Tobacco carotenoid monooxygenase BCM for catalytic synthesis of beta-ionone and application of tobacco carotenoid monooxygenase BCM in tobacco leaf quality improvement

Abstract

The invention discloses tobacco carotenoid monooxygenase BCM for catalytic synthesis of beta-ionone and application of the tobacco carotenoid monooxygenase BCM in tobacco leaf quality improvement, and belongs to the technical field of genetic engineering. The amino acid sequences of the tobacco carotenoid monooxygenase BCM are as shown in SEQ ID NO. 1 to SEQ ID NO. 10. The carotenoid monooxygenase BCM disclosed by the invention has relatively good enzymatic activity and can be applied to catalyzing beta-carotene to generate beta-ionone, and the production capacity of an expression strain disclosed by the invention is stable and sustainable; when the recombinant engineering bacterium expressing the BCM is applied to tobacco leaf fermentation, the content of beta-ionone in tobacco leaves can be increased, the smoking quality of the tobacco leaves is improved, and the recombinant engineering bacterium shows a wide application prospect in the fields of tobacco leaf fermentation and quality improvement and is expected to promote transformation and upgrading of the tobacco industry.

Description

Technical Field

[0001] The invention relates to a tobacco carotenoid monooxygenase BCM for catalyzing the synthesis of beta-ionone and application thereof in improving the quality of tobacco leaves, belonging to the technical field of genetic engineering. Background Art

[0002] In the tobacco industry, the quality and flavor of tobacco leaves are one of the key factors that determine its market value. Carotenoids in tobacco leaves, especially β-carotene, as important precursors, have a significant impact on the color, aroma and taste of tobacco leaves. However, under natural conditions, the conversion efficiency of β-carotene in tobacco leaves is low, and it is difficult to directly meet the demand for high-quality tobacco products. Therefore, the development of an enzyme catalyst that can efficiently catalyze the conversion of β-carotene into more valuable compounds, such as β-ionone, is of great significance for improving the quality of tobacco leaves and enriching the flavor of tobacco. In the prior art, research on carotenoid converting enzymes has made certain progress, but enzyme catalysts for tobacco-specific environments and needs are still relatively scarce.

[0003] As a microorganism widely present in the natural environment, Enterobacter holmii has rich biodiversity and potential production capacity of bioactive substances. Its genome contains a large number of underexplored genetic resources, including genes that may encode efficient enzymes. Since Enterobacter holmii has evolved a unique metabolic mechanism in the process of adapting to different ecological environments, the enzymes it produces are often efficient, specific and highly adaptable to the environment. At present, there is still a lack of knowledge about the functions of certain enzyme sequences in Enterobacter holmii, especially how these enzymes can be used to improve the quality of tobacco leaves, and there is even less research. Summary of the invention

[0004] In order to overcome the problems existing in the prior art, the present invention discovered a specific sequence from Enterobacter hallii, which was classified as unknown in the NCBI database. Through the innovative research and functional analysis of the present invention, it was revealed that the sequence encodes an enzyme with the activity of cleaving β-carotene, which was named BCM. The enzyme can catalyze the cleavage reaction of β-carotene to generate β-ionone, an important aroma substance, and has broad application prospects in optimizing tobacco leaf quality and improving the flavor of tobacco products.

[0005] The present invention provides an enzyme derived from Enterobacter hormaechei (named BCM), a recombinant plasmid containing the enzyme, or a recombinant engineered bacterium expressing the enzyme for use in improving the quality of cut tobacco. The enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics:

[0006] (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0007] (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0008] (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0009] (4) any one of the amino acid sequences in SEQ ID NOs. 1 to 10;

[0010] The BCM may be a carotenoid monooxygenase.

[0011] As an optional method of the present invention, the improving of tobacco leaf quality is to improve any one of the β-ionone content, aroma quality, aroma quantity, permeability or body fluid production of the tobacco leaves.

[0012] As an optional method of the present invention, the application is to use the recombinant engineered bacteria expressing BCM to spray and ferment the tobacco.

[0013] As an optional method of the present invention, the ratio of the recombinant engineered bacteria to the tobacco in the application is 0.05-0.20 g of bacterial mud / 100 g of tobacco, wherein the concentration of the bacterial mud is (1.30-3.55)×10 10 CFU / g.

[0014] As an optional mode of the present invention, the fermentation conditions in the application are a temperature of 25 to 35° C., a humidity of 65 to 75%, and a fermentation time of 12 to 20 hours.

[0015] The present invention also provides an enzyme derived from Enterobacter hormaechei, a recombinant plasmid containing the enzyme, or a recombinant engineered bacterium expressing the enzyme for use in preparing β-ionone, wherein the enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics:

[0016] (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0017] (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0018] (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0019] (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

[0020] As an optional mode of the present invention, the use is to generate β-ionone by converting β-carotene.

[0021] The present invention also provides a recombinant plasmid expressing an enzyme that catalyzes the production of β-ionone from β-carotene, wherein the recombinant plasmid is inserted with a nucleotide sequence encoding an enzyme derived from Enterobacter hormaechei, wherein the enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics:

[0022] (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0023] (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0024] (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0025] (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

[0026] As an optional mode of the present invention, the recombinant plasmid is obtained by cloning the gene encoding BCM into the multiple cloning site of the expression vector.

[0027] As an optional mode of the present invention, the expression vector is pPICZαA.

[0028] As an optional mode of the present invention, the recombinant plasmid is obtained by cloning the gene encoding the enzyme between the EcoRI and NotI restriction sites of the vector pPICZαA, connecting the target gene sequence with the expression regulatory sequence, and obtaining the expression recombinant plasmid.

[0029] The present invention also provides a recombinant engineered bacterium producing β-ionone, wherein the recombinant engineered bacterium expresses an enzyme derived from Enterobacter hormaechei, wherein the enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics:

[0030] (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0031] (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0032] (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0033] (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

[0034] As an optional mode of the present invention, the host cell of the recombinant engineered bacteria is yeast.

[0035] As an optional mode of the present invention, the host cell of the recombinant engineered bacteria is Saccharomyces cerevisiae or Pichia pastoris.

[0036] The present invention also provides a biological preparation for catalyzing β-carotene to produce β-ionone, wherein the active ingredient of the biological preparation contains an enzyme derived from Enterobacter hormaechei or a recombinant engineered bacterium expressing the enzyme, wherein the enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics:

[0037] (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0038] (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0039] (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0040] (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

[0041] Beneficial effects:

[0042] The present invention reveals for the first time the key role of the BCM sequence derived from Enterobacter hormaechei in the biosynthesis of β-ionone, filling the gap in research in this field; the recombinant engineered bacteria constructed using the BCM sequence have efficient and stable β-ionone production capacity, providing the possibility for industrial production; the application of recombinant engineered bacteria expressing BCM in tobacco leaf fermentation can increase the β-ionone content in tobacco leaves and improve the smoking quality of tobacco leaves, including aroma quality, aroma quantity, transparency and body fluid production, etc., showing broad application prospects in the field of tobacco leaf fermentation and quality improvement, and is expected to promote the transformation and upgrading of the tobacco industry.

[0043] In summary, the present invention not only enriches the functional cognition of the monooxygenase family from Enterobacter hallii, but also provides strong biotechnological support for the aroma improvement and quality enhancement of tobacco products, and has significant theoretical value and practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a graph showing the expression level of tobacco carotenoid monooxygenase detected by Western-Blot electrophoresis.

[0045] Figure 2 Diagram for the degradation of β-carotene, a substrate catalyzed by monooxygenase.

[0046] Figure 3 This is a diagram showing the degradation of β-carotene, a substrate in the reaction catalyzed by tobacco carotenoid monooxygenase, detected by HPLC.

[0047] Figure 4 This is a diagram showing the conversion of β-carotene to β-ionone by monooxygenase BCM.

[0048] Figure 5 A graph was generated for HPLC detection of the conversion of β-carotene by carotenoid monooxygenase BCM.

[0049] Figure 6 Diagram for the production of β-ionone in tobacco leaves by BCM fermentation with monooxygenase. β-ionone: β-ionone. DETAILED DESCRIPTION

[0050] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0051] The amino acid sequences SEQ ID NO. 1 to 10 used in the present invention are derived from Enterobacter hallii.

[0052] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The various expression elements or sequences described in the present invention are shown and connected in the order of 5' to 3' unless otherwise specified.

[0053] 1. The experimental materials and instruments involved in the following embodiments are as follows:

[0054] Restriction endonucleases EcoRI and NotI, and DNA ligase were purchased from ThermoFisher Scientific;

[0055] Enterobacter hormaechei F8-1 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the accession number CGMCC 22156;

[0056] Plasmid vector pPICZαA was purchased from Novagen;

[0057] ClonExpress Ultra One Step Cloning Kit was purchased from Sangon Biotechnology Co., Ltd.;

[0058] Restriction endonuclease BglII was purchased from Thermo Fisher Scientific;

[0059] DiaSpin column PCR product purification kit was purchased from Sangon Biotechnology Co., Ltd.;

[0060] The bacterial genomic DNA extraction kit was purchased from TAKARA;

[0061] The chassis cell Pichia pastoris X33 strain was purchased from Invitrogen;

[0062] Tobacco raw material Gansu C3L-AB 2021 comes from Gansu Tobacco Industry Co., Ltd.;

[0063] Glucose, FeSO4, dichloromethane, and methanol were purchased from Tianjin Kaitong Chemical Reagent Co., Ltd.; yeast extract and peptone were purchased from Beijing Aoboxing Biotechnology Co., Ltd.; Tween-80 was purchased from Beijing Solebow Technology Co., Ltd.; all the reagents involved were of analytical grade.

[0064] β-Carotene N805181-5 g (purity > 96%) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0065] β-ionone B65386-100 mg (purity>98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0066] 1260 high performance liquid chromatography system was purchased from Agilent Technologies, USA; gas chromatography-tandem mass spectrometer (GC-MS / MS) was purchased from Agilent Technologies, USA; three-dimensional pressure steam sterilizer LDZX-50KBS was purchased from Shanghai Shen'an Medical Equipment Factory; constant temperature oscillator was purchased from Shanghai Fuma Experimental Equipment Co., Ltd.; three-dimensional pressure steam sterilizer LDZX-50KBS was purchased from Shanghai Shen'an Medical Equipment Factory; high-speed refrigerated centrifuge was purchased from Herolab, Germany; electrophoresis apparatus DYY-6C was purchased from Beijing Liuyi Instrument Factory; fully automatic gel imaging analyzer JB-680B was purchased from Shanghai Peiqing Technology Co., Ltd.

[0067] 2. The culture medium involved in the following embodiments is as follows:

[0068] YPD liquid medium: 1% yeast powder, 2% peptone, 2% glucose.

[0069] YP liquid medium: 1% yeast powder, 2% peptone, 1% acid hydrolyzed casein.

[0070] Antibiotic plates with a working concentration of 100 μg / mL Zeocin (bleomycin): 1% yeast powder, 2% peptone, 2% glucose, 2% agar powder, and a working concentration of 100 μg / mL Zeocin (bleomycin).

[0071] 3. The experimental methods involved in the following embodiments are as follows:

[0072] (1) β-carotene content detection method (high performance liquid chromatography detection)

[0073] Preparation of standard substances: Take β-carotene standard substance and prepare it into 1 mg / mL mother solution with dichloromethane, and dilute it into 0, 5, 10, 50, 100, 200 mg / L standard solutions respectively. Protect from light during the preparation process and store the mother solution in a brown bottle or a reagent bottle wrapped in tin foil.

[0074] A linear regression was performed on the peak area according to the concentration of the β-carotene standard, and the β-carotene content in the sample was calculated using the external standard method.

[0075] Preparation of β-carotene stock solution: Under light-proof conditions, weigh 50 mg of β-carotene and dissolve it in 12 mL of dichloromethane. After the β-carotene is completely dissolved, add 0.4 g of Tween-80 for emulsification, concentrate under reduced pressure to remove dichloromethane, and add 10 mL of sterile water to dissolve to obtain 5 mg / mL β-carotene stock solution for standby use.

[0076] Liquid chromatography conditions for β-carotene content: 1260 high performance liquid chromatography system (Agilent, USA), C18 chromatographic column (4.6×250 mm; diameter 5 μm; USNH017518, USA), ethyl acetate as solvent A and acetonitrile: water (88:12) as solvent B, concentrations of 2% A-60% A, running 0-24 min, 60% A-2% A, running 24-26 min, 2% A, running 26-41 min. Flow rate 1.0 mL / min, column temperature 25°C, injection volume 10 μL, absorbance monitoring at 450 nm for detection.

[0077] (2) β-ionone content detection method (high performance liquid chromatography detection)

[0078] Preparation of standards: Take β-ionone standard and prepare it into a 1 mg / mL stock solution with methanol, and dilute it into 0, 0.5, 1, 2, 5, and 10 μg / mL standard solutions respectively. Protect from light during the preparation process and store the stock solution in a brown bottle or a reagent bottle wrapped in tin foil.

[0079] The peak area was linearly regressed according to the concentration of β-ionone standard, and the β-ionone content in the sample was calculated by external standard method.

[0080] Liquid chromatography conditions for β-ionone content: 1260 high performance liquid chromatography system (Agilent, USA), C18 chromatographic column (4.6×250 mm; diameter 5 μm; USNH017518, USA), distilled water (A) and methanol (B) as solvents, concentrations of 50% B-100% B for 0-25 min, 100% B-50% for 25-26 min, and 50% B for 26-35 min. The flow rate was 1.2 mL / min, the column temperature was 40° C., the injection volume was 20 μL, and the absorbance was monitored at 300 nm for detection.

[0081] Example 1: PCR amplification of BCM gene and construction of recombinant plasmid

[0082] The whole genome sequencing results of Enterobacter hallii F8-1 established a gene set of carbohydrate active enzymes, oxygenases, proteases, esterases, etc. Each sequence in the gene set was subjected to blast analysis, and combined with the oxygenase annotation results of other Enterobacter hallii in NCBI, ten sequences were screened as SEQ ID NO.1-10, which were compared with SEQ ID NO.1, with a similarity of more than 98%, and the functions were not annotated in the database. The present invention preliminarily speculated that the sequence function was a monooxygenase, and it was named BCM.

[0083] The sequence used in this example is SEQ ID NO.3.

[0084] The BCM gene cloning and vector construction process are briefly described as follows:

[0085] (1) BCM gene cloning

[0086] The specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows (SEQ ID NO.11-12):

[0087] BCM-F(SEQ ID NO.11):GAATTCATGGACGAGCTTTACATCCTTGG

[0088] BCM-R (SEQ ID NO. 12): GCGGCCGCTTTTTCTTCTCCTGCTTTAGGCGG

[0089] The genomic DNA from E. hallii F8-1 was extracted using a bacterial genomic DNA extraction kit and used as a template for PCR amplification of the BCM gene. The PCR amplification program was: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 30 s, and after 34 cycles, 72°C full extension for 5 min. The PCR amplification product was detected by agarose gel electrophoresis, and the electrophoresis product was recovered.

[0090] (2) Construction of recombinant expression plasmid BCM-pPICZαA

[0091] The electrophoresis product recovered in step (1) was double-digested with EcoRI and NotI, and the empty vector pPICZαA was double-digested with EcoRI and NotI at the same time. The digestion products were recovered and connected using ClonExpress Ultra One Step Cloning Kit to obtain the recombinant expression plasmid BCM-pPICZαA.

[0092] (3) Electrotransformation of BCM-pPICZαA into Pichia pastoris X33 strain

[0093] The recombinant expression plasmid BCM-pPICZαA was linearized with restriction endonuclease BglII. The 80 μL enzyme digestion system was as follows: 10X buffer: 8 μL; BCM-pPICZαA plasmid: 5-6 mg (cut 25 μL); BglII: 1 μL; add water to: 80 μL to obtain a linearized recombinant vector.

[0094] React at 37℃ for 1 hour, and use DiaSpin column PCR product purification kit to recover the linearized recombinant vector. The linearized recombinant vector was precipitated with 400μL Buffer B3, then passed through the column at 12000r / min, and then washed twice with PW buffer at 12000r / min, spun and dried in air, added 40μL preheated water, let stand for 3min, and spun in air at 12000r / min for 2min to obtain the purified linearized plasmid.

[0095] Take 10 μg of the purified linearized plasmid and mix it with 100 μL of X33 competent cells (Pichia pastoris X33 strain), transfer it to a 0.2 cm ice-precooled electroporation cup, wipe the electroporation cup clean with paper to ensure that there are no water droplets on the outside, and electroporate at 2kV. After electroporation, immediately resuspend the bacteria with 1 mL of ice-precooled sorbitol, transfer it to a 1.5 mL EP tube, culture it at 200 rpm and 30 ° C for 2 hours, centrifuge it at 3000 rpm and 4 ° C for 5 minutes, retain 100 μL of the supernatant suspension competent state and spread it on an antibiotic plate containing 100 μg / mL Zeocin (bleomycin), culture it at 30 ° C for 3 days until a single colony appears, select the positive clones, and obtain the recombinant engineering strain to be verified.

[0096] Example 2: Recombinant yeast fermentation expression of BCM

[0097] The recombinant engineering bacteria constructed in Example 1 were picked on the plate with smooth edges and vigorous monoclones and inoculated into 20mL YPD liquid culture medium, 20μL of Zeocin was added to make its working concentration 100μg / mL, and the first-level seed solution was obtained at 30°C and 200rpm for 24h. The first-level seed solution was inoculated into 100mL YPD liquid culture medium without antibiotics at a ratio of 1:50, and cultured at 30°C and 200rpm for 24h. After the shake flask was left to stand for 3h, the supernatant was poured out to retain the bacterial precipitate. Resuspended in 100mL YP liquid culture medium, 0.5% methanol was subsequently added, and induced at 25°C and 200rpm for 24h. After adding 0.5% methanol again for induction for 16h, the expressed recombinant engineering bacteria fermentation liquid was obtained.

[0098] 1 mL of fermentation liquid was taken for detection of the extracellular secretion expression of the exogenous gene, and the remaining fermentation liquid was crushed in a homogenizer (90 MPa) for 3 min, and 1 mL of the crushed supernatant was taken for detection of the intracellular expression of the exogenous gene.

[0099] Western-blot technology was used to verify whether protein expression was present, with creatinine carrying a His-tag as a positive control. Figure 1 The present invention utilizes the BCM strain to conduct stability testing research on recombinant engineering bacteria, verification analysis and evaluation of target substrates.

[0100] Example 3: Study on the degradation ability of BCM on β-carotene

[0101] The specific experimental steps for studying the degradation ability of BCM on β-carotene are as follows:

[0102] (1) The fermentation liquid of the recombinant engineered bacteria obtained in Example 2 was centrifuged at 4°C and 7000 r / min for 10 min, and the culture medium was discarded to obtain bacterial sludge. The bacterial sludge was suspended in sterile water, and the excess culture medium was removed. The bacterial sludge was centrifuged at 4°C and 7000 r / min for 10 min to obtain bacterial sludge (2.80×10 10 The above bacterial sludge was prepared into a bacterial suspension of 0.4 g / mL using 0.1 M PBS buffer (pH 7.0).

[0103] (2) Pipette 18mL of BCM bacterial suspension into a 50mL sterile Erlenmeyer flask, add 2mL of β-carotene stock solution to make the final concentration of the substrate 500mg / L, add 20μL of 100mM FeSO4 solution to a final concentration of 0.1mM, and prepare a total system of 20mL of bacterial suspension reaction mixture. Incubate at 30℃ and 200r / min for 0h, 8h, and 24h respectively. The incubated sample was transferred to a 50mL centrifuge tube, extracted with 20mL of dichloromethane, centrifuged at 12000g for 10min at 4℃, and the lower organic phase was transferred to a new 50mL centrifuge tube. The upper aqueous phase was extracted again with 20mL of dichloromethane, centrifuged at 12000g for 10min at 4℃, and the lower organic phase was transferred again and merged into the 50mL centrifuge tube where the dichloromethane for the first extraction was stored. The sample after dichloromethane extraction was diluted 10 times, passed through a 0.22 μm organic phase filter, and then tested by high performance liquid chromatography.

[0104] (3) The implementation method of the control group is the same as step (2), except that the BCM bacterial suspension is adjusted to 0.1 M PBS buffer.

[0105] The retention time of β-carotene is 26 minutes. Figure 2 and Figure 3 As shown in Table 1, the content of β-carotene gradually decreased with the extension of time. The degradation rate of BCM after 24 hours of degradation by recombinant engineering bacteria was 41.98% (Table 1).

[0106] Table 1 β-carotene concentration and degradation rate

[0107] time PBS control group BCM Group 0hβ-carotene concentration (mg / L) 574.07 448.76 0hβ-carotene degradation rate 1.02％ 22.63％ 8hβ-carotene concentration (mg / L) 578.58 315.38 8hβ-carotene degradation rate 0.24％ 45.62％ 24h β-carotene concentration (mg / L) 533.84 241.70 24h β-carotene degradation rate 7.96％ 58.33％

[0108] Note: The time here (0h, 8h, 24h) is the shaking incubation time in the above-mentioned method for determining the degradation ability of BCM on β-carotene.

[0109] Example 4: Conversion of β-carotene to β-ionone by BCM

[0110] The specific experimental steps for studying the ability of BCM to convert β-carotene to β-ionone are as follows:

[0111] (1) Pipette 18 mL of the BCM bacterial suspension obtained in Example 3 into a 50 mL sterile Erlenmeyer flask, add 2 mL of β-carotene stock solution to make the final concentration of the substrate 500 mg / L, add 20 μL of 100 mM FeSO4 solution to a final concentration of 0.1 mM, and prepare a total system of 20 mL of bacterial suspension reaction mixture. Add 5% (M / V) n-dodecane phase to capture β-ionone, which floats on the upper layer of the sample as an organic phase. Incubate at 30°C and 200 r / min for 0 h, 8 h, and 24 h, respectively. The incubated sample is transferred to a 50 mL centrifuge tube, centrifuged at 12000 g for 10 min at 4°C, and the n-dodecane organic phase on the upper layer of the sample is aspirated. The dodecane phase is mixed with dichloromethane (V / V=1:1) and then passed through a 0.22 μm organic phase membrane for high performance liquid chromatography detection.

[0112] (2) The implementation method of the control group is the same as step (1), except that the BCM bacterial suspension is adjusted to 0.1 M PBS buffer.

[0113] like Figure 4-5 As shown, in the mass spectrum of the recombinant engineered bacteria, the chromatographic peak at 21.87 min is consistent with the mass spectrum of β-ionone Figure 1 As the reaction time increases, the concentration of β-ionone basically shows a trend of continuous increase. Therefore, BCM has the activity of cleaving β-carotene, and the amount of β-ionone produced after 8 hours of degradation is 14.976 mg / L. The amount of β-ionone produced after 24 hours of degradation is 18.256 g / L (Table 2).

[0114] In summary, BCM derived from Enterobacter hallii has good catalytic activity, can efficiently catalyze the degradation of β-carotene to produce β-ionone, and has good industrial application prospects.

[0115] Table 2 β-ionone production (mg / L)

[0116] time PBS control group BCM Group 0h 3.680 4.853 8h 5.903 14.976 24h 7.380 18.256

[0117] Note: The time here (0h, 8h, 24h) is the shaking incubation time in the above-mentioned determination method for converting β-carotene into β-ionone by BCM.

[0118] Example 5: Detection of β-ionone, a key aroma component of fermented tobacco leaves sprayed with BCM bacterial suspension

[0119] 100 mL of the fermentation broth of the recombinant engineered bacteria obtained in Example 2 was centrifuged at 4°C, 7000 r / min for 10 min, and the culture medium was discarded to obtain bacterial sludge (2.80 × 10 10CFU / g). The bacterial sludge was suspended in sterile water, the excess culture medium was removed, and the bacterial sludge was obtained by centrifugation at 4°C and 7000r / min for 10min. The above bacterial sludge was resuspended in 50mL sterile water to prepare a 0.1g / mL BCM bacterial suspension.

[0120] A sterile tray was used to place Gansu C3L-AB 2021 tobacco leaves, and 1 mL of BCM bacterial suspension was evenly sprayed on 100 g of tobacco leaves, and a sterile glass rod was used to stir while spraying to ensure uniform spraying. The sprayed tobacco leaves were placed in a constant temperature and humidity incubator at 30°C and 70% humidity for 24 hours of fermentation.

[0121] The β-ionone content detection method (high performance liquid chromatography detection) was used to detect the content of β-ionone in tobacco leaves before and after treatment with BCM bacterial suspension.

[0122] The test results show that the β-ionone content in the tobacco leaves before treatment was 0.5 μg / g, and the β-ionone content in the tobacco leaves after treatment was 0.57 μg / g, which was 14% higher than that before fermentation ( Figure 6 ).

[0123] Example 6: Expert smoking verification of cigarettes after BCM bacterial suspension was sprayed on fermented tobacco leaves

[0124] 100 mL of the fermentation broth of the recombinant engineered bacteria obtained in Example 2 was centrifuged at 4°C and 7000 r / min for 10 min, and the culture medium was discarded to obtain bacterial sludge (2.80 × 10 10 CFU / g). Use sterile water to suspend the bacterial sludge, remove the excess culture medium, and centrifuge at 4°C and 7000r / min for 10min to obtain bacterial sludge. Resuspend the above bacterial sludge in 50mL sterile water to prepare a 0.1g / mL bacterial suspension.

[0125] A sterile tray was used to place Gansu C3L-AB 2021 tobacco leaves, and 1 mL of bacterial suspension was evenly sprayed on 100 g of tobacco leaves, and a sterile glass rod was used to stir while spraying to ensure uniform spraying. The sprayed tobacco leaves were placed in a constant temperature and humidity incubator and fermented at 30°C and 70% humidity for 18 hours to obtain fermented tobacco samples.

[0126] The fermented tobacco samples obtained in the above steps were dried at 90-120°C for 30-90s, and then balanced for 24h under a relative humidity of 60%±2% and a temperature of 22±2°C, and then rolled using the same tobacco tube. The sensory quality of the single-ingredient tobacco was evaluated by professional smoking evaluation personnel with reference to the sensory evaluation method for tobacco and tobacco products YC / T 138-1998. The sensory quality was scored by professional smoking evaluation experts, and the sensory evaluation results of the fermented tobacco leaves are shown in Table 3.

[0127] The results showed that the taste of tobacco leaves after fermentation with BCM recombinant engineered bacteria was significantly improved, the aroma quality and aroma quantity were significantly increased, the sense of transparency and salivation was increased, the irritation was reduced, the impurities were reduced, the sense of strength was enhanced, and it had a good quality improvement effect.

[0128] This indirectly reflects that the results of aroma component detection are consistent with the results of sensory evaluation.

[0129]

[0130]

[0131] Table 3 Sensory evaluation of tobacco leaves

[0132] Example 7: Application of BCM sequence in synthesizing β-ionone and improving tobacco leaf quality

[0133] The amino acid sequences SEQ ID NO. 1 to 10 are all derived from Enterobacter hormaechei, and after homology comparison, the similarity is more than 98.10%.

[0134] In this example, the amino acid sequences SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.4 to 10 were subjected to PCR amplification of the BCM sequence and construction of the recombinant plasmid according to the methods of Examples 1 to 6 to obtain a recombinant engineering strain. The degradation ability of β-carotene and the synthesis ability of β-ionone of the recombinant engineering strain carrying BCM were measured. The degradation rate of β-carotene of the recombinant engineering bacteria obtained using these 9 sequences was more than 50%, and the conversion rate of β-ionone was more than 6.84%.

[0135] The recombinant engineered bacterial suspension obtained from these 9 sequences was sprayed on fermented tobacco to detect the key aroma-causing component β-ionone and conduct sensory evaluation. The β-ionone content in the treated tobacco leaves increased by about 14% compared with before fermentation; the aroma quality, aroma volume and aroma concentration increased, the permeability and salivation sense were enhanced, the irritation and impurities were reduced, and the quality of the tobacco leaves was significantly improved.

[0136] The sequence involved in the present invention is as follows:

[0137] Enzymes encoded by the BCM nucleotide sequence from Enterobacter hormaechei, including 869

[0138] amino acids, the amino acid sequences are shown in SEQ ID NOs. 1 to 10, specifically as follows:

[0139] SEQ ID NO.1:

[0140] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTPPVDPWRPEPPVTEAEPALVPAAKRPSAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPIELRLAATALFAMVLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSVVALFSFYLMLSIGILAISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVAQLGASQLFLKQELSVLEGVMALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAAFSLLGLVVFYREVDRYYPASFAQSRPWPAVVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0141] SEQ ID NO.2:

[0142] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTPPVDPWRPEPPVTEAEPALVPAAKRPSAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPIELRLAATALFAMVLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSVVALFSFYLMLSIGILAISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVISLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVAQLGASQLFLKQELSVLEGVMALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTN LQDGVVYQQTWLPLVNPLEEGAAFALLGLVMFYREVDRYYPASFAQSRPWPAVVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0143] SEQ ID NO.3:

[0144] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTPPVDPWRPEPPVTEAEPALVPAAKRPSAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPIELRLAATALFAMVLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSVVALFSFYLMLSIGILAISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVAQLGASQLFLKQELSVLEGVMALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAAFALLGLVVFYREVDHYYPASFAQSRPWPAVVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0145] SEQ ID NO.4:

[0146] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTAPVDPWRPEPPVTEAEPALVPAAKRPSAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPLELRLAATALFAMVLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSVVALFSFYLLLSIGILAISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVALLGASQLFLKQELSVLEGVMALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWTILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAALALLGLVVFYREVDRYYPASFAQSRPWPAIVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMFCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0147] SEQ ID NO.5:

[0148] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTPPVDPWRPEPPVTEAEPALVPAAKRPSAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPLELRLAATALFSMVLLAVGWRLRHRQRNYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSFVALFSFYLLLSIGILAISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGSFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVAQLGASQLFLKQELSVLAGVLALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAAFALLGLVVFYREVDRYYPASLAQSRPWPAVVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0149] SEQ ID NO.6:

[0150] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTAPVDPWRPEPPVTEAEPALVPAAKRPYAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPLELRLAATALFAMVLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSFVALFSFYLLLSIGILAISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVALLGASQLFLKQELSVLAGVMALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAAFALLGLVVFYREVDRYYPASFAQSRPWPAVVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0151] SEQ ID NO.7:

[0152] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTAPVDPWRPEPPVTEAEPALVPAAKRPSAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPLELRLAATALFAMGLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSFVALFSFYLLLSIGILVISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVAQLGASQLFLKQELSVLEGVMALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAAFALLGLVVFYREVDRYYPASFAQSRPWPAVVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0153] SEQ ID NO.8:

[0154] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPAPVPEAVTAPVDPWRPEPPVTEAEPALIPAAKRPSAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPIELRLAATALFAMVLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSVVALFSFYLMLSIGILAISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVAQLGASQLFLKQELSVLEGVMALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAAFALLGLVVFYREVDRYYPASLAQSRPWPAVVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0155] SEQ ID NO.9:

[0156] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTPPVDPWRPEPPVTEAEPALVPAAKRPSAFGGILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPIELRLAATALFAMVLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVICAASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSVVALFSFYLMLSIGILAISIWQHWRELNL LGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVALSLRAQEKGKHIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLGVQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQLGSWALLAGGLLFWLVAQLGASQLFLKQELSVLEGVMALTAASAWGWRQAAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVGGGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAAFSLLGLVVFYREVDRYYPASFAQSRPWPAVVLMALGFWWLNGALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0157] SEQ ID NO.10:

[0158] MDELYILGCLFLVFALVVAPVLAVIGFNRSTAARQEIARLRQRIEALEQRGVTEKAPEPVQTAAPVETPASVAEAVTPPVDPWRPEPPVTEAEPALVPAAKRPSAFGG ILSSLTRWFMQGNPLAKLGIVLLFLGLSFLLRYTVEHSLFPIELRLAATALFAMVLLAVGWRLRHRQRIYALILQGGATGVLYLTVFGAFRLWQMLPMTLAFALLVVIC AASVGLAVLQKALSLAMLASLGGYLAPLLLSTGGGSVVALFSFYLMLSIGILAISIWQHWRELNLLGLLFTFGVGGVWGLSDYQPEDYAICQLFLIANTLIFGVFSVA LSLRAQEKGKQIIDGALLFAPPLIGFGMQYGMTSHWTYGPALSALGYGAFYLSLAFLALRRYPSVGRPLVMAALAIGGGFATLAIPLALSARWTAMAWALEGLGILWLG VQQQQRRMSYSGTALLVLALGSAIWAQMDGVTSLSLLLIFAVLSLCWLAAAWLWQKIAQPGSWALLAGGLLFWLVAQLGASQLFLKQELSVLEGVMALTAASAWGWRQ AAARLAWRELDASKWLLWPVMLLMVLYQIWQQQILAAGWANLAWAIALPAALMLLRRDEDKLLPRISMGLHLSLVWMILLAIAAELYWFARSLPWGMAAWGSGIAMAVG GGVIMALSAAVRRRGWPFRVWPALYACLAAIPVVVALVVLLVVTNLQDGVVYQQTWLPLVNPLEEGAAFALLGLVVFYREVDRYYPASFAQSRPWPAVVLMALGFWWLN GALLRALAWYGEVAWNWESLWHSRLIQTTFALFWMLCALVVMIRATRHASRQQWLGGAALLGVVMLKLMLVDSAGGGGLARAVAFIGVAILVLIVGYFSPLPPKAGEEK

[0159] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An application of an enzyme derived from Enterobacter hormaechei, a recombinant plasmid containing the enzyme, or a recombinant engineered bacterium expressing the enzyme in improving tobacco leaf quality, characterized in that: The enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics: (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

2. The application according to claim 1, characterized in that: The application is to use the recombinant engineering bacteria expressing the enzyme to spray and ferment tobacco shreds.

3. The application according to claim 2, characterized in that: In the method, the ratio of the recombinant engineered bacteria to the tobacco shreds is 0.05-0.20 g of bacterial sludge / 100 g of tobacco shreds, wherein the concentration of the bacterial sludge is (1.30-3.55)×10 10 CFU / g; Preferably, the fermentation conditions in the method are a temperature of 25 to 35° C., a humidity of 65 to 75%, and a fermentation time of 12 to 20 hours.

4. Use of an enzyme derived from Enterobacter hormaechei, a recombinant plasmid containing the enzyme, or a recombinant engineered bacterium expressing the enzyme in the preparation of β-ionone, characterized in that: The enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics: (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

5. The application according to claim 4, characterized in that: The application generates β-ionone by converting β-carotene.

6. A recombinant plasmid expressing an enzyme that catalyzes the production of β-ionone from β-carotene, characterized in that: The recombinant plasmid is inserted with a nucleotide sequence encoding an enzyme derived from Enterobacter hormaechei, wherein the enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics: (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

7. The recombinant plasmid according to claim 6, which is obtained by cloning the gene encoding the enzyme into the multiple cloning site of an expression vector; Preferably, the expression vector is pPICZαA; Preferably, the recombinant plasmid is obtained by cloning the gene encoding the enzyme between the EcoRI and NotI restriction sites of the vector pPICZαA, and connecting the target gene sequence with the expression regulatory sequence.

8. A recombinant engineered bacterium producing β-ionone, characterized in that: The recombinant engineered bacteria express an enzyme derived from Enterobacter hormaechei, wherein the enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics: (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

9. The recombinant engineered bacterium according to claim 8, characterized in that The host cell of the recombinant engineered bacteria is a yeast; preferably, the host cell of the recombinant engineered bacteria is Saccharomyces cerevisiae or Pichia pastoris.

10. A biological agent for catalyzing the production of β-ionone from β-carotene, characterized in that: The active ingredient of the biological preparation contains an enzyme derived from Enterobacter hormaechei or a recombinant engineered bacterium expressing the enzyme, wherein the enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics: (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 869th amino acid residues starting from the amino terminus of any of the sequences shown in SEQ ID NOs. 1 to 10; and having a homology of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10; (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

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