Expression cassette, expression vector and application thereof in tobacco leaf fermentation

Abstract

The invention relates to the field of tobacco, in particular to an expression cassette, an expression vector and application thereof in tobacco fermentation. The invention provides an expression cassette. The expression cassette comprises a gene for coding carotenoid cleavage dioxygenase and a promoter, the gene for coding the carotenoid cleavage dioxygenase has a nucleotide sequence as shown in SEQ ID NO: 2. Probiotic escherichia coli E. coli Nissle 1917 is used as an expression host, genetically engineered bacteria for heterologously expressing carotenoid cleavage dioxygenase are constructed, endotoxin is prevented from being introduced in the tobacco leaf fermentation process, and a biological preparation capable of increasing aroma substances of tobacco leaves can be obtained through simple cell disruption. Meanwhile, the content of aroma substances such as geranyl acetone and ionone in the tobacco leaves can be remarkably increased through tobacco leaf fermentation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tobacco, in particular to an expression cassette, an expression vector and application thereof in tobacco fermentation. BACKGROUND

[0002] Carotenoids and their derivatives in tobacco are important precursors for the formation of plant volatile aroma components, and have an important influence on the appearance quality and aroma quality of tobacco. Carotenoid biodegradation 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 and the like. The tobacco leaf without fermentation treatment has defects such as heavy odor, strong irritation, and insufficient aroma. Fermentation helps to degrade carotenoids in tobacco, produce various aroma substances, and significantly improve the industrial usability of tobacco.

[0003] Tobacco fermentation is an important method for improving the quality of tobacco, which is divided into natural aging and artificial fermentation. Natural aging is a natural fermentation process for about 2 years under the condition of low moisture content of tobacco (the moisture content of tobacco is usually 11%~13%). Artificial fermentation usually refers to a method of piling tobacco 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. At present, most cigarette industry enterprises adopt the natural aging method, but the natural aging has problems such as low efficiency, high cost, poor quality of tobacco, etc., and artificial fermentation gradually attracts the attention of researchers and the industry. SUMMARY

[0004] Therefore, the present application provides an expression cassette, an expression vector and application thereof in tobacco fermentation. The present application takes probiotic E. coli Nissle 1917 as an expression host, constructs a genetically engineered bacterium for heterologous expression of carotenoid cleavage dioxygenase, avoids the introduction of endotoxin in the process of tobacco fermentation, and obtains a biological preparation capable of improving tobacco aroma substances by simple cell disruption. At the same time, through tobacco fermentation, the content of aroma substances such as geranylacetone and ionone in tobacco 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 carotenoid cleavage dioxygenase and a promoter; the gene encoding carotenoid cleavage dioxygenase has:

[0007] (1) the nucleotide sequence shown in SEQ ID NO: 2;

[0008] (2) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (1);

[0009] (3) a sequence having at least 80% homology to the nucleotide sequence shown in (1);

[0010] (4) a complementary sequence of the sequence shown in (1), (2) or (3).

[0011]

[0012] In some embodiments of the present application, the promoter in the above expression cassette comprises: pSyn1 and / or P43.

[0013] In some embodiments of the present application, the expression cassette in the above expression cassette further comprises: a signal peptide penp.

[0014] In some embodiments of the present application, the promoter in the above expression cassette has:

[0015] (5) a nucleotide sequence as shown in SEQ ID NO: 3 or as shown in SEQ ID NO: 7;

[0016] (6) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence as shown in (5);

[0017] (7) a sequence having at least 80% homology with the nucleotide sequence as shown in (5);

[0018] (8) a complementary sequence of the sequence as shown in (5), (6) or (7); and / or

[0019] The signal peptide penp has:

[0020] (9) a nucleotide sequence as shown in SEQ ID NO: 6;

[0021] (10) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence as shown in (9);

[0022] (11) a sequence having at least 80% homology with the nucleotide sequence as shown in (9);

[0023] (12) a complementary sequence of the sequence as shown in (9), (10) or (11).

[0024] In some embodiments of the present application, the sequence of SEQ ID NO: 3 in the above expression cassette is: TTTTATTGACAATCTTGTACATAGTCTTTATAATGCTATACTCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACAT. (pSyn1)

[0025] In some embodiments of the present application, in the above expression cassette, the sequence of SEQ ID NO: 7 is: TGATAGGTGGTATGTTTTCGCTTGAACTTTTAAATACAGCCATTGAACATACGGTTGATTTAATAACTGACAAACATCACCCTCTTGCTAAAGCGGCCAAGGACGCCGCCGCCGGGGCTGTTTGCGTTCTTGCCGTGATTTCGTGTACCATTGGTTTACTTATTTTTTTGCCAAGGCTGTAATGGCTGAAAATTCTTACATTTATTTTACATTTTTAGAAATGGGCGTGAAAAAAAGCGCGCGATTATGTAAAATATAAAGTGATAGCGGTACCATTATAGGTAAGAGAGGAATGTACAC. (P43)

[0026] In some embodiments of the present application, in the above expression cassette, the sequence of SEQ ID NO: 6 is: ATGAAGTTGAAAACTAAAGCGTCAATAAAATTCGGAATATGTGTTGGGCTTTTATGTTTAAGCATTACTGGTTTCACACCTTTTTTCAACTCAACACATGCCGAAGCA.

[0027] In some embodiments of the present application, the above expression cassette has:

[0028] (13) a nucleotide sequence as shown in SEQ ID NO: 4 or as shown in SEQ ID NO: 8;

[0029] (14) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (13);

[0030] (15) a sequence having at least 80% homology to the nucleotide sequence shown in (13);

[0031] (16) a complementary sequence of the sequence shown in (13), (14) or (15).

[0032]

[0033]

[0034] In some embodiments of the present application, in the above-mentioned expression cassette, the amino acid sequence of the carotenoid cleavage dioxygenase is as shown in SEQ ID NO: 1: MGKKEEEVAMERSERGWVVVKPKANKKFGAKAIDWLEKVIVKLMYNSGSNSKACHFLSGNFAPVDETPPCKDLPVKGYLPECLNGEFVRVGPNPKFAPVAGYHWFDGDGMIHGLQIKDGKATYVSRFVRTSRLKQEEFFGGAKFMKIGDLKGLFGLFSVYIYMLREKLKVLDTSYGNGTANTAMIYHHGKLLALHEGDKPYVVKVLEDGDLQTLGMLDYDKRLQHSFTAHPKVDPVTGEMFTFGYSQTPPYATYRVISKDGVMQDPVPITIPASVMMHDFAITENYSIMMDLPLYFKPKEMVKNKKLAYSFDPTKKACFGILPRYAKNESLIKWFELPNCFIFHNANAWEEGDDVVLITCRLQNPDLDAINGTEKEQQRDGFTNELYEMRFNMKTGLASQKKLSESAVDFPRVNENYTGRKQRYVYGTILNHMAKITGVVKFDLHGEPETGKTKLEVGGNVLGIFDLGPGRFGSEAIFVPRQPGTECEEDG YLILFVHDENTGKSSVNVIDAKTMSAEPVAVVELPKRVPYGFHAFFVTEEQIQEQAKL.

[0035] The present application also provides an expression vector comprising the above-mentioned expression cassette and a backbone plasmid.

[0036] In some embodiments of the present application, in the above-mentioned expression vector, the backbone plasmid comprises pCOLADuet-1 and / or pMA5.

[0037]

[0038]

[0039] The present invention also provides a host cell into which the above expression vector is transformed and / or transfected into chassis cells.

[0040] In some embodiments of the present invention, the chassis cells in the host cells include: Escherichia coli and / or Bacillus subtilis.

[0041] The present invention also provides an enzyme or a preparation thereof, which is prepared directly or indirectly using the above-described expression cassette, the above-described expression vector and / or the above-described host cells.

[0042] The present invention also provides the use of the above-described expression cassette, expression vector, host cell and / or enzyme or preparation thereof in any of the following:

[0043] ( ), tobacco fermentation; and / or

[0044] ( ), aroma enhancement of tobacco leaves; and / or

[0045] ( ), degrade carotenoids in tobacco leaves; and / or

[0046] ( ), and increase the content of aroma compounds in tobacco leaves.

[0047] The present invention also provides a method for degrading carotenoids, wherein the above-mentioned enzyme or its preparation is mixed with carotene mother liquor and incubated.

[0048] The present invention also provides a method for fermenting tobacco leaves, wherein the above-mentioned enzyme or its preparation is mixed with tobacco leaves and incubated.

[0049] This invention utilizes the probiotic *Escherichia coli* Nissle 1917 as an expression host to construct a genetically engineered bacterium that heterologously expresses carotenoid-lysin dioxygenase. This avoids the introduction of endotoxins during tobacco fermentation, and allows for the acquisition of a biological agent that enhances the aroma compounds in tobacco leaves through simple cell disruption. Simultaneously, tobacco fermentation significantly increases the content of aroma compounds such as geraniol acetone and ionone in tobacco leaves. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0051] Figure 1 The pMA-CCD1 and pCOLADuet-CCD1 plasmid maps are shown.

[0052] Figure 2 The results of in vitro degradation of carotene using different expression systems are shown.

[0053] Figure 3 WB map of carotenoid cleavage dioxygenase; wherein: lane M: protein Marker; lane 1: protein band of control EcN-pCOLADuet; lane 2: protein band of B. subtilis expression strain BS-CCD1 of CCD1; lane 3: protein band of E. coli expression strain EcN-CCD1 of CCD1. DETAILED DESCRIPTION

[0054] The application discloses an expression cassette, an expression vector and application thereof in tobacco leaf fermentation.

[0055] It should be understood that the expression "one or more of" individually includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in connection with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0056] The use of the terms "including," "has," "having" or "contains" and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements not specifically recited, unless otherwise specified or understood from the context.

[0057] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0058] The use of any and all examples, or exemplary language herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0059] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are only used to convey generally understood precision. Numerical parameters are only expected to be useful as definitions in any judicial or administrative ruling, and are not to be used in a literal and / or strict sense unless expressly so defined by the parties. Unless otherwise expressly provided, all ranges, quantities, numerical values and percentages used herein are modified to be "about" modified unless otherwise expressly provided. Herein, "about" is generally intended to refer to within 10%, 5%, 1% and 0.5% of a given value or range.

[0060] The application provides a method for determining the degradation rate of carotenes in vitro, comprising the following steps:

[0061] Mix 0.9 mL of the crude enzyme solution with 0.1 mL of the carrot mother liquor thoroughly, and incubate at 37 DEG C for 2 hours with shaking, and then take 0.2 mL of the reaction solution to measure the absorbance at 450 nm, and calculate the degradation rate of the carotene according to the standard curve.

[0062] Further, the carrot mother liquor contains 1-1.5 g / L of carotene, 200-250 g / L of Tween 40, and 40-60 mL of dimethyl sulfoxide, and is mixed uniformly by ultrasonic oscillation.

[0063] Further, the crude enzyme solution is the fermentation supernatant or the broken supernatant of the genetically engineered bacteria.

[0064] Further, the standard curve is configured by using the carrot mother liquor with a carotene concentration of 0.25-1.5 g / L, and the initial strain is used instead of the genetically engineered bacteria to perform the reaction.

[0065] The application further provides a method for fermenting tobacco leaves to increase the content of aroma-producing substances, which comprises the following steps:

[0066] The tobacco leaves are added into the crude enzyme solution, and incubated at 37 DEG C and 220 rpm for 4-8 hours, and then the tobacco leaves are collected and the content of the aroma-producing substances in the tobacco leaves is determined.

[0067] Further, the tobacco leaves are added in an amount of 15-25 g / L.

[0068] Further, the crude enzyme solution is from the recombinant strains of E. coli Nissle 1917 and B. subtilis SCK6.

[0069] In the examples 1-6 of the application, the raw materials and reagents used can be purchased from the market.

[0070] The application is further described below in combination with the examples:

[0071] Example 1

[0072] The application provides a carotene-cleaving dioxygenase, which comprises 550 amino acids, and the amino acid sequence is shown as SEQ ID NO: 1, and the gene encoding the enzyme has the nucleotide sequence shown as SEQ ID NO: 2, and the full length is 1650 nucleotides.

[0073] The application further provides a recombinant plasmid for expressing carotenoid cleavage dioxygenase, comprising 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: 3, and the full length is 84 nucleotides; the nucleotide sequence of the expression cassette is shown as SEQ ID NO: 4, and the full length is 1734 nucleotides; the plasmid used is pCOLADuet-1, and the nucleotide sequence is shown as SEQ ID NO: 5, and the full length is 3719 nucleotides. Figure 1 ).

[0074] Embodiment 2

[0075] The application provides an E. coli-dry grass shuttle plasmid for expressing carotenoid cleavage dioxygenase, comprising a carotenoid cleavage dioxygenase sequence, a signal peptide penP for extracellular expression of the carotenoid cleavage dioxygenase and a promoter P43 for expression of the carotenoid cleavage dioxygenase, wherein the nucleotide sequence of the signal peptide penP is shown as SEQ ID NO: 6, and the full length is 108 nucleotides, and the signal peptide encodes 36 amino acids; the nucleotide sequence of the promoter P43 is shown as SEQ ID NO: 7, and the full length is 300 nucleotides; the nucleotide sequence of the expression cassette is shown as SEQ ID NO: 8, and the full length is 2058 nucleotides; and the plasmid used is pMA5, and the nucleotide sequence is shown as SEQ ID NO: 9, and the full length is 7202 nucleotides. Figure 1 ).

[0076] Embodiment 3

[0077] The embodiment provides a method for determining the degradation rate of carotenes in vitro, and the specific steps are as follows:

[0078] 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 oscillation reaction is carried out on a vibration type constant temperature mixing instrument at 37 DEG C for 2 h; after the reaction is completed, 200 μL supernatant is taken out, and the absorbance of the reaction solution is measured at 450 nm; and the residual carotene concentration is calculated according to the standard curve.

[0079] The degradation rate is defined as: (initial carotene concentration-reaction carotene concentration) ÷ initial carotene concentration x 100.

[0080] Embodiment 4

[0081] The embodiment provides a method for constructing a recombinant strain EcN-CCD1, and the specific steps are as follows:

[0082] (1) Amplification of carotenoid cleavage dioxygenase coding sequence

[0083] The recombinant plasmid pET-28a(+)-CCD1 containing carotenoid cleavage dioxygenase coding gene (SEQ ID NO: 2) synthesized by GenScript was used as a template, and primers (P1, P2) were designed to amplify the carotenoid cleavage dioxygenase coding sequence containing the promoter homologous arm:

[0084] Primer P1: 5'- TTTATAATGCTATACTccatcttagtatattagttaagtataagaaggagatatacatATGGGCAAAAAGGAAGAGGAAG- 3' (as shown in SEQ ID NO: 10)

[0085] Primer P2: 5'- cagcggtttctttaccagactcgag- 3' (as shown in SEQ ID NO: 11)

[0086] The PCR amplification reaction was performed in a 40 μL system, and 20 μL Phanta ® Max, 17 μL ddH2O, 1 μL template DNA, 1 μL of each of the upstream and downstream primers were added. The reaction conditions were as follows: pre-denaturation at 95°C for 3 min, followed by 33 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 1 min; and final extension at 72°C for 5 min. The PCR product was subjected to nucleic acid electrophoresis identification and gel recovery and purification.

[0087] (2) Linearization of pCOLADuet-1 plasmid

[0088] The plasmid pCOLADuet-1 was used as a template, and primers (P3, P4) were designed for reverse PCR amplification of the linearized plasmid containing the promoter:

[0089] Primer P3: 5'- ctcgagtctggtaaagaaaccgctg- 3' (as shown in SEQ ID NO: 12)

[0090] Primer P4: 5'- atactaagatggAGTATAGCATTATAAAGACTATGTACAAGATTGTCAATAAAAtaatttcgattatgcggccgt- 3' (as shown in SEQ ID NO: 13)

[0091] The PCR amplification reaction was performed in a 40 μL system, and 20 μL Phanta ®Max, 17 μL ddH2O, 1 μL template DNA, 1 μL each of upstream and downstream primers. The reaction conditions were as follows: after pre-denaturation at 95 °C for 3 min, start the cycle: 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; final extension at 72 °C for 5 min. The PCR product was subjected to nucleic acid electrophoresis identification and gel recovery and purification. The product was further subjected to single enzyme digestion with Dpn I, and the linearized plasmid was recovered after 3 h of enzyme digestion at 37 °C.

[0092] (3) Construction of plasmid pCOLADuet-CCD1

[0093] In the PCR tube, 0.02 pmol of the recovered product of step (1) and 0.06 pmol of the linearized plasmid of step (2) were added, and an equal volume of Hieff Clone ® Universal Enzyme Premix was added. After mixing, the ligation was performed at 50 °C for 30 min, and the ligation product was transformed into E. coli JM 109 competent cells, which were cultured overnight on solid LB medium containing kanamycin resistance (50 mg / L). Multiple transformants were picked and cultured in LB liquid medium (Kanr) at 37 °C and 220 rpm for 10-12 h. After plasmid extraction, PCR verification was performed, and the sequence was determined by Tianlin Sequencing Company. The recombinant plasmid with correct sequence was named pCOLADuet-CCD1.

[0094] (4) Construction of recombinant strain EcN-CCD1

[0095] The verified plasmid of step (3) was transformed into E. coli Nissle 1917. Positive transformants were determined by colony PCR and sequence determination by Tianlin Sequencing Company. The recombinant strain with correct sequence was named EcN-CCD1. At the same time, pCOLADuet-1 was transformed according to the same steps to construct the control strain EcN-pCOLADuet. Positive transformants were picked and inoculated in 10 mL LB liquid medium (Kanr) at 37 °C and 220 rpm for 12 h, and then inoculated in 30 mL TB liquid medium (Kanr) at 2% inoculation amount and cultured at 30 °C and 220 rpm for 12 h. The fermentation broth was centrifuged at 12000 rpm for 10 min at 4 °C to collect the bacterial cells, which were resuspended with PBS buffer (pH 7.0) and subjected to ultrasonic disruption to obtain crude enzyme solution for determination of carotene degradation rate. The carotene degradation rate was about 43.5%, which was 15.0 times that of the unmodified strain Figure 2 、 3 ).

[0096] Example 5

[0097] The present embodiment provides a method for constructing a recombinant strain BS-CCD1, and the specific steps are as follows:

[0098] (1) The genome of B. subtilis 168 (purchased from Moli Bio) was used as a template, and primers (P5, P6) were designed to amplify the penP signal peptide sequence, and primers (P7, P8) were designed to amplify the P43 promoter sequence:

[0099] Primer P5: 5'-ATGAAGTTGAAAACTAAAGCGTC-3' (as shown in SEQ ID NO: 14)

[0100] Primer P6: 5'-TTGCTTCGGCATGTGTTGAG-3' (as shown in SEQ ID NO: 15)

[0101] Primer P7: 5'-CCTAAAAAGGAGCGATTTACATATGCGGTTGATTTAATAACTGACAAAC-3' (as shown in SEQ ID NO: 16)

[0102] Primer P8: 5'-GACGCTTTAGTTTTCAACTTCATTCCTCTCTTACCTATAA-3' (as shown in SEQ ID NO: 17)

[0103] The PCR amplification reaction was performed in a 40 μL system, and 20 μL Phanta®, 17 μL ddH2O, 1 μL template DNA, 1 μL upstream and downstream primers were added to the reaction system. The reaction conditions were as follows: pre-denaturation at 95°C for 3 min, followed by 33 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s; and final extension at 72°C for 5 min. The PCR product was subjected to nucleic acid electrophoresis identification and gel recovery and purification.

[0104] (2) Amplification of carotenoid cleavage dioxygenase coding sequence

[0105] The recombinant plasmid pET-28a(+)-CCD1 containing the carotenoid cleavage dioxygenase coding gene was used as a template, and primers (P9, P10) were designed to amplify the carotenoid cleavage dioxygenase coding sequence containing homologous arms:

[0106] Primer P9: 5'-CTCAACACATGCCGAAGCAAATGGGCAAAAAGGAAGAGGAAG-3' (as shown in SEQ ID NO: 18)

[0107] Primer P10: 5'-GCTTGAGCTCGACTCTAGAGGATCCCAGTTTCGCCTGCTCCTGAAT-3' (as set forth in SEQ ID NO: 19)

[0108] (3) Linearization of pMA5 plasmid

[0109] The pMA5 plasmid was single-cut with Nde I, and after 3 h of enzyme cutting at 37 °C, nucleic acid electrophoresis was performed to verify and cut the gel to recover the linearized plasmid.

[0110] (4) Construction of multimeric plasmid

[0111] In the PCR tube, 0.2 pmol of the recovered products of steps (1), (2), and (3) was added, and an equal volume of Phanta ® Max was connected by POE-PCR reaction. The reaction conditions were as follows: after pre-denaturation at 95 °C for 30 s, start the cycle: denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 7 min, a total of 30 cycles; and final extension at 72 °C for 5 min.

[0112] (5) Transformation of B. subtilis SCK6 with the multimeric plasmid

[0113] The multimeric plasmid in step (4) was transformed into B. subtilis WB600 (purchased from Moli Bio). Positive transformants were determined by colony PCR and sequence determination by Tianlin Sequencing Company, and the recombinant strain with correct sequence was named BS-CCD1. At the same time, the control strain BS-pMA5 was constructed by transforming pMA5 according to the same steps. The positive transformants were inoculated in 10 mL LB liquid medium (Kanr) and cultured at 37 °C and 220 rpm for 12 h, and then inoculated in 30 mL TB liquid medium (Kanr) at a 2% inoculation amount and cultured for 36 h. The fermentation broth was centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected to determine the carotene degradation rate. The carotene degradation rate was about 16.7%, which was 2.1 times that of the unmodified strain Figure 2 、 3 ).

[0114] Example 6

[0115] This example provides a tobacco leaf fermentation method of the enzyme preparation, and the specific steps are as follows:

[0116] 20 g / L tobacco leaves were added to 30 mL of E. coli crude enzyme solution, and incubated at 37 °C and 220 rpm for 6 h. 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).

[0117] Table 1. Leaf fermentation gas chromatography results

[0118]

[0119] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the scope of protection of the present application.

Claims

1. An expression cassette characterized in that, The expression cassette comprises: a gene encoding carotenoid cleavage dioxygenase and a promoter; the gene encoding carotenoid cleavage dioxygenase has: (1) a nucleotide sequence as shown in SEQ ID NO: 2; (2) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases of the nucleotide sequence shown in (1); (3) a sequence having at least 80% homology with the nucleotide sequence shown in (1); (4) a complementary sequence of the sequence shown in (1), (2) or (3); the promoter comprises: pSyn1 and / or P43.

2. The expression cassette of claim 1, wherein, The expression cassette further comprises: a signal peptide penp.

3. The expression cassette of claim 2, wherein, The promoter has: (5) a nucleotide sequence as shown in SEQ ID NO: 3 or as shown in SEQ ID NO: 7; (6) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases of the nucleotide sequence shown in (5); (7) a sequence having at least 80% homology with the nucleotide sequence shown in (5); (8) a complementary sequence of the sequence shown in (5), (6) or (7); and / or The signal peptide penp has: (9) a nucleotide sequence as shown in SEQ ID NO: 6; (10) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases of the nucleotide sequence shown in (9); (11) a sequence having at least 80% homology with the nucleotide sequence shown in (9); (12) a complementary sequence of the sequence shown in (9), (10) or (11).

4. The expression cassette of claim 3, wherein, The expression cassette has: (13) a nucleotide sequence as shown in SEQ ID NO: 4 or as shown in SEQ ID NO: 8; (14) a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases of the nucleotide sequence shown in (13); (15) a sequence having at least 80% homology with the nucleotide sequence shown in (13); (16) a complementary sequence of the sequence shown in (13), (14) or (15).

5. An expression vector, characterized in that, The expression cassette and backbone plasmid as claimed in any one of claims 1 to 4; The backbone plasmid comprises: pCOLADuet-1 and / or pMA5. The expression vector as claimed in claim 5 is transformed and / or transfected into a chassis cell; 6. A host cell characterized in that, The chassis cell comprises: Escherichia coli and / or Bacillus subtilis. The expression cassette as claimed in any one of claims 1 to 4, the expression vector as claimed in claim 5 and / or the host cell as claimed in claim 6 are directly or indirectly prepared.

7. Enzyme or preparation thereof, characterized in that, 8. Use of the expression cassette as claimed in any one of claims 1 to 4, the expression vector as claimed in claim 5, the host cell as claimed in claim 6 and / or the enzyme or preparation thereof as claimed in claim 7 in any one of: (I) tobacco leaf fermentation; and / or (II) tobacco leaf flavoring; and / or (III) degrading carotenoids in tobacco leaf; and / or (IV) increasing the content of flavoring substances in tobacco leaf. The enzyme or preparation thereof as claimed in claim 7 is mixed with a carrot mother liquor and incubated.

9. A method for the degradation of carotenoids, characterized in that, ​ 10. A method of fermentation of tobacco leaves, characterized in that, Mixing the enzyme or preparation thereof as claimed in claim 7 with tobacco leaves and incubating.

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