtained engineered bacterial strain B to target the synthetic pathway to the peroxisome and optimize it, thereby obtaining an engineered bacterial strain C;
[0008] Step 2: increasing the host to increase the precursor of sclareol synthesis - digoxin pyrophosphate (GGPP), thereby allowing more GGPP to flow into the synthesis process of sclareol, wherein the host is brewing mother liquor;
[0009] Step 3: recombining Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica is constructed by overexpressing the mutant gene of lisdendial pyrophosphate synthase CalLPPS, the gene of sclareol synthase SSTPS, and the gene of geranylgeranyl pyrophosphate synthase SaGGPP in Yarrowia lipolytica po1f, and expressing the gene of 3-hydroxy-3-methylglutaryl coester A reductase tHMGR and the gene of isopentenyl pyrophosphate isomerase idi;
[0010] Step 4: Fermentation of sclareol. The engineered bacteria are connected to the seed tank, and the culture medium is added to obtain the seed liquid. The seed liquid first enters the first-level tank body, and the culture medium is added. After preliminary fermentation, the culture liquid is obtained. The culture liquid in the first-level tank body first enters the first group of second-level tank bodies, and is fermented at a high density after batch feeding. The culture liquid after preliminary fermentation in the first-level tank body then enters the next group of second-level tank bodies in turn, and is fermented at a high density after batch feeding.
[0011] Step 5: Using other flower buds to ferment and produce sclareol, the other flower buds are selected from cigar tobacco buds, mixing cigar tobacco bud powder with deionized water, adding edible yeast after sterilization, and then fermenting at 25-35° C. and 100-300 rpm / min for 24-30 hours; the mass ratio of the cigar tobacco bud powder to deionized water is (1-15):100;
[0012] Step 6: Purify sclareol from the fermentation broth of the strain.
[0013] Preferably, in the step 1, the engineered bacterial strain A is regulated by integrating the sclareol synthase gene (SsLPPS) and the sclareol synthase gene (SSTPS) into the chromosome PNSII-5 site in the host strain, optimizing the integration of at least one or more synthase genes or isoenzymes with the same / similar functions in the host strain mevalonate pathway into the host strain chromosome, weakening the competitive pathway for squalene synthesis, increasing the supply of precursor acetyl-CoA, and increasing the supply of reducing power NADPH.
[0014] Preferably, in the step 2, the process of constructing the engineered strain of the brewer's yeast is as follows: 1) expressing the key enzyme gene for sclareol synthesis in Saccharomyces cerevisiae; thereby allowing more GGPP to flow into the synthesis process of sclareol; the key enzyme genes are: lysene diol pyrophosphate synthase gene (LPPs) and sclareol synthase gene (Tps); 2) overexpressing the related enzyme genes for GGPP synthesis in Saccharomyces cerevisiae: thereby increasing the precursor substances for the host sclareol synthesis; the related enzyme genes are: a combination of two or more enzyme genes among the hydroxymethyl-CoA reductase gene (HMG1), the farnesyl pyrophosphate synthase gene (ERG20) and the dioxinyl pyrophosphate synthase gene (BTS1).
[0015] Preferably, in the step three, a single colony of the recombinant Yarrowia lipolytica is picked and inoculated into 25 mL / 250 ml of YPD liquid medium, cultured at 30 ° C and 220 rpm for 1 day to obtain a seed solution, and the seed solution is inoculated into a 5L fermentor at an inoculum volume ratio of 2%, wherein the fermentor contains 4L of YPD fermentation medium, and the fermentation is carried out at 30 ° C, the stirring speed is 800 rpm, the ventilation ratio is 1 vvm, the dissolved oxygen is 10%-20%, 5M sodium hydroxide is added to adjust the pH to 5.5, and the glucose content is maintained at 5 g / L by feeding. The fermentation is 168-192 hours to obtain sclareol.
[0016] Preferably, in step four, the culture medium in the feed tank first enters the dissolved oxygen tank, mixes with the air introduced by the air supply mechanism, and then enters the secondary tank body: during fermentation, the speed of the high-speed stirring shaft is 2000r / min, the speed of the low-speed stirring shaft is 200r / min, the filling coefficient of the primary tank body after the culture medium is added is 0.8, the initial filling coefficient of the secondary tank body is 0.3, and the filling coefficient is 0.8 when the fermentation is completed.
[0017] Preferably, in step 5, the preparation method of the cigar tobacco bud powder is: washing and drying the cigar tobacco buds, grinding them, and passing them through a 40-80 mesh sieve to obtain the cigar tobacco bud powder.
[0018] Preferably, in the step 5, the cigar tobacco buds are washed and dried and then ground, and passed through a 40-80 mesh sieve to obtain cigar tobacco bud powder; the tobacco bud powder is then mixed with deionized water in a mass ratio of (1-15):100, and the mixture is sterilized by moist heat at 110-130° C. for 15-20 min to obtain a cigar tobacco bud culture medium; edible yeast is added to the above-obtained cigar tobacco bud culture medium at an inoculum amount of 0.5%-3%, and the mixture is fermented at 25-35° C. and 100-300 rpm / min for 24-30 h to obtain a sclareol sample.
[0019] Preferably, in step six, the fermentation broth is centrifuged at 8000 rpm for 15 minutes, the upper bacterial liquid is removed, the lower bacterial body is collected, and dried; the bacterial body is crushed into 100-200 mesh powder for standby use; the bacterial body powder is mixed with an organic solvent in a certain proportion, and the sclareol contained in the bacterial body is extracted with microwave-assisted extraction at a certain temperature. After extraction for a certain time, liquid-solid separation is performed to collect the extract, and the extract is concentrated to obtain an extract; the sclareol in the extract is extracted with a certain amount of acetone aqueous solution to remove impurities; the collected filtrate is crystallized at a certain temperature for a period of time to obtain purified sclareol.
[0020] In view of this, compared with the prior art, the beneficial effects of the present invention are:
[0021] (1) In the present application, the construction method involves expressing two key enzymes for the synthesis of plant-derived sclareol - lysene diol pyrophosphate synthase and sclareol synthase, and overexpressing a combination of one or more endogenous enzyme genes in yeast, including the key enzyme of the mevalonate pathway - hydroxymethyl-CoA reductase, and two enzymes of the isopentenyl pyrophosphate pathway - farnesyl pyrophosphate synthase and dioxyl pyrophosphate synthase, which can efficiently produce sclareol.
[0022] (2) In the present application, the utilization rate of the fermentation tank can be improved, the yield of the fermentation product can be increased, and the dissolved oxygen content of the fermentation liquid can be increased during high-density fermentation under low-speed stirring conditions to ensure the yield of the strain after fermentation, thereby increasing the yield of sclareol.
[0023] (3) In the present application, by comparison, tobacco buds are used as the only raw material, no additional nutrients are added, and edible yeast is used as the fermentation agent to ferment the tobacco buds, thereby achieving green and efficient synthesis of sclareol: 175 mg / ml of sclareol can be synthesized within 24 hours of fermentation. The synthesis efficiency of sclareol is extremely high, the operation is simple, the cost is extremely low, the controllability is high, and the yield of sclareol is high. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Shown is a flow chart of the strain fermentation method for synthesizing sclareol provided by the present invention. DETAILED DESCRIPTION
[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0026] See also Figure 1 , the present invention provides the following embodiments:
[0027] A strain fermentation method for synthesizing sclareol comprises the following steps:
[0028] Step 1: constructing a strain, constructing a biosynthetic pathway for sclareol in a host strain, and optimizing the intracellular mevalonate metabolic pathway and the central metabolic pathway, thereby obtaining an engineered bacterial strain A; the host strain is Pichia pastoris; or, overexpressing or knocking out metabolic regulatory factors in the above-obtained engineered bacterial strain A, thereby obtaining an engineered bacterial strain B; or, using a cell compartmentalization strategy in the above-obtained engineered bacterial strain B to target the synthetic pathway to the peroxisome and optimize it, thereby obtaining an engineered bacterial strain C;
[0029] Step 2: increasing the host to increase the precursor of sclareol synthesis - digoxin pyrophosphate (GGPP), thereby allowing more GGPP to flow into the synthesis process of sclareol, wherein the host is brewing mother liquor;
[0030] Step 3: recombining Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica is constructed by overexpressing the mutant gene of lisdendial pyrophosphate synthase CalLPPS, the gene of sclareol synthase SSTPS, and the gene of geranylgeranyl pyrophosphate synthase SaGGPP in Yarrowia lipolytica po1f, and expressing the gene of 3-hydroxy-3-methylglutaryl coester A reductase tHMGR and the gene of isopentenyl pyrophosphate isomerase idi;
[0031] Step 4: Fermentation of sclareol. The engineered bacteria are connected to the seed tank, and the culture medium is added to obtain the seed liquid. The seed liquid first enters the first-level tank body, and the culture medium is added. After preliminary fermentation, the culture liquid is obtained. The culture liquid in the first-level tank body first enters the first group of second-level tank bodies, and is fermented at a high density after batch feeding. The culture liquid after preliminary fermentation in the first-level tank body then enters the next group of second-level tank bodies in turn, and is fermented at a high density after batch feeding.
[0032] Step 5: Using other flower buds to ferment and produce sclareol, the other flower buds are selected from cigar tobacco buds, mixing cigar tobacco bud powder with deionized water, adding edible yeast after sterilization, and then fermenting at 25-35° C. and 100-300 rpm / min for 24-30 hours; the mass ratio of the cigar tobacco bud powder to deionized water is (1-15):100;
[0033] Step 6: Purify sclareol from the fermentation broth of the strain.
[0034] Specifically, in step 1, the engineered bacterial strain A is regulated by integrating the sclareol synthase gene (SsLPPS) and the sclareol synthase gene (SSTPS) into the chromosome PNSII-5 site in the host strain, optimizing the integration of at least one or more synthase genes or isoenzymes with the same / similar functions in the host strain mevalonate pathway into the host strain chromosome, weakening the competitive pathway for squalene synthesis, increasing the supply of precursor acetyl-CoA, and increasing the supply of reducing power NADPH.
[0035] Specifically, in the step 2, the process of constructing the engineered strain of the brewer's yeast is as follows: 1) expressing the key enzyme gene for sclareol synthesis in Saccharomyces cerevisiae; thereby allowing more GGPP to flow into the synthesis process of sclareol; the key enzyme genes are: lysene diol pyrophosphate synthase gene (LPPs) and sclareol synthase gene (Tps); 2) overexpressing the related enzyme genes for GGPP synthesis in Saccharomyces cerevisiae: thereby increasing the precursor substances for the host's sclareol synthesis; the related enzyme genes are: a combination of two or more enzyme genes among the hydroxymethyl-CoA reductase gene (HMG1), the farnesyl pyrophosphate synthase gene (ERG20) and the dioxinyl pyrophosphate synthase gene (BTS1).
[0036] Specifically, in the step three, a single colony of the recombinant Yarrowia lipolytica was picked and inoculated into 25 mL / 250 ml of YPD liquid medium, cultured at 30 ° C and 220 rpm for 1 day to obtain a seed solution, and the seed solution was inoculated into a 5 L fermentor at an inoculum volume ratio of 2%. The fermentor contained 4 L of YPD fermentation medium, and fermented at 30 ° C, with a stirring speed of 800 rpm, an aeration ratio of 1 vvm, and a dissolved oxygen of 10%-20%. 5 M sodium hydroxide was added to adjust the pH to 5.5, and the glucose content was maintained at 5 g / L by feeding. The fermentation was carried out for 168-192 hours to obtain sclareol.
[0037] Specifically, in step four, the culture medium in the feed tank first enters the dissolved oxygen tank, mixes with the air introduced by the air supply mechanism, and then enters the secondary tank body: during fermentation, the speed of the high-speed stirring shaft is 2000r / min, the speed of the low-speed stirring shaft is 200r / min, the filling coefficient of the primary tank body after the culture medium is added is 0.8, the initial filling coefficient of the secondary tank body is 0.3, and the filling coefficient is 0.8 when the fermentation is completed.
[0038] Specifically, in step 5, the preparation method of the cigar tobacco bud powder is: washing and drying the cigar tobacco buds, grinding them, and passing them through a 40-80 mesh sieve to obtain the cigar tobacco bud powder.
[0039] Specifically, in the step 5, the cigar tobacco buds are cleaned and dried, then ground and passed through a 40-80 mesh sieve to obtain cigar tobacco bud powder; the tobacco bud powder is then mixed with deionized water in a mass ratio of (1-15):100, and the mixture is sterilized by wet heat at 110-130° C. for 15-20 minutes to obtain a cigar tobacco bud culture medium; edible yeast is added to the above-obtained cigar tobacco bud culture medium at an inoculum amount of 0.5%-3%, and the mixture is fermented at 25-35° C. and 100-300 rpm / min for 24-30 hours to obtain a sclareol sample.
[0040] Specifically, in step six, the fermentation broth is centrifuged at 8000 rpm for 15 minutes, the upper bacterial liquid is removed, the lower bacterial body is collected, and the cells are dried; the bacterial body is crushed into a 100-200 mesh powder for standby use; the bacterial body powder is mixed with an organic solvent in a certain proportion, and sclareol contained in the bacterial body is extracted with microwave assistance at a certain temperature. After extraction for a certain time, liquid-solid separation is performed to collect the extract, and the extract is concentrated to obtain an extract; the sclareol in the extract is extracted with a certain amount of acetone aqueous solution to remove impurities; the collected filtrate is crystallized at a certain temperature for a period of time to obtain purified sclareol.
[0041] The specific implementation of this embodiment is as follows: the construction method involves expressing two key enzymes for the synthesis of plant-derived sclareol, namely lysostigmine diol pyrophosphate synthase and sclareol synthase, and overexpressing a combination of one or more endogenous enzyme genes in yeast, including a key enzyme of the mevalonate pathway, namely hydroxymethyl-CoA reductase, and two enzymes of the isopentenyl pyrophosphate pathway, namely farnesyl pyrophosphate synthase and dioxyl pyrophosphate synthase. This method can efficiently produce sclareol. In contrast, the method uses tobacco buds as the only raw material, does not add any additional nutrients, and ferments the tobacco buds with edible yeast as a starter, thereby achieving green and efficient synthesis of sclareol: 175 mg / ml of sclareol can be synthesized within 24 hours of fermentation. The synthesis efficiency of sclareol is extremely high, the operation is simple, the cost is extremely low, the controllability is high, and the yield of sclareol is high.
[0042] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
[0043] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.
Claims
1. A strain fermentation method for synthesizing sclareol, characterized in that: The following steps are involved: Step 1: constructing a strain, constructing a biosynthetic pathway for sclareol in a host strain, and optimizing the intracellular mevalonate metabolic pathway and the central metabolic pathway, thereby obtaining an engineered bacterial strain A; the host strain is Pichia pastoris; or, overexpressing or knocking out metabolic regulatory factors in the above-obtained engineered bacterial strain A, thereby obtaining an engineered bacterial strain B; or, using a cell compartmentalization strategy in the above-obtained engineered bacterial strain B to target the synthetic pathway to the peroxisome and optimize it, thereby obtaining an engineered bacterial strain C; Step 2: increasing the host to increase the precursor of sclareol synthesis - digoxin pyrophosphate (GGPP), thereby allowing more GGPP to flow into the synthesis process of sclareol, wherein the host is brewing mother liquor; Step 3: recombining Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica is constructed by overexpressing the mutant gene of lisdendial pyrophosphate synthase CalLPPS, the gene of sclareol synthase SSTPS, and the gene of geranylgeranyl pyrophosphate synthase SaGGPP in Yarrowia lipolytica po1f, and expressing the gene of 3-hydroxy-3-methylglutaryl coester A reductase tHMGR and the gene of isopentenyl pyrophosphate isomerase idi; Step 4: Fermentation of sclareol. The engineered bacteria are connected to the seed tank, and the culture medium is added to obtain the seed liquid. The seed liquid first enters the first-level tank body, and the culture medium is added. After preliminary fermentation, the culture liquid is obtained. The culture liquid in the first-level tank body first enters the first group of second-level tank bodies, and is fermented at a high density after batch feeding. The culture liquid after preliminary fermentation in the first-level tank body then enters the next group of second-level tank bodies in turn, and is fermented at a high density after batch feeding. Step 5: Using other flower buds to ferment and produce sclareol, the other flower buds are selected from cigar tobacco buds, mixing cigar tobacco bud powder with deionized water, adding edible yeast after sterilization, and then fermenting at 25-35° C. and 100-300 rpm / min for 24-30 hours; the mass ratio of the cigar tobacco bud powder to deionized water is (1-15):100; Step 6: Purify sclareol from the fermentation broth of the strain.
2. The method for synthesizing sclareol by fermentation according to claim 1, wherein: In the step 1, the engineered bacterial strain A is regulated by one or more of the following: integrating the sclareol synthase gene (SsLPPS) and the sclareol synthase gene (SSTPS) into the chromosome PNSII-5 site in the host strain, optimizing the integration of at least one or more synthase genes or isoenzymes with the same / similar functions in the host strain mevalonate pathway into the host strain chromosome, weakening the competitive pathway toward squalene synthesis, increasing the supply of precursor acetyl-CoA, and increasing the supply of reducing power NADPH.
3. The method for synthesizing sclareol by fermentation according to claim 1, wherein: In the step 2, the process of constructing the engineered strain of the brewer's yeast is as follows: 1) expressing the key enzyme gene for sclareol synthesis in Saccharomyces cerevisiae; thereby allowing more GGPP to flow into the synthesis process of sclareol; the key enzyme genes are: lysene diol pyrophosphate synthase gene (LPPs) and sclareol synthase gene (Tps); 2) overexpressing the related enzyme genes for GGPP synthesis in Saccharomyces cerevisiae: thereby increasing the precursor substances for the host's sclareol synthesis; the related enzyme genes are: a combination of two or more enzyme genes among the hydroxymethyl-CoA reductase gene (HMG1), the farnesyl pyrophosphate synthase gene (ERG20) and the dioxinyl pyrophosphate synthase gene (BTS1).
4. The method for synthesizing sclareol by fermentation according to claim 1, wherein: In the step three, a single colony of the recombinant Yarrowia lipolytica was picked and inoculated into 25 mL / 250 ml of YPD liquid culture medium, and cultured at 30° C. and 220 rpm for 1 day to obtain a seed solution. The seed solution was inoculated into a 5 L fermentor at an inoculum volume ratio of 2%, and the fermentor contained 4 L of YPD fermentation medium. The fermentation was carried out at 30° C., a stirring speed of 800 rpm, an aeration ratio of 1 vvm, a dissolved oxygen content of 10%-20%, 5 M sodium hydroxide was added to adjust the pH to 5.5, and the glucose content was maintained at 5 g / L by feeding. The fermentation was carried out for 168-192 hours to obtain sclareol.
5. The fermentation method for synthesizing sclareol according to claim 1, wherein: In the step 4, the culture medium in the feed tank first enters the dissolved oxygen tank, mixes with the air introduced by the air supply mechanism, and then enters the secondary tank body: during fermentation, the speed of the high-speed stirring shaft is 2000r / min, the speed of the low-speed stirring shaft is 200r / min, the filling coefficient of the primary tank body after the culture medium is added is 0.8, the initial filling coefficient of the secondary tank body is 0.3, and the filling coefficient is 0.8 when the fermentation is completed.
6. The fermentation method for synthesizing sclareol according to claim 1, wherein: In the step 5, the preparation method of the cigar tobacco bud powder is as follows: the cigar tobacco buds are washed and dried, then ground, and passed through a 40-80 mesh sieve to obtain the cigar tobacco bud powder.
7. The fermentation method for synthesizing sclareol according to claim 6, characterized in that: In the step 5, the cigar tobacco buds are cleaned and dried, then ground and passed through a 40-80 mesh sieve to obtain cigar tobacco bud powder; the tobacco bud powder is then mixed with deionized water in a mass ratio of (1-15):100, and the mixture is sterilized by wet heat at 110-130° C. for 15-20 minutes to obtain a cigar tobacco bud culture medium; edible yeast is added to the obtained cigar tobacco bud culture medium at an inoculum amount of 0.5%-3%, and the mixture is fermented at 25-35° C. and 100-300 rpm / min for 24-30 hours to obtain a sclareol sample.
8. The bacterial strain fermentation method for synthesizing sclareol according to claim 1, characterized in that: In the sixth step, the fermentation broth is centrifuged at 8000 rpm for 15 minutes, the upper bacterial liquid is removed, the lower bacterial body is collected, and the cells are dried; the bacterial body is crushed into a 100-200 mesh powder for standby use; the bacterial body powder is mixed with an organic solvent in a certain proportion, and sclareol contained in the bacterial body is extracted with microwave assistance at a certain temperature. After extraction for a certain time, liquid-solid separation is performed to collect the extract, and the extract is concentrated to obtain an extract; the sclareol in the extract is extracted with a certain amount of acetone aqueous solution to remove impurities; and the collected filtrate is crystallized at a certain temperature for a period of time to obtain purified sclareol.