Enterobacter hormaechei-derived dioxygenase (BCD) sequence, gene and application thereof in improving tobacco leaf quality by biologically producing aroma

Abstract

The invention discloses a dioxygenase (BCD) sequence from enterobacter hormaechei, a gene and application of the gene in improving tobacco leaf quality by biologically producing aroma, and belongs to the field of biotransformation aroma production. The dioxygenase BCD has good enzymatic activity, the production capacity of an expression strain is stable and sustainable, and the dioxygenase BCD can be applied to catalyzing beta-carotene to generate beta-ionone; the content of the aroma product beta-ionone in the tobacco leaves fermented by spraying the dioxygenase BCD recombinant engineering strain is greatly increased, and the taste is obviously improved.

Description

Technical Field

[0001] The invention relates to a dioxygenase (BCD) sequence from Enterobacter hallii, a gene and application of the same in bio-aroma production to improve tobacco leaf quality, and belongs to the field of bio-conversion aroma production, in particular to the field of carotenoid oxidation aroma production. Background Art

[0002] Tobacco (Nicotiana tabacum) is an important economic crop and a model species for biological research. As an aromatic plant, tobacco contains a large amount of carotenoids and their degradation products. Carotenoid degradation products play an important role in the aroma of tobacco leaves due to their relatively low threshold, good aroma quality, and low irritation. Studies have shown that increasing the content of carotenoid degradation products within the range of 0.01% to 0.5% can make the smoke fuller, the smoke aroma thicker, and improve the alcohol and mildness. The increase in the content of carotenoids and their degradation products in tobacco leaves is conducive to improving the aroma quality of tobacco leaves and can reduce the content of tar and free radicals in smoke. At present, the carotenoids in tobacco leaves are usually degraded by modulation, processing, aging, etc., and the double bonds at different positions are broken to produce aroma substances such as β-damascenone, β-ionone, megastigmatrienone, and dihydroactinolide, thereby achieving the purpose of increasing the aroma of tobacco leaves, bright color, and improving quality. However, there are still many problems in this process. The natural degradation cycle of carotenoids is long, with many influencing factors and difficult to control. Insufficient degradation rate of carotenoids during curing and aging will lead to insufficient aroma of tobacco leaves. Therefore, it is extremely important to find a substance that can degrade carotenoids to generate aroma substances to improve the quality of tobacco leaves.

[0003] Enterobacter hormaechei, as an intestinal bacillus widely present in the natural environment, has a rich genome encoding genes for bioactive substances, including a variety of enzymes with catalytic functions. Although studies have shown that some biologically derived oxidases can participate in the formation of aroma components and can be applied to the field of biotransformation aroma production, there is still insufficient research on the functions of certain enzyme sequences in Enterobacter hormaechei and their application in improving tobacco leaf quality. In-depth research on specific sequences in Enterobacter hormaechei and their application potential in tobacco leaf bioaroma production still needs to be explored. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention discovered a specific sequence derived from Enterobacter hallii, which was previously marked as having an unknown function in the NCBI database. Through in-depth research and functional identification work of the present invention, it was revealed that the sequence encodes an enzyme with the activity of cleaving β-carotene, which is named BCD. The enzyme can synthesize β-ionone by cleaving β-carotene, and has potential application value in the fields of tobacco microbial fermentation and improvement of tobacco quality.

[0005] The first object of the present invention is to provide a method for improving the quality of tobacco leaves, the method comprising using an enzyme derived from Enterobacter hormaechei (named BCD), a recombinant plasmid containing the enzyme, or a recombinant engineered bacterium expressing the enzyme to improve any one of the β-ionone content, aroma quality, aroma quantity, permeability, or salivation of tobacco leaves;

[0006] The enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics:

[0007] (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0008] (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 459th 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;

[0009] (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0010] (4) any one of the amino acid sequences in SEQ ID NOs. 1 to 10;

[0011] The BCD may be a carotenoid dioxygenase.

[0012] In one embodiment of the present invention, the method comprises spraying and fermenting the tobacco shreds using recombinant engineered bacteria expressing BCD.

[0013] In one embodiment of the present invention, the ratio of the recombinant engineered bacteria to the tobacco shreds in the method 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;

[0014] In one embodiment of the present invention, 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.

[0015] The second object of the present invention is to provide an enzyme derived from Enterobacter hormaechei, a recombinant plasmid containing the enzyme, or a recombinant engineered bacterium expressing the enzyme for use in synthesizing 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 459th 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] In one embodiment of the present invention, the application is to use the enzyme, the recombinant plasmid or the recombinant engineered bacteria to catalyze the production of β-ionone from β-carotene.

[0021] The third object of the present invention is to provide a biological preparation for catalyzing the production of β-ionone from β-carotene, wherein the active ingredient of the biological preparation contains an enzyme derived from Enterobacter hormaechei or a recombinant engineered bacterium expressing the enzyme;

[0022] The enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics:

[0023] (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0024] (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 459th 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;

[0025] (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0026] (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

[0027] In one embodiment of the present invention, 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.

[0028] The fourth object of the present invention is to provide a recombinant plasmid expressing an enzyme capable of catalyzing 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;

[0029] The enzyme has the activity of degrading carotenoids, and the amino acid sequence 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 459th 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] In one embodiment of the present invention, the recombinant plasmid is obtained by cloning the gene encoding BCD into the multiple cloning site of the expression vector.

[0035] In one embodiment of the present invention, the expression vector is pPICZαA.

[0036] In one embodiment of the present invention, the recombinant plasmid is obtained by cloning the gene encoding BCD 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.

[0037] A fifth object of the present invention is to provide a recombinant engineered bacterium expressing an enzyme capable of catalyzing the production of β-ionone from β-carotene, wherein the recombinant engineered bacterium expresses an enzyme derived from Enterobacter hormaechei;

[0038] The enzyme has the activity of degrading carotenoids, and the amino acid sequence of the enzyme has at least one of the following characteristics:

[0039] (1) having a sequence similarity of more than 80% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0040] (2) An amino acid sequence formed by substituting, deleting or adding one or more amino acids from the 1st to the 459th 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;

[0041] (3) having a sequence similarity of more than 98% with any of the amino acid sequences shown in SEQ ID NOs. 1 to 10;

[0042] (4) is any one of the amino acid sequences in SEQ ID NOs. 1 to 10.

[0043] In one embodiment of the present invention, the recombinant engineered bacteria is yeast.

[0044] In one embodiment of the present invention, the recombinant engineered bacteria is Saccharomyces cerevisiae or Pichia pastoris.

[0045] In one embodiment of the present invention, the carotenoid is β-carotene.

[0046] Beneficial effects:

[0047] The present invention provides a dioxygenase (BCD) sequence from Enterobacter hallii and its application. Experiments have shown that the recombinant engineered bacteria constructed using the sequence of the present invention can cleave β-carotene to produce the aroma substance β-ionone; after spraying it on fermented tobacco shreds, the aroma of the tobacco shreds can be significantly improved and the quality of tobacco leaves can be improved. The protein of the present invention and its encoding gene have important theoretical and practical significance for the research on the synthesis of β-ionone and the improvement of tobacco leaf quality. They will play an important role in tobacco leaf fermentation and the improvement of tobacco leaf quality, and have broad application prospects. The present invention not only fills the gap in the functional cognition of the sequence, but also provides new biological tools and strategies for technological progress and product development in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1This is the expression level diagram of BCD, PhCCD1, NtCCD1 and NtCCD4 detected by Western-Blot.

[0049] Figure 2 This is a diagram of the degradation of β-carotene, a substrate in a dioxygenase-catalyzed reaction.

[0050] Figure 3 This is a diagram showing the degradation of β-carotene, a substrate in a PBS-catalyzed reaction, detected by HPLC.

[0051] Figure 4 This is a diagram showing the degradation of β-carotene, a substrate in the PhCCD1 catalytic reaction, detected by HPLC.

[0052] Figure 5 This is a diagram showing the degradation of β-carotene, a substrate in the NtCCD1 catalytic reaction, detected by HPLC.

[0053] Figure 6 This is a diagram showing the degradation of β-carotene, a substrate in the NtCCD4 catalytic reaction, detected by HPLC.

[0054] Figure 7 This is a diagram showing the degradation of β-carotene, a substrate in the BCD catalytic reaction, detected by HPLC.

[0055] Figure 8 Diagram showing the conversion of β-carotene to β-ionone by dioxygenase.

[0056] Fig. 9 This is a diagram showing the conversion of PBS to β-carotene to β-ionone using HPLC.

[0057] Fig.10 This is a diagram showing the HPLC detection of PhCCD1 converting β-carotene to β-ionone.

[0058] Fig.11 This is a diagram showing the HPLC detection of NtCCD1 converting β-carotene to β-ionone.

[0059] Fig.12 This is a diagram showing the HPLC detection of NtCCD4 converting β-carotene to β-ionone.

[0060] Fig.13 This is a diagram showing the HPLC detection of BCD converting β-carotene to β-ionone.

[0061] Fig.14 Diagram for the production of β-ionone from tobacco leaves by dioxygenase BCD fermentation. β-ionone: β-ionone. DETAILED DESCRIPTION

[0062] 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.

[0063] The amino acid sequences SEQ ID NO. 1 to 10 used in the present invention are derived from Enterobacter hallii.

[0064] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are 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'.

[0065] 1. The experimental materials and instruments involved in the following embodiments are as follows:

[0066] 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 above reagents were analytical grade;

[0067] β-Carotene N805181-5g (purity>96%) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; β-ionone B65386-100mg (purity>98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0068] Tobacco raw material Gansu C3L-AB 2021 comes from Gansu Tobacco Industry Co., Ltd.;

[0069] The chassis cell Pichia pastoris X33 strain was purchased from Invitrogen;

[0070] Bacterial genomic DNA extraction kit ver.3.0 was purchased from TAKARA;

[0071] Enterobacter hormaechei F8-1 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the accession number CGMCC 22156;

[0072] Plasmid vector pPICZαA was purchased from Novagen;

[0073] ClonExpress Ultra One Step Cloning Kit was purchased from Sangon Biotechnology Co., Ltd.;

[0074] Restriction enzymes EcoRI and NotI, DNA ligase, and restriction enzyme BglII were purchased from Thermo Fisher Scientific;

[0075] DiaSpin column PCR product purification kit was purchased from Sangon Biotechnology Co., Ltd.;

[0076] Zeocin (bleomycin) was purchased from Thermo Fisher Scientific;

[0077] 1260 high performance liquid chromatography system was purchased from Agilent Technologies, USA; LDZX-50KBS three-dimensional pressure steam sterilizer was purchased from Shanghai Shen'an Medical Equipment Factory; constant temperature oscillator was purchased from Shanghai Fuma Experimental Equipment Co., Ltd.; LDZX-50KBS three-dimensional pressure steam sterilizer 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.

[0078] 2. The culture medium involved in the following embodiments is as follows:

[0079] YPD liquid medium: 1% yeast powder, 2% peptone, 2% glucose.

[0080] YP liquid medium: 1% yeast powder, 2% peptone, 1% acid hydrolyzed casein.

[0081] 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).

[0082] 3. The experimental methods involved in the following embodiments are as follows:

[0083] (1) β-carotene content detection method (high performance liquid chromatography detection)

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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, acetonitrile: water (88:12) as solvent B, concentration 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 monitored at 450 nm.

[0088] (2) β-ionone content detection method (high performance liquid chromatography detection)

[0089] 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.

[0090] 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.

[0091] 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 from 50% B to 100% B were run from 0 to 25 min, from 100% B to 50% B were run from 25 to 26 min, and 50% B was run from 26 to 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.

[0092] Example 1: PCR amplification of BCD sequence, construction of recombinant plasmid and acquisition of recombinant engineering strain

[0093] 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.5, with a similarity of more than 98%. The function was not annotated in the database. The present invention preliminarily speculated that the sequence function was dioxygenase, and it was named BCD.

[0094] The sequence used in this example is SEQ ID NO.7.

[0095] The BCD gene cloning and vector construction process are briefly introduced as follows.

[0096] (1) BCD gene cloning

[0097] The specific coding sequence was selected as the target fragment, and the PCR amplification primer sequences were designed as follows (SEQ ID NO.11-12):

[0098] BCD-F(SEQ ID NO.11):GAATTCATGAAATGGAATGAACCGG

[0099] BCD-R (SEQ ID NO.12): GCGGCCGCTGCTGCGCCGTCTT

[0100] The genomic DNA from Enterobacter hallii F8-1 was extracted using a bacterial genomic DNA extraction kit and used as a template for PCR amplification of the BCD 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.

[0101] The sequences of NtCCD1 (amino acid sequence: SEQ ID NO.13), NtCCD4 (amino acid sequence: SEQ ID NO.14) derived from tobacco, and PhCCD1 (amino acid sequence: SEQ ID NO.15) derived from petunia were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0102] (2) Construction of recombinant expression plasmid

[0103] 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, and the digestion products were recovered and connected using ClonExpress Ultra One Step Cloning Kit to obtain the recombinant expression plasmid BCD-pPICZαA. PhCCD1, NtCCD1, and NtCCD4 sequences were constructed into the pPICZαA vector through the EcoRI and NotI double digestion sites to obtain the recombinant expression plasmids PhCCD1-pPICZαA, NtCCD1-pPICZαA, and NtCCD4-pPICZαA, respectively.

[0104] (3) Electroporation of the recombinant expression plasmid into Pichia pastoris X33 strain

[0105] The recombinant expression plasmids BCD-pPICZαA, PhCCD1-pPICZαA, NtCCD1-pPICZαA, and NtCCD4-pPICZαA were linearized with the restriction endonuclease BglII, 80 μL enzyme cutting system: 10X buffer: 8 μL; (BCD-pPICZαA, PhCCD1-pPICZαA, NtCCD1-pPICZαA, NtCCD4-pPICZαA) plasmid: 5-6 mg (25 μL each); BglII: 1 μL; add water to 80 μL.

[0106] React at 37℃ for 1 hour, and use DiaSpin column PCR product purification kit to recover the linearized recombinant vector. Precipitate the linearized recombinant vector with 400μL Buffer B3, then pass through the column at 12000r / min, then wash twice with PW buffer at 12000r / min, spin dry, add 40μL preheated water, let stand for 3min, spin at 12000r / min for 2min, and obtain the purified linearized plasmid.

[0107] 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 apply 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 strains of BCD-pPICZαA, PhCCD1-pPICZαA, NtCCD1-pPICZαA, and NtCCD4-pPICZαA.

[0108] Example 2: Recombinant yeast induced expression of BCD, PhCCD1, NtCCD1, NtCCD4

[0109] The recombinant engineering bacteria constructed in Example 1 were picked on the plate with smooth edges and vigorous monoclones, inoculated in 20mL YPD liquid medium, and 20μL of Zeocin was added to make its working concentration 100μg / mL, and cultured at 30°C and 200rpm for 24h to obtain a primary seed solution. The primary seed solution was inoculated in 100mL YPD liquid 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 to retain the bacterial precipitate. Resuspended in 100mL YP liquid medium, followed by addition of 0.5% methanol, induced at 25°C and 200rpm for 24h. 0.5% methanol was added again for induction for 16h to obtain the fermentation broth of recombinant engineering bacteria expressing BCD, PhCCD1, NtCCD1, and NtCCD4, respectively.

[0110] 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.

[0111] Western-blot technology was used to verify whether protein expression was present, with creatinine carrying a His-tag as a positive control.

[0112] Protein expression results Figure 1 The present invention utilizes the BCD, PhCCD1, NtCCD1, and NtCCD4 strains to conduct stability testing of recombinant engineering bacteria, verification analysis of target substrates, and evaluation of absorption.

[0113] Example 3: Study on the Degradability of β-Carotene

[0114] The specific experimental steps for studying the degradation ability of BCD on β-carotene are as follows:

[0115] (1) The fermentation broth of the recombinant engineered bacteria BCD, PhCCD1, NtCCD1, and NtCCD4 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 (3.04×10 10 The above bacterial sludges were prepared into 0.4 g / mL bacterial suspensions using 0.1 M PBS buffer (pH 7.0), which were BCD, PhCCD1, NtCCD1 and NtCCD4 bacterial suspensions.

[0116] (2) Pipette 18 mL of BCD bacterial suspension 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. Incubate at 30°C and 200 r / min for 0 h, 8 h, and 24 h, respectively. After incubation, transfer the sample to a 50 mL centrifuge tube, add 20 mL of dichloromethane for extraction, centrifuge at 12000 g for 10 min at 4°C, and transfer the lower organic phase to a new 50 mL centrifuge tube. Add 20 mL of dichloromethane to the upper aqueous phase and extract until colorless, centrifuge at 12000 g for 10 min at 4°C, and transfer the lower organic phase again and merge it into the 50 mL 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.

[0117] (3) The implementation method of the control group is the same as step (2), except that the bacterial suspension is adjusted to 0.1 M PBS buffer.

[0118] Table 1 β-carotene concentration and degradation rate

[0119] time PBS control group BCD PhCCD1 NtCCD1 NtCCD4 0hβ-carotene concentration (mg / L) 574.07 448.76 460.92 402.23 427.62 0hβ-carotene degradation rate 1.02％ 21.78％ 20.53％ 30.65％ 26.27％ 8hβ-carotene concentration (mg / L) 578.58 315.38 429.68 222.66 290.63 8hβ-carotene degradation rate 0.24％ 47.70％ 25.92％ 61.61％ 49.89％ 24h β-carotene concentration (mg / L) 533.84 241.70 258.73 214.98 263.36 24h β-carotene degradation rate 7.96％ 62.98％ 55.39％ 62.94％ 54.59％

[0120] Note: The time here (0h, 8h, 24h) is the shaking incubation time in the β-carotene degradation ability determination method.

[0121] The retention time of β-carotene is 26 minutes. Figure 2 to Figure 7As shown, the content of β-carotene gradually decreased with the extension of time. After 24 hours of degradation by recombinant engineering bacteria, the degradation rates of PhCCD1, NtCCD1, NtCCD4, and BCD were 55.39%, 62.94%, 54.59%, and 62.98%, respectively. The degradation rates of NtCCD1 and BCD strains were higher (Table 1).

[0122] In summary, BCD has the function of cleaving β-carotene and has a higher β-carotene degradation rate compared with other carotenoid cleavage dioxygenase (CCD) genes from tobacco and petunia.

[0123] Example 4: Study on the ability to convert β-carotene into β-ionone

[0124] The specific experimental steps for studying the ability of BCD to convert β-carotene into β-ionone are as follows:

[0125] (1) 18 mL of the bacterial suspension of BCD, PhCCD1, NtCCD1, and NtCCD4 obtained in Example 3 were respectively taken into a 50 mL sterile conical flask, 2 mL of β-carotene stock solution was added to make the final concentration of the substrate 500 mg / L, 20 μL of 100 mM FeSO4 solution was added to make the final concentration 0.1 mM, and a total system of 20 mL of bacterial suspension reaction mixture was prepared, and 5% (M / V) n-dodecane phase was added to capture β-ionone, which was floated 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 was 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 was taken. The dodecane phase was mixed with dichloromethane (V / V=1:1) and then passed through a 0.22 μm organic phase membrane for high performance liquid chromatography detection.

[0126] (2) The implementation method of the control group is the same as step (1), except that the bacterial suspension is adjusted to 0.1 M PBS buffer.

[0127] like Figure 8 to Figure 13 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 1As the reaction time increases, the concentration of β-ionone basically shows a trend of continuous increase. From the results of degradation to 8h, the β-ionone production of PhCCD1, NtCCD1, NtCCD4 and BCD were 13.820mg / L, 13.741mg / L, 10.364mg / L, and 17.279mg / L, respectively. After 24h of degradation, the β-ionone production of PhCCD1, NtCCD1, NtCCD4 and BCD were 16.910mg / L, 17.197mg / L, 16.433mg / L, and 19.100mg / L, respectively (Table 2).

[0128] Table 2 β-ionone production (mg / L)

[0129] time PBS control group BCD PhCCD1 NtCCD1 NtCCD4 0h 3.680 7.927 3.291 3.575 4.989 8h 5.903 17.279 13.820 13.741 10.364 24h 7.380 19.100 16.910 17.197 16.433

[0130] Note: The time here (0h, 8h, 24h) is the shaking incubation time in the determination method of converting β-carotene to β-ionone.

[0131] In summary, BCD from Enterobacter hallii has good catalytic activity and can efficiently catalyze the degradation of β-carotene to produce β-ionone. It is a carotenoid dioxygenase and has good industrial application prospects.

[0132] Example 5: Detection of β-ionone, a key aroma component of fermented tobacco shreds sprayed with BCD bacterial suspension

[0133] 100 mL of the fermentation liquid of the BCD recombinant engineering 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 (3.04×10 10 CFU / 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 BCD bacterial suspension.

[0134] A sterile tray was used to place Gansu C3L-AB 2021 tobacco, and 1 mL of BCD suspension was evenly sprayed on 100 g of tobacco, stirring with a sterile glass rod 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 24 hours.

[0135] The β-ionone content detection method (high performance liquid chromatography detection) was used to detect the β-ionone content in tobacco leaves before and after treatment with BCD bacterial suspension.

[0136] 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 0.62 μg / g, which was increased by 24.5% ( Fig.14 ).

[0137] Example 6: Expert smoking verification of cigarettes after spraying fermented tobacco with BCD, PhCCD1, NtCCD1, and NtCCD4 bacterial suspensions

[0138] 100 mL of the fermentation broth of the recombinant engineered bacteria BCD, PhCCD1, NtCCD1, and NtCCD4 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 (3.04×10 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 0.1g / mL BCD, PhCCD1, NtCCD1, and NtCCD4 bacterial suspensions.

[0139] A sterile tray was used to place Gansu C3L-AB 2021 tobacco, and 1 mL of bacterial suspension was evenly sprayed on 100 g of tobacco, and a sterile glass rod was used to stir the tobacco while spraying to ensure uniform spraying. The sprayed tobacco was placed in a constant temperature and humidity incubator and fermented at 30°C and 70% humidity for 18 hours to obtain fermented tobacco samples.

[0140] The fermented tobacco samples obtained in the above steps were dried at 90-120°C for 30-90s, then balanced at a relative humidity of 60±2% and a temperature of 22±2°C for 24h, 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.

[0141] The results showed that the tobacco cuts fermented by spraying with recombinant engineering bacteria expressing BCD, PhCCD1, NtCCD1 and NtCCD4 had improved taste, among which the tobacco cuts fermented by BCD had the most obvious improvement in quality, with increased aroma volume and aroma concentration, enhanced permeability and salivation, reduced irritation and impurities, and a clean and comfortable aftertaste, which had a good quality improvement effect. This indirectly reflects that the results of the aroma component detection are consistent with the results of the sensory evaluation.

[0142] Table 3 Sensory evaluation of tobacco cut after dioxygenase treatment

[0143]

[0144] Example 7: Application of BCD sequence in synthesizing β-ionone and improving tobacco leaf quality

[0145] The amino acid sequences SEQ ID NO. 1 to 10 are all derived from Enterobacter hormaechei, and after homology comparison, the similarity is 98.09%.

[0146] In this example, the amino acid sequences SEQ ID NO. 1 to 6 and SEQ ID NO. 8 to 10 were subjected to PCR amplification of the BCD sequence and construction of the recombinant plasmid according to the method 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 BCD were measured. The degradation rate of β-carotene of the recombinant engineering bacteria obtained by using the remaining 9 sequences was more than 50%, and the conversion rate of β-ionone was more than 7.40%.

[0147] The recombinant engineered bacterial suspension obtained from the remaining 9 sequences was used to spray 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 20% compared with before fermentation; the 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.

[0148] The sequence involved in the present invention is as follows:

[0149] Enzymes encoded by the BCD nucleotide sequence from Enterobacter hormaechei, including 459

[0150] amino acids, the amino acid sequences are shown in SEQ ID NOs. 1 to 10, specifically as follows:

[0151] SEQ ID NO.1:

[0152] MKWMNAVVCAALLAVAGPAFSEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQPAPTTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHAAAAGQYYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSALTLTGVNAINATEQPESEHISLAGEGERLSESEAVWRWKHPQPLSAIGFMLNDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPLPGGLVEAVKIKTLNARLPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMVLAGMAWRIWREVKKDGAA

[0153] SEQ ID NO.2:

[0154] MKWMNAVVCAALLAVAGPAFCEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQLAPTTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHATAAGQHYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSALTLTGVNAINATEQPESEHISLAGEGERLSESEAVWRWKHPQPLSAIGFMLNDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPLPGGLVEAVKIKTLNARLPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMVLAGMAWRIWREVKKDGAA

[0155] SEQ ID NO.3:

[0156] MKWMNAVVCAALLAVAGPAFCEGAPAESPRDYAYGLSLDTSASSPWYRVMPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQPAPTTTALRLFALNASPGGEEGSGDKIMLRARNGVEIVLEGQHAAAAGQHYLLTLPENASEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSA LTLTGVNAINATAQPESEHISLAGEGERLSEAVWRWKHPQPLSAIGFMLNGDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPPLPGGLVEAVKITLNARPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVEDTMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMVLAGMAWRIWREVKKDGAA

[0157] SEQ ID NO.4:

[0158] MKWMNAVVCTALLAVAGPAFCEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQPAPTTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHAAAAGQHYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSALTLTGVNAINATEQPESEHISLAGEGERLSESEAVWRWKHPQPLSAIGFMLNDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPLPGGLVEAVKIKTLNARLPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGGMVLAGMAWRIWREVKKDGAA

[0159] SEQ ID NO.5:

[0160] MKWMNAVVCAALLAVAGPAFCEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQPAPNTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHAAAAGQHYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSALTLTGVNAINATEQPESEHISLAGEGERLSESEAVWRWKHPQPLSAIGFMLNDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPLPGGLVEAVKIKTLNARLPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMLAGMAWRIWREVKKDGAA

[0161] SEQ ID NO.6:

[0162] MKWMNAVVCAALLAVAGPAFCEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQPAPTTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHAAAAGQHYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSALTLTGVNAINATEQPESEHISLAGEGERLSESEAVWRWKHPQPLSAIGFMLNGDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPLPGGLVETVKIKTLNARLPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMVLAGMAWRIWREVKKDGAA

[0163] SEQ ID NO.7:

[0164] MKWMNAVVCTALLAVAGPAFCEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQPAPTTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHAAAAGQHYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSALTLTGVNAINATEQPESEHISLAGEGERLSESEAVWRWKHPQPLSAIGFMLNDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPLPGGLVEAVKIKTLNARLPEN LPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMVLAGMAWRIWREVKKDGAA

[0165] SEQ ID NO.8:

[0166] MKWMNAVVCAALLAVAGPAFCEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTMRPQPAPTTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHAAAAGQHYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSALTLTGVNAINATEQPESEHISLAGEGERLSESEAVWRWKHPQPLSAIGFMLNDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPLPGGLVEAVKIKTLNARLPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMVLAGMAWRIWREVKKDGAA

[0167] SEQ ID NO.9:

[0168] MKWMNAVVCAALLAVAGPAFCEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQPAPTTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHAAAAGQHYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEVPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSA LTLTGVNAINATAQPESEHISLAGEGERLSEAVRWWKHPQPLSAIGFMLNGDGVLPAEIAWREADKDRWHTLKKEVIYQLGGKTSEPVPPLPGGLVEAVKITLNARPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPIELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMVLAGMAWRIWREVKKDGAA

[0169] SEQ ID NO.10:

[0170] MKWMNAVVCAALLAVAGPAFSEGAPAESPRDYAYGLSLDTSASSPWYRVMLPLAVYQQSTSPDLRDVRVFNQSGEPVPFSLVTTTRPQPAPTTTALRLFALNASPGGEEDSGDKIMLRARNGVEIVLEGQHAAAAGQHYLLTLPENATEVPLSQLQLFWDTPQGSWQGTASVYYSEDLKNWYTLREEMPLLDVMSGQDRLKLDRIDTDLVLSPEANRYLLLVLNTGRSALTLTGVNAINATAQPESEHISLAGEGERLSESEAVWRWKHPQPLSAIGFMLNGDGVLPAEIAWREADKDRWHMLKKEVIYQLGGKTSEPVPLPGGLVEAVKIKTLNARLPENLPGVTGQRVRYDLVFNAQGKGPYLLAWGNGAAKPASVETDMLIPTELRKTYDLAALPMADTLEPVPLGGEARLTATSAAEQESRMKTLMVWGVLIAGVMVLAGMAWRIWREVKKDGAA

[0171] Amino acid sequence of NtCCD1 (SEQ ID NO. 13):

[0172] MGRKEEDDTVERTEGGVVVVNPKPKKGVIGKAIDLLEAVIIKLMHDSTKPLHYLSGNFAPTDETPPLKDLSVTGHLPECLNGEFVRVGPNPKFAPVAGYHWFDGDGMIHGLRIKDGKATYVSRYVRTSRLKQEEFFGGAKFMKIGDLKGLFGLFTVYMQVLRAKLKVLDITYGNGTANTALVYHHGKLLALSEADKPYALKVMEDGDLQTLGMLDYDKRLAHSFTAHPKVDPVTGEMFTFGYSQNPPYITYRVISKGGIMQDPVPITIPEPIMMHDFAITENYAIMMDLPLCFRPKEMVKNNQLAFFFDATKNARFGVLPRYAQSEALIKWFELPNCFIFHNANAWEEGDEVVLITCRVQNPNLDMVNGVVKEKLENFSNELYEMRFNMKSGAASQKKLSESAVDFPRINENYTGRKQRYVYGTTLDSIAKVTGIIKFDLHAEPETGKAQLEVGGNVQGIFDLGPGRFGSEAVFVPRQPGTECEEDGGYLIFFVHDENTGKSAVNVIDAKTMSAEPVAVVELPKRVPYGFHAFFVTEEQIQEQAKL

[0173] Amino acid sequence of PhCCD1 (SEQ ID NO.14):

[0174] MGRKESDDGVERIEGGVVVVNPKPKKGITAKAIDLLEKVIIKLMHDSSKPLHYLSGNFAPTDETPPLNDLPIKGHLPECLNGEFVRVGPNPKFAPVAGYHWFDGDGMIHGLRIKDGKATYVSRYVRTSRLKQEEFFEGAKFMKIGDLKGLFGLFTVYMQMLRALLKILDTSYGNGTANTALVYHHGKLLALSEADKPYALKVLEDGDLQTLGMLDYDKRLLHSFTAHPKVDPVTGEMFTFGYAHEPPYITYRVISKDGIMQDPVPITIPEAIMMHDFAITENYAIMMDLPLCFRPKEMVKNNQLAFTFDTTKKARFGVLPRYAKSEALIRWFELPNCFIFHNANAWEEGDEVVLITCRLPHPDLDMVNGEVKENLENFSNELYEMRFNMKSGAASQKKLSESSVDFPRINENYTGRKQRYVYGTTLNSIAKVTGIIKFDLHAEPETGKKQLEVGGNVQGIFDLGPGRFGSEAVFVPSQPGTECEEDDGYLIFFVHDENTGKSAVNVIDAKTMSAEPVAVVELPKRVPYGFHAFFVTEEQIQEQAKL

[0175] Amino acid sequence of NtCCD4 (SEQ ID NO.15):

[0176] MDAFSSSTFLSTLSQHPKSLLSPNYSPNTSSSPILKVSSVRIEERPQTTTTTTRTKPQEKPTPSPYTPPKDTPKRQLPTKSITTKKPVEPSFPSVIFNAFDDFVNTFI DPPLKPCVDPKYILSNNFAPVDELPPTECEVVVGSLPPCLDGAYIRNGPNPQYLPRGPYHLFDGDGMLHSIKISQSKATLCSRYVKTYKYTIEREAGSPVFPNVFSGF NGLTASAARGAITAARAIAAGQFNPTNGIGLANTSLALFGGKLFALGESDLPYAVKLAPDGDIITLGRYDFDGKLFMSMTAHPKIDPDTNEAFAFRYGPMPPFLTYFR IEPNGTKTPDVPIFSMTRPSFLHDFAITNKFAIFSDIQIGMNPLEFITGGSPVSSDSGKIPRLGVIPRYAKNESEMKWFDVPGINIVHAINAWDEDDGDTIVMVAPNI

[0177] LSVEHTLERDMMIHASVEKVKIDLKSGMVSRQP

[0178] LSTRNLDFGVINPAYVGKKNKYVYAAIGDPMPKIAGIAKLDVSVAEADRRDCIVACRLFGEGCFGGEPYF

[0179] VANNSAADEDDGYVVSFVHNEKTGESRFLVMDAKSPNLDIVAAVKLPRRVPYGFHGLFVRETDLRKLM

[0180] 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. A method for improving tobacco leaf quality, characterized in that: The method uses an enzyme derived from Enterobacter hormaechei, a recombinant plasmid containing the enzyme, or a recombinant engineered bacterium expressing the enzyme to increase the β-ionone content in tobacco leaves, 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 459th 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 method according to claim 1, characterized in that: The method comprises the steps of using recombinant engineering bacteria expressing the enzyme to spray and ferment tobacco shreds.

3. The method 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 synthesizing 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 459th 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 use according to claim 4, characterized in that: The application is to utilize the enzyme, the recombinant plasmid or the recombinant engineering bacteria to catalyze the production of β-ionone from β-carotene.

6. 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 459th 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. A recombinant plasmid expressing an enzyme capable of catalyzing 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 459th 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.

8. The recombinant plasmid according to claim 7, 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, connecting the target gene sequence with the expression regulatory sequence, and obtaining the expression recombinant plasmid.

9. A recombinant engineered bacterium expressing an enzyme capable of catalyzing the production of β-ionone from β-carotene, 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 459th 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.

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

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