Expression cassette, expression vector and preparation for improving tobacco leaf quality and application of expression cassette, expression vector and preparation
By constructing genetically engineered bacteria that produce carotenoid-lysin dioxygenase, the problems of low fermentation efficiency and high cost of tobacco leaves have been solved, resulting in a significant improvement in the aroma and quality of tobacco leaves and providing an efficient enzyme preparation and fermentation method.
Patent Information
- Application Number
- CN202511238210.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-01
- Publication Date
- 2025-11-18
AI Technical Summary
Existing tobacco fermentation technologies suffer from low efficiency, high cost, and poor tobacco quality, especially the natural aging method, which is difficult to effectively improve the aroma quality of tobacco leaves.
Using the probiotic Escherichia coli Nissle 1917 as the expression host, a genetically engineered bacterium was constructed to heterologously express carotenoid lysin dioxygenase. The enzyme preparation was obtained through simple cell disruption and used in tobacco fermentation to increase the content of aroma compounds.
It significantly increases the content of aroma-producing substances such as geraniol acetone and ionone in tobacco leaves, with a degradation rate of 54.7%. It has a short fermentation cycle, no endotoxins, and low cost, making it suitable for the tobacco and food industries.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of tobacco, in particular to an expression cassette, an expression vector, a preparation and application thereof for improving the quality of tobacco leaves. BACKGROUND
[0002] Carotenoids and their derivatives in tobacco are important precursors for the formation of volatile aroma components of plants, and have an important influence on the appearance quality and aroma quality of tobacco leaves. The biodegradation of carotenoids is a relatively important method for generating aroma, which relies on the action of microbial peroxidase, carotenoid cleavage dioxygenase, lipoxygenase and other oxidases to decompose carotenoids into relatively small aroma molecules and some aroma precursors, etc. The tobacco leaves without fermentation treatment have defects such as heavy odor, strong irritation, and insufficient aroma. Fermentation helps to degrade carotenoids in tobacco leaves, produce various aroma substances, and significantly improve the industrial usability of tobacco leaves.
[0003] Tobacco fermentation is an important method for improving the quality of tobacco leaves, which is divided into natural aging and artificial fermentation. Natural aging is a natural fermentation process that lasts for about 2 years under the condition of low moisture content of tobacco leaves (tobacco moisture content is usually 11%~13%). Artificial fermentation usually refers to a method of piling tobacco leaves in a fermentation room, introducing humid hot steam, controlling the temperature in the fermentation room at 45℃~55℃ and maintaining for 3~6 weeks to improve the internal quality of tobacco leaves. For example, carotenoids in tobacco fermentation are degraded by biological or non-biological pathways to produce many aroma substances, such as β-ionone, geranylacetone, linalool and 3-hydroxy-β-dihydrodamascenone, etc. Studies have shown that carotenoid cleavage products account for about 16% of tobacco aroma components, with a contribution rate of up to 85%. At present, most cigarette industrial enterprises adopt natural aging method, but natural aging has problems such as low efficiency, high cost and poor quality of tobacco leaves, and artificial fermentation has gradually attracted the attention of researchers and the industry. SUMMARY
[0004] Therefore, the present application provides an expression cassette, an expression vector, a preparation and application thereof for improving the quality of tobacco leaves. The present application uses probiotic E. coli Nissle 1917 as an expression host to construct a genetically engineered bacterium for heterologous expression of carotenoid cleavage dioxygenase, avoids the introduction of endotoxin in the fermentation process of tobacco leaves, and can obtain a biological preparation capable of improving the aroma substances of tobacco leaves by simple cell disruption. At the same time, through tobacco fermentation, the content of aroma substances such as geranylacetone and ionone in tobacco leaves can be significantly improved.
[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:
[0006] The present application provides an expression cassette comprising: a gene encoding a carotenoid cleavage dioxygenase and a promoter.
[0007] In some embodiments of the present application, the gene encoding a carotenoid cleavage dioxygenase in the above expression cassette has:
[0008] (1) the nucleotide sequence shown in SEQ ID NO: 1;
[0009] (2) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (1);
[0010] (3) a sequence having at least 80% homology with the nucleotide sequence shown in (1);
[0011] (4) a complementary sequence of the sequence shown in (1), (2) or (3); and / or
[0012] The promoter has:
[0013] (5) the nucleotide sequence shown in SEQ ID NO: 2;
[0014] (6) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (5);
[0015] (7) a sequence having at least 80% homology with the nucleotide sequence shown in (5);
[0016] (8) a complementary sequence of the sequence shown in (5), (6) or (7).
[0017]
[0018] In some embodiments of the present application, the sequence of SEQ ID NO: 2 in the above expression cassette is: TTTTATTGACAATCTTGTACATAGTCTTTATAATGCTATACTCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACAT.
[0019] In some embodiments of the present application, the above expression cassette has:
[0020] (9) a nucleotide sequence as shown in SEQ ID NO: 3;
[0021] (10) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (9);
[0022] (11) a sequence having at least 80% homology to the nucleotide sequence shown in (9);
[0023] (12) a complementary sequence of the sequence shown in (9), (10) or (11).
[0024]
[0025] The present application also provides an expression vector comprising: the above expression cassette and a backbone plasmid.
[0026] In some embodiments of the present application, the backbone plasmid in the above expression vector comprises: pCOLADuet-1.
[0027]
[0028] In some embodiments of the present application, the amino acid sequence of the carotenoid cleavage dioxygenase is as shown in SEQ ID NO: 5: MVEKEKLENDVVARNGIVAVDPKPKNGVTSKVIDFVEKLIVKWMYDSSQPLHYLSGNFAPVPDETPPTKDLHVIGHLPDCLNGEFVRVGPNPKFAPVAGYHWFDGDGMIHGLRIKDGKATYVSRFVRTSRLKQEEFFGGAKFMKIGDLKGLFGLLMVNMQMLRAKLKVLDVSYGNGTANTALVYHHGKLLALSEADKPYAVKVLEDGDLQTLGMLDYDKRLQHSFTAHPKVDPYTGEMFTFGYAHTPPYITYRVISKDGFMHDPVPITVSDPIMMHDFAITENYAIFMDLPLYFRPKEMVKENKLIFTFDDTKKARFGILPRYAKNEAQMKWFELPNCFIFHNANAWEEEDEVVLITCRLDKPDLDMVNGAVKEKLENFSNELYEMRFNLKTGLASQKRLSASAVDFPRVNECYTGRKQRYVYGTILDSIAKVTGIIKFDLHAEPDEEKTKLEVGGNVRGIFDLGPGRFGSEAVFVPREPGTSSEEDDGYLIFFSHDEKTGKSSVNVIDAKTMSADPVAVVELPQRVPYGFHAFFVSEEQLEEQAKL.
[0029] The present application also provides a host cell transformed and / or transfected with the above-mentioned expression vector.
[0030] In some embodiments of the present application, the host cell is transformed and / or transfected with the above-mentioned expression vector, wherein the chassis cell is E. coli.
[0031] In some embodiments of the present application, the host cell is transformed and / or transfected with the above-mentioned expression vector, wherein the chassis cell is E. coli Nissle1917.
[0032] The present application also provides a preparation prepared directly or indirectly from the above-mentioned expression cassette, the above-mentioned expression vector and / or the above-mentioned host cell.
[0033] The present application also provides a preparation prepared directly or indirectly from the above-mentioned expression cassette, the above-mentioned expression vector and / or the above-mentioned host cell.
[0034] In some embodiments of the present application, in the above preparation method, the inoculation amount of the inoculation is 1-3%; the temperature of the inoculation is 35-38°C, and the rotation speed is 200-220 rpm.
[0035] In some embodiments of the present application, in the above preparation method, the fermentation culture is carried out at a temperature of 25-35°C, a rotation speed of 200-220 rpm, and for a time of 15-20 h.
[0036] In some embodiments of the present application, in the above preparation method, the collection of the precipitate comprises a centrifugation step; and the centrifugation is carried out at a rotation speed of 8000-12000 rpm.
[0037] In some embodiments of the present application, in the above preparation method, after the collection of the precipitate, a resuspension step is further included before the disruption; and the resuspension is carried out using a PB buffer at a pH of 7-7.5.
[0038] In some embodiments of the present application, in the above preparation method, the disruption is carried out using ultrasonic disruption or a high-pressure homogenizer.
[0039] The present application further provides the use of the above expression cassette, the above expression vector, the above host cell, the above preparation, and / or the above preparation method in any of the following:
[0040] fermentation of tobacco leaves; and / or
[0041] enhancing the flavor of tobacco leaves; and / or
[0042] degrading carotenoids in tobacco leaves; and / or
[0043] increasing the content of aroma-producing substances in tobacco leaves.
[0044] The present application further provides a fermentation method of tobacco leaves, which comprises mixing the tobacco leaves with the above preparation and / or the preparation obtained by the above preparation method, and incubating.
[0045] In some embodiments of the present application, in the above fermentation method, the temperature of the mixing is 35-38°C, the rotation speed is 200-220 rpm, and the time is 3-6 h.
[0046] In some embodiments of the present application, in the above fermentation method, the concentration of the tobacco leaves is 15-25 g / L.
[0047] In some embodiments of the present application, in the above fermentation method, the amount of the enzyme preparation added is 30-50 mL.
[0048] The application is based on probiotic E. coli Nissle 1917 to construct a carotenoid cleavage dioxygenase expression strain, realize efficient expression of carotenoid cleavage dioxygenase, and the degradation rate of carotenoid in vitro reaches 54.7%. The enzyme preparation is obtained by cell disruption, and the enzyme preparation is used for fermentation of tobacco leaves, which significantly improves the content of carotenoid degradation products in tobacco leaves and improves the content of aroma substances. The fermentation cycle of the genetically engineered bacteria is short, and the genetically engineered bacteria are non-endotoxin and non-pathogenic. The enzyme preparation is simple to prepare and low in cost, and can be widely used in the tobacco industry and the food industry. BRIEF DESCRIPTION OF DRAWINGS
[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description.
[0050] Figure 1 Comparison of aroma substances in tobacco treated by genetically engineered bacteria EcN-CCD1 and control strain EcN-pCOLADuet. DETAILED DESCRIPTION
[0051] The application discloses an expression cassette, an expression vector, a preparation and application thereof for improving quality of tobacco leaves.
[0052] It should be understood that the expression "one or more of something" includes each of the objects recited after the expression and various combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.
[0053] The use of the terms "including", "has", or "contains", including their grammatical synonyms, should generally be understood to be open and non-limiting, for example, not excluding other non-recited elements or steps, unless specifically stated otherwise or understood from the context.
[0054] It should be understood that the order of steps or the order of performing certain actions is not important as long as the present application is still operable. In addition, two or more steps or actions can be performed simultaneously.
[0055] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is intended merely to better illustrate the present application and does not in any way limit the scope of the present application, unless otherwise claimed. Any language in this specification should not be interpreted as indicating any non-claimed element is essential to the practice of the present application.
[0056] Also, the numerical ranges and parameters setting forth the broadest scope of equivalents of the application are approximations because some variations and permutations of the quantities, concentrations, and other values are inevitable as a result of normal tolerances and measurement variations. Accordingly, unless explicitly stated otherwise, all numerical ranges are approximate and are understood to be preceded by the word "about". It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.
[0057] The present application provides an E. coli Nissle 1917 expression system for realizing safe and rapid heterologous expression of carotenoid cleavage dioxygenase.
[0058] The present application provides an enzyme preparation preparation method and a liquid fermentation tobacco leaf method for increasing the content of aroma substances in the tobacco leaf to solve the problem of application of carotenoid cleavage dioxygenase in tobacco leaf flavoring.
[0059] The present application also provides an enzyme preparation preparation method, which is made of the genetically engineered bacteria and includes the following steps:
[0060] The seed liquid of the genetically engineered bacteria is inoculated into the TB culture medium at an inoculation amount of 1-3%, the culture temperature is 25-35 DEG C, the rotation speed is 200-220 rpm, and the fermentation is carried out for 15-20 h, then the bacteria are collected by centrifugation at 8000-12000 rpm at 4 DEG C and resuspended with a buffer, and the bacteria are broken to obtain a crude enzyme liquid.
[0061] Further, the seed liquid is obtained by inoculating the genetically engineered bacteria into the LB culture medium and culturing at 35-38 DEG C and 200-220 rpm until the logarithmic growth phase.
[0062] Further, the buffer is a PB buffer with a pH of 7-7.5.
[0063] Further, the bacteria breaking method is ultrasonic breaking or high-pressure homogenizer.
[0064] The present application also provides a tobacco leaf fermentation method, which includes the following steps:
[0065] The tobacco leaf is added into the enzyme preparation and incubated at 35-38 DEG C and 200-220 rpm for 3-6 h, the tobacco leaf is collected, and the content of aroma substances in the tobacco leaf is determined by headspace solid-phase microextraction and gas chromatography-mass spectrometry.
[0066] Further, the tobacco leaf powder is added in an amount of 15-25 g / L.
[0067] Further, the enzyme preparation is used in an amount of 30-50 mL.
[0068] In Embodiment 1 to Embodiment 5 of the present application, the raw materials and reagents used can be purchased from the market.
[0069] The present application is further described below in combination with examples:
[0070] Embodiment 1
[0071] The present application provides a carotenoid cleavage dioxygenase, which comprises 547 amino acids, the amino acid sequence is shown as SEQ ID NO: 5, and the gene encoding the enzyme has a nucleotide sequence shown as SEQ ID NO: 1, with a full length of 1641 nucleotides.
[0072] The present application also provides a recombinant plasmid for expressing a carotenoid cleavage dioxygenase, which comprises a carotenoid cleavage dioxygenase sequence and a constitutive promoter pSyn1 for expression, wherein the nucleotide sequence of the constitutive promoter pSyn1 is shown as SEQ ID NO: 2, with a full length of 84 nucleotides; the nucleotide sequence of the expression cassette is shown as SEQ ID NO: 3, with a full length of 1725 nucleotides; and the plasmid used is pCOLADuet-1, with a nucleotide sequence shown as SEQ ID NO: 4, with a full length of 3719 nucleotides.
[0073] Embodiment 2
[0074] The present embodiment provides a method for determining the degradation rate of carotenes in vitro, and the specific steps are as follows:
[0075] The prepared 900 μL substrate carotene mother liquor (1.25 g / L carotene, 220 g / L Tween 40, 50 mL dimethyl sulfoxide, ultrasonic oscillation and uniform mixing) and 100 μL carotenoid cleavage dioxygenase enzyme solution are fully mixed, and then oscillated on a vibration type constant temperature homogenizer at 37℃ for 2 h. After the reaction is completed, 200 μL supernatant is taken to measure the absorbance of the reaction solution at 450 nm, and the residual carotene concentration is calculated according to the standard curve.
[0076] The degradation rate is defined as: (initial carotene concentration-reaction carotene concentration) ÷ initial carotene concentration x 100.
[0077] Embodiment 3
[0078] (1) Amplification of carotenoid cleavage dioxygenase coding sequence
[0079] The recombinant plasmid pET-28a(+)-CCD1 containing a carotenoid cleavage dioxygenase coding gene (SEQ ID NO. 2) synthesized by Kingsway is used as a template, and primers (P1, P2) are designed to amplify the carotenoid cleavage dioxygenase coding sequence containing a promoter homologous arm:
[0080] Primer P1 : 5'- TTTATAATGCTATACTccatcttagtatattagttaagtataagaaggagatatacatATGGTTGAAAAAGAAAAACTGG- 3' (as set forth in SEQ ID NO: 6);
[0081] Primer P2: 5'- cagcggtttctttaccagactcgagCAGCTTCGCCTGTTCTTCCAGC- 3' (as set forth in SEQ ID NO: 7);
[0082] PCR amplification reaction was performed in a 40 μL system, and the reaction system was added with 20 μL Phanta® Max, 17 μL ddH2O, 1 μL template DNA, 1 μL of the upper and lower primers respectively. The reaction condition was that after pre-denaturation at 95°C for 3 min, the cycle was started: denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, for a total of 33 cycles; and final extension at 72°C for 5 min. The PCR product was subjected to nucleic acid electrophoresis identification and gel cutting and purification.
[0083] (2) Linearization of pCOLADuet-1 plasmid
[0084] The primers (P3, P4) were designed to reverse PCR amplify the linearized plasmid containing the promoter, with the plasmid pCOLADuet-1 as the template:
[0085] Primer P3: 5'- ctcgagtctggtaaagaaaccgctg- 3' (as set forth in SEQ ID NO: 8);
[0086] Primer P4: 5'- atactaagatggAGTATAGCATTATAAAGACTATGTACAAGATTGTCAATAAAAtaatttcgattatgcggccgt- 3' (as set forth in SEQ ID NO: 9);
[0087] PCR amplification reaction was performed in a 40 μL system, and the reaction system was added with 20 μL Phanta® Max, 17 μL ddH2O, 1 μL template DNA, 1 μL of the upper and lower primers respectively. The reaction condition was that after pre-denaturation at 95°C for 3 min, the cycle was started: denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, for a total of 33 cycles; and final extension at 72°C for 5 min. The PCR product was subjected to nucleic acid electrophoresis identification and gel cutting and purification. The product was further subjected to single enzyme digestion with Dpn I, and the linearized plasmid was recovered after enzyme digestion at 37°C for 3 h.
[0088] (3) Plasmid pCOLADuet-CCD1 construction
[0089] In the PCR tube, add 0.02 pmol of the product recovered in step (1) and 0.06 pmol of the linearized plasmid in step (2), and add an equal volume of Hieff Clone® Universal Enzyme Premix. After mixing, connect at 50°C for 30 min, and transform the connection product into E. coli JM 109 competent cells, and culture overnight on solid LB medium containing kanamycin resistance (50 mg / L), pick multiple transformants into LB liquid medium (Kanr), culture at 37°C, 220 rpm for 10-12 h, extract the plasmid, and then perform PCR verification and send to Tianlin Sequencing Company for sequence determination. The recombinant plasmid with correct sequence is named pCOLADuet-CCD1.
[0090] (4) Recombinant strain EcN-CCD1 construction
[0091] The verified plasmid in step (3) is transformed into E. coli Nissle1917 competent cells purchased from Moli Biotech. Positive transformants are determined by colony PCR and sequence determination by Tianlin Sequencing Company, and the recombinant strain with correct sequence is named EcN-CCD1. At the same time, pCOLADuet-1 is transformed according to the same steps to construct the control strain EcN-pCOLADuet. Positive transformants are inoculated in 10 mL LB liquid medium (Kanr) and cultured at 37°C, 220 rpm for 12 h, and then inoculated in 30 mL TB liquid medium (Kanr) at a 2% inoculation amount (OD600 is about 4-5) and cultured at 30°C, 220 rpm for 12 h. The fermentation broth is centrifuged at 10000 rpm for 10 min at 4°C to collect the bacterial cells, which are resuspended with 10 mL PB buffer (pH 7.0) and subjected to ultrasonic crushing to obtain crude enzyme solution for determination of carotene degradation rate (according to the determination method of Example 2). The carotene degradation rate is about 54.7%, which is 18.9 times that of the control strain.
[0092] Example 4
[0093] The present embodiment provides a method for determining the content of aroma substances in tobacco leaves, and the specific steps are as follows:
[0094] (1) Sample treatment
[0095] For liquid samples, 6 mL of fermentation broth is added to a headspace extraction bottle, 3 g of sodium chloride is added, and 10 μL of 20 mg / L 2-octanol (dissolved in chromatographic grade methanol) is added as an internal standard.
[0096] (2) Headspace solid-phase microextraction
[0097] The volatile compounds were extracted from the headspace of the sample using a PAL3 autosampler (CTC Analytics AG, Switzerland) with SPME fiber head (thickness 80 pm, length 10 mm, DVB / C-WR / PDMS) (Agilent Technology Co., ltd, USA). The sample was pre-heated at 50 °C for 5 min, then extracted at 50 °C for 30 min. After extraction, the extraction head was inserted into the gas chromatograph mass spectrometer for analysis (250 °C, 5 min).
[0098] (3) GC-MS chromatographic conditions
[0099] A DB-Wax capillary column (column length 60 m, inner diameter 0.32 mm, liquid film thickness 0.25 pm) was used, with an injection port temperature of 250 °C, and a temperature program of 50 °C for 2 min, then 3 °C / min to 145 °C, then 15 °C / min to 230 °C, and held for 3 min. Helium (purity 99.99%) was used as the carrier gas at a flow rate of 1.0 mL / min, with a split ratio of 20:1. Mass spectrometry conditions: EI ion source, ion source temperature 260 °C, electron impact energy: 70 eV, scan mass range: 33-350 m / z. The content was calculated using the internal standard method, and the relative content of each substance was calculated by comparing the peak area values.
[0100] Example 5
[0101] This example provides a tobacco leaf fermentation method using the enzyme preparation, and the specific steps are as follows:
[0102] 20 g / L tobacco leaves were added to 30 mL of crude enzyme solution, incubated at 37 °C, 220 rpm for 6 h, and the tobacco leaves were collected and the content of aroma compounds in the tobacco leaves was determined by headspace solid phase microextraction and gas chromatography mass spectrometry (Table 1, Figure 1 ).
[0103] Table 1. GC-MS results of tobacco leaf fermentation
[0104]
[0105] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.
Claims
1. An expression box, characterized in that, include: Gene and promoter encoding carotenoid cleavage dioxygenase.
2. The expression box as described in claim 1, characterized in that, The gene encoding carotenoid cleavage dioxygenase has the following characteristics: (1) The nucleotide sequence shown in SEQ ID NO:1; (2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (1); (3) A sequence that has at least 80% homology with the nucleotide sequence shown in (1); (4) Complementary sequences to sequences shown in (1), (2) or (3); and / or The promoter has: (5) The nucleotide sequence shown in SEQ ID NO:2; (6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (5); (7) A sequence that has at least 80% homology with the nucleotide sequence shown in (5); (8) Complementary sequences to sequences shown in (5), (6) or (7).
3. The expression box as described in claim 1 or 2, characterized in that, The expression box has: (9) The nucleotide sequence shown in SEQ ID NO:3; (10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (9); (11) A sequence that has at least 80% homology with the nucleotide sequence shown in (9); (12) Complementary sequences to sequences shown in (9), (10) or (11).
4. An expression carrier, characterized in that, include: The expression cassette and backbone plasmid as described in any one of claims 1 to 3.
5. The expression vector as described in claim 4, characterized in that, The backbone plasmid includes pCOLADuet-1.
6. A host cell, characterized in that, Transformation and / or transfection with the expression vector as described in claim 4 or 5.
7. A formulation, characterized in that, It is prepared directly or indirectly using the expression cassette as described in any one of claims 1 to 3, the expression vector as described in claim 4 or 5, and / or the host cell as described in claim 6.
8. The method for preparing the formulation according to claim 7, characterized in that, After inoculating the host cells with fermentation culture, the precipitate is collected, broken up, and the preparation is obtained.
9. The use of the expression cassette as described in any one of claims 1 to 3, the expression vector as described in claim 4 or 5, the host cell as described in claim 6, the formulation as described in claim 7, and / or the formulation obtained by the preparation method as described in claim 8 in any of the following: ( ), tobacco fermentation; and / or ( ), aroma enhancement of tobacco leaves; and / or ( ), degrade carotenoids in tobacco leaves; and / or ( ), and increase the content of aroma compounds in tobacco leaves.
10. A method for fermenting tobacco leaves, characterized in that, The tobacco leaves are mixed with the formulation as described in claim 7 and / or the formulation obtained by the preparation method as described in claim 8, and then incubated.