A carotenoid cleavage enzyme or carotenoid cleavage enzyme mutant and use thereof

Abstract

The application provides a carotenoid cleavage enzyme or carotenoid cleavage enzyme mutant and application, and belongs to the technical field of bioengineering.The purpose of the application is to efficiently prepare carotenoid cleavage aroma compounds and develop a preparation method of a fresh and sweet cigarette flavor based on a biological method.The application provides a carotenoid cleavage enzyme or carotenoid cleavage enzyme mutant, an amino acid sequence of the carotenoid cleavage enzyme is shown in SEQ ID NO.1, and the mutant is obtained by mutating A at the 187th position into V and D at the 426th position into G in SEQ ID NO:1. A187V / D426G The application of the fresh and sweet flavor prepared by the biological method to improving the fresh and sweet flavor and the sense of moistening and sweetness of a cigarette product and improving the yield of beta-ionone.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a carotenoid lyase or a carotenoid lyase mutant and its applications. Background Technology

[0002] Carotenoids are important pigments and aroma precursors in tobacco, tea, flowers, fruits, and various microbial cell factories. Oxidative cleavage of carotenoids yields a variety of carotenoid cleavage aroma compounds that contribute significantly to aroma, such as β-ionone, α-ionone, β-cyclocitral, pseudoionone, geranylacetone, and precursors related to dihydroactinolone. These compounds have significant applications in tobacco aroma quality, food flavorings and fragrances, and biomanufacturing.

[0003] In plants, carotenoids are degraded mainly through two pathways: (1) through carotenoid cleavage dioxygenase (CCD), which mainly catalyzes the cleavage of β-carotene to produce precursors of plant hormones strigolactone and apocarotenoids, as well as a variety of small molecule compounds; (2) through 9-cis-epoxycarotenoid dioxygenase (NCED), which mainly catalyzes the degradation of neoxanthin to produce precursors of plant hormone abscisic acid. Among these, CCD is a type of metalloenzyme that can catalyze the oxidative cleavage of carotenoids at specific sites, and can cleave specific sites of carotenoids such as β-carotene to produce aroma substances such as β-ionone, α-ionone, β-cyclocitral, and dihydroactinolone. Based on homology comparison and sequence conservation analysis, researchers have predicted more than 30 CCD-type and NCED-type genes that may be involved in the degradation of tobacco carotenoids from the tobacco genome. However, it remains unclear whether these CCD and NCED genes possess catalytic activity, what their specific catalytic substrates are, and whether they exhibit substrate selectivity and specificity. Currently, only a few CCD genes in tobacco have been reported to potentially participate in the degradation of tobacco carotenoids, and a large number of functional genes involved in the degradation of tobacco carotenoids still await further discovery and identification. Summary of the Invention

[0004] The purpose of this invention is to efficiently prepare carotenoid pyrolysis aroma compounds, increase the yield of carotenoid pyrolysis aroma compounds, and develop a biological method for preparing a fresh, sweet, and aromatic tobacco flavoring.

[0005] This invention provides a carotenoid lyase or a carotenoid lyase mutant, wherein the amino acid sequence of the carotenoid lyase is shown in SEQ ID NO:1; the mutant is based on SEQ ID NO:1, with the A at position 187 mutated to V and the D at position 426 mutated to G.

[0006] The present invention provides a gene encoding the above-mentioned carotenoid lyase or carotenoid lyase mutant.

[0007] The present invention provides a recombinant vector containing the above-mentioned carotenoid lyase or carotenoid lyase mutant gene.

[0008] The present invention provides a recombinant microbial cell expressing the above-mentioned carotenoid lyase or carotenoid lyase mutant.

[0009] The present invention provides the application of the above-mentioned carotenoid lyase or carotenoid lyase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cells in lysing β-carotene or producing carotenoid lysing aroma compounds.

[0010] Further specifying, the carotenoid cleavage aroma compounds are β-ionone and β-cyclocitral.

[0011] This invention provides the application of the above-mentioned carotenoid lyase or carotenoid lyase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cell in the preparation of cigarettes, the improvement of cigarette quality, and the preparation of fragrant, sweet flavorings and fragrances.

[0012] This invention provides a method for preparing carotenoid lysin-based aroma compounds, wherein the above-mentioned carotenoid lyase or carotenoid lyase mutant is added to a reaction system containing β-carotene emulsion and reacted at 40°C in the dark for at least 2 hours.

[0013] Further specifying, the reaction system also includes 0.2 mM FeSO4 and 2 mM ascorbic acid.

[0014] This invention provides a method for preparing carotenoid lysate aroma compounds, wherein the above-mentioned gene is expressed in β-carotene-accumulating Yersinia lipolytica and fermented for 72 hours.

[0015] Beneficial effects: (1) This invention provides a novel tobacco-derived enzyme NtCCD11 and its mutant NtCCD11 that are highly efficient at degrading carotenoids to generate aroma compounds. A187V / D426G It also provides NtCCD11 and NtCCD11 A187V / D426GIts application as an enzyme for the preparation of aroma compounds by cleaving carotenoids provides a new enzymatic tool for the biosynthesis of aroma compounds such as β-ionone.

[0016] (2) This invention establishes a method for preparing a fresh and sweet flavoring based on in vitro enzyme catalysis and β-carotene-accumulating Yersinia lipolytica. On one hand, NtCCD11 or NtCCD11 A187V / D426G It can catalyze the formation of β-ionone and / or β-cyclocitral from carotenoid substrates such as β-carotene in a buffer system under mild reaction conditions. The products can be detected and quantified by GC-MS, GC-FID, HPLC, or LC-MS. On the other hand, NtCCD11 A187V / D426G It can be introduced into β-carotene-accumulating Yersinia lipolytica as a carotenoid cleavage functional module, utilizing the β-carotene accumulated endogenously in the host cell to produce aroma substances such as β-ionone, providing a new technical solution for constructing a high-yield β-ionone microbial cell factory.

[0017] (3) The present invention provides a method using NtCCD11 or NtCCD11 A187V / D426G The application of a fresh and sweet flavoring prepared by biological methods in enhancing the fresh and sweet aroma and smoothness of cigarette products. Adding this fresh and sweet flavoring to cigarette products can significantly improve the aroma quality, making the smoke delicate and mellow, with a marked increase in the fresh and sweet aroma and the amount of aroma. Attached Figure Description

[0018] Figure 1 β-Ionone Standard and Expression of NtCCD11 A187V / D426G GC-MS results of fermentation products from *Yarrowia lipolytica* engineered strains that accumulate β-carotene; A is β-ionone standard; B is NtCCD11. A187V / D426G Engineered bacteria. Detailed Implementation

[0019] The following examples are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Unless otherwise stated, the reagents and consumables used in the examples are commercially available, and molecular cloning, bacterial culture, protein purification, and GC-MS detection can be performed using conventional methods in the art.

[0020] Example 1: NtCCD11 and its mutant NtCCD11 A187V / D426G Amino acid sequence and E. coli preferred codon sequence This embodiment provides tobacco-derived carotenoid lyase NtCCD11 and its mutant NtCCD11. A187V / D426G The amino acid sequence of NtCCD11 is shown in SEQ ID NO:1. A187V / D426GThe amino acid sequences are shown in SEQ ID NO:2, both of which are 581 aa in length. Based on the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2, the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 were designed according to the codon preferences of E. coli. Both sequences are 1746 bp in length, have a GC content of approximately 47.5%, and contain a stop codon. These sequences can be directly synthesized using commercial gene technology or used as templates for PCR amplification.

[0021] Example 2: NtCCD11 and its mutant NtCCD11 A187V / D426G Construction of Nucleic Acid Encoding and Expression Vectors Using the optimized codon sequences of *E. coli* shown in SEQ ID NO:3 and SEQ ID NO:4 as templates, amplification primers containing NdeI and XhoI restriction endonuclease sites were designed. The forward primer had a protective base and an NdeI site added to its 5' end. The ATG at the NdeI site served as NtCCD11 and its mutant NtCCD11. A187V / D426G The start codon; the reverse primer has a protective base, an XhoI site and a TAA stop codon reverse complementary sequence added to the 5' end, so that the expression product retains the N-terminal 6×His tag of the pET-28a(+) vector and does not fuse with the C-terminal His tag of the vector.

[0022] Table 1. Construction of pET-28a(+)-NtCCD11 and its mutant NtCCD11 A187V / D426G primers

[0023] The PCR reaction system can use a high-fidelity DNA polymerase system. Taking a 50 μL reaction system as an example, it includes: 10 μL of 5× high-fidelity buffer, 4 μL of dNTP mixture, 1 μL of NdeI-F primers, 1 μL of XhoI-R primers, 100 ng of template (SEQ ID NO:3 or SEQ ID NO:4), 1 μL of high-fidelity DNA polymerase, and nuclease-free water to a final volume of 50 μL. The PCR program can be: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 56-60℃ annealing for 15 s, 72℃ extension for 90-120 s, 30 cycles; 72℃ final extension for 5 min.

[0024] PCR products were recovered after detection by agarose gel electrophoresis. The pET-28a(+) vector and PCR products were double-digested with NdeI and XhoI, respectively. After purification, the digested products were ligated using T4 DNA ligase at 16℃ for 4–16 h, or using an equivalent rapid ligation system. The ligation products were transformed into *E. coli* DH5α, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated upside down at 37℃ for 12–16 h. Single colonies were picked for colony PCR, restriction enzyme digestion identification, and Sanger sequencing to obtain the correctly sequenced pET-28a(+)-NtCCD11 and pET-28a(+)-NtCCD11. A187V / D426G Recombinant plasmid.

[0025] Example 3: NtCCD11 and its mutant NtCCD11 A187V / D426G Expression and purification in Escherichia coli BL21(DE3) The correctly sequenced pET-28a(+)-NtCCD11 and pET-28a(+)-NtCCD11 were respectively... A187V / D426G The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing 50 μg / mL kanamycin, and cultured overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium containing kanamycin, and cultured at 37°C with shaking at 220 rpm for 16 h to obtain the seed culture. The seed culture was then transferred at a 1% inoculum to 500 mL of LB medium containing kanamycin and cultured at 37°C with shaking at 220 rpm until OD500 reached. 600 The concentration should be 0.6-0.8. Add IPTG to a final concentration of 0.2 mM and incubate at 22℃ for 16-18 h. Alternatively, incubate at 16℃ for 12-20 h to improve soluble expression. After induction, collect the cells by centrifugation at 10000g for 20 min at 4℃.

[0026] Resuspend the bacterial cells at a ratio of 5 mL of binding buffer per 1 g of wet bacterial cells. The binding buffer can be 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.5. The bacterial suspension is sonicated under ice bath conditions or homogenized by high-pressure autoclaving. The lysate is centrifuged at 10000 g for 30 min at 4 °C, and the supernatant is collected and filtered through a 0.45 μm low-protein adsorption filter. The filtered supernatant is loaded onto a Ni-NTA resin column pre-equilibrated with binding buffer. After loading, the column is washed with 10 column volumes of binding buffer, followed by washing with a wash buffer containing 50 mM imidazole to remove non-specifically binding proteins. Finally, NtCCD11 is eluted with an elution buffer containing 250 mM imidazole. A187V / D426GCollect the eluted fraction and analyze it by SDS-PAGE. A target protein band should appear at approximately 66-67 kDa. The eluted protein can be concentrated using ultrafiltration tubes with a molecular weight cutoff of 10-30 kDa, and the buffer should be changed with a buffer solution of 50 mM Tris-HCl, 20 mM NaCl, pH 8.0. Protein concentration can be determined using the Bradford method or the BCA method. Purified protein should be stored for a short period at 4°C, or stored at -80°C after adding 10%-20% glycerol.

[0027] Example 4: NtCCD11 and its mutant NtCCD11 A187V / D426G In vitro catalytic β-carotene cleavage The β-carotene emulsion substrate was prepared as follows: 5 mg of β-carotene standard was weighed under light-protected conditions and dissolved completely in 10 mL of dichloromethane; 1 g of Tween 80 was added for emulsification; the solution was transferred to a brown round-bottom flask, and the dichloromethane was removed by rotary evaporation under reduced pressure at room temperature; the residual solution was transferred to a 100 mL brown volumetric flask, diluted to volume with sterile water, and thoroughly mixed to obtain a 50 mg / L β-carotene emulsion, which was stored at 4°C protected from light for later use.

[0028] The in vitro catalytic reaction can be carried out in a 20 mL headspace vial or a glass reaction flask. A 5 mL reaction system consists of: 500 μL of β-carotene emulsion, NtCCD11 or NtCCD11 A187V / D426G The final protein concentration was 0.5 mg / mL, and the volume was made up with 50 mM Tris-HCl buffer (pH 8.0). 0.2 mM FeSO4 and 2 mM ascorbic acid could be added to the reaction system to maintain the metal center and reducing environment. After mixing, the reaction system was incubated at 40°C in the dark for 2 h. Control groups included an enzyme-free control, a heat-inactivated enzyme control, an empty vector BL21(DE3) purification solution control, and a substrate-free control.

[0029] After the reaction, volatile pyrolysis products were analyzed by GC-MS. The reaction solution was placed in a chromatographic vial and equilibrated at 50°C for 15 min, followed by solid-phase microextraction (SPE) fiber extraction for 30 min, and desorption at 230°C for 5 min at the GC-MS inlet. Mass spectrometry was performed using EI ionization with an ionization energy of 70 eV and a scan range of m / z 30-550. Products such as β-ionone and β-cyclocitral were identified by matching the retention times of standards with the mass spectra, and quantified using the external standard method. β-carotene consumption could also be detected by absorbance at 450 nm or by HPLC.

[0030] Table 2 lists NtCCD11 and its mutant NtCCD11. A187V / D426GIn vitro catalysis results are presented as mean ± standard deviation of three independent replicates. After the reaction, the reaction products were detected by GC-MS. Compared with the enzyme-free control, heat-inactivated enzyme control, and empty vector purification solution control, His-NtCCD11 and His-NtCCD11... A187V / D426G The reaction group detected β-ionone and a small amount of β-cyclocitral, and His-NtCCD11 A187V / D426G The contents of β-ionone and β-cyclocitral in the reaction group were significantly higher than those in the His-NtCCD11 reaction group.

[0031] Table 2 NtCCD11 and its mutant NtCCD11 A187V / D426G Results of in vitro catalysis of β-carotene

[0032] Example 5: NtCCD11 A187V / D426G Application in β-carotene-accumulating Yersinia lipolytica Based on the in vitro catalysis results of Example 4, NtCCD11 with higher catalytic activity was selected. A187V / D426G Expression in *Yersinia lipolytica*. Using *Yersinia lipolytica* engineered strains capable of accumulating β-carotene as a chassis, NtCCD11 was expressed. A187V / D426G The expression cassette is introduced into the host, causing the β-carotene accumulated in the host cells to be converted by NtCCD11. A187V / D426GLysis produces β-ionone. The β-carotene-producing engineered strain was constructed using *Yarrowia lipolytica* Po1f Δku70 (obtained from Kretzschmar A, et al., Current Genetics, 2013, 59(1-2):63-72) as the host. Using CRISPR-Cas9 gene editing technology, the key genes CarB (GenBank: CAB40843.1) and CarRP (GenBank: CAB60272.1) of the β-carotene synthesis pathway were integrated into the D17 site of the Po1f Δku70 genome. First, a dual plasmid system was constructed: one was D17-pCRISPRyl, used to express the Cas9 nuclease and a specific sgRNA targeting the D17 site (sequence: TCCGTAATATAGGTGACGAC); the other was the donor plasmid pHR-D17-CarB-CarRP, providing arms homologous to the sequences flanking the D17 site and the target genes CarB and CarRP. Two plasmids were co-transformed into host cells using the conventional lithium acetate transformation method. Positive transformants were screened using SD-Leu-Ura deficient medium. After successful integration was verified by colony PCR, plasmids were eliminated using YPD medium containing 5-FOA. Plasmid-loss strains that grew on YPD plates but not on SD-Leu / Ura plates were screened, and finally, label-free, genotype-stable β-carotene engineered bacteria were obtained.

[0033] Yarrowia lipolyticaPo1fΔku70(MatA,Δku70::hisG,leu2 270,ura3 302, xpr2 322, axp1 2), abbreviated as Yarrowia lipolytica Po1fΔku70. Yarrowia lipolytica Po1fΔku70 was constructed by knocking out the ku70 gene responsible for non-homologous recombination from Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1)). 2):63-72).

[0034] NtCCD11 A187V / D426G The expression cassette contains the Yersinia lipophila promoter and NtCCD11. A187V / D426GThe coding sequence and the terminator of *Yarrowia lipophila*. The promoter can be pGPD, pTEF, pEXP1, or hp4d, and the terminator can be tCYC1, tXPR2, or tLip2. The promoter is preferably pTEF (SEQ ID NO:8), and the terminator is preferably tCYC1 (SEQ ID NO:9). According to NtCCD11 A187V / D426G The amino acid sequence was designed according to the preferred codons of *Yarrowia lipolytica*, resulting in the nucleotide sequence shown in SEQ ID NO:7. The expression cassette was integrated into the *Yarrowia lipolytica* genome AXP site via CRISPR-Cas9 mediation.

[0035] Single clones of *Yarrowia lipolyticis* positive engineered strains were obtained, activated in YPD, and inoculated into fermentation medium. The cultures were incubated at 28-30℃ and 200 rpm for 72 hours. During cultivation, a 5%-10% (v / v) dodecane capping layer was added to capture volatile β-ionones. After fermentation, the dodecane phase was collected, or aroma products were extracted from the cells and culture medium for qualitative and quantitative analysis using GC-MS. The control group included strains without NtCCD11 introduction. A187V / D426G The expression cassette included β-carotene-accumulating Yersinia lipolytica and Yersinia lipolytica without the β-carotene accumulation module. The NtCCD11 expression was validated by comparing the β-ionone content in the dodecane phase or extract of each group. A187V / D426G Contribution to β-carotene cleavage and β-ionone formation. Figure 1 The GC-MS chromatograms are of β-ionone standards and fermentation products of engineered Yersinia lipolytica β-ionone strain.

[0036] NtCCD11 A187V / D426G As a carotenoid cleavage module, it can be used in conjunction with the mevalonate pathway, carotenoid synthesis pathway, and β-carotene accumulation module in host cells to construct a microbial cell factory for producing β-ionone. In this embodiment, compared with in vitro pure enzyme catalysis, the Yersinia lipolytica system can directly utilize β-carotene synthesized in host cells as a substrate, making it suitable for constructing a β-ionone cell factory.

[0037] Example 6: NtCCD11 A187V / D426G Sensory evaluation of catalytic products added to cigarettes The reaction products of Example 4 and the fermentation products of Example 5 were extracted with ethyl acetate and concentrated by nitrogen blowing. The concentrated product solution was prepared into a 20 mL / L spray solution using anhydrous ethanol as the solvent. B2F grade standard tobacco samples were selected and sprayed evenly onto the tobacco at a weight ratio of 1:100, serving as the flavoring treatment group. The in vitro catalytic product treatment of Example 4 was designated as flavoring treatment group 1, and the in vivo fermentation product treatment of Example 5 was designated as flavoring treatment group 2. Anhydrous ethanol was sprayed evenly onto the tobacco at the same ratio as the control group. The treated tobacco samples were equilibrated in a constant temperature and humidity chamber (25℃, 60% relative humidity) for 48 hours before being rolled into cigarettes. A sensory evaluation panel of seven tobacco industry professionals was organized to evaluate the sensory quality of the samples. The results are shown in Tables 3 and 4. Compared with the control group cigarettes, the flavoring treatment group 1 and flavoring treatment group 2 showed a significant increase in aroma, smoothness, and sweetness, with noticeable increases in sweetness, smoothness, and aroma.

[0038] Table 3. Qualitative description of sample evaluation results

[0039] Table 4 Evaluation of the Flavoring Effect and Sensory Quality of Cigarettes

[0040] The above description is merely an embodiment of the present invention. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

[0041] SEQ ID NO:1(NtCCD11) ; SEQ ID NO:2(NtCCD11 A187V / D426G ) MHTILPNPSLPKPHNSPSFSCKILSNPSKKNTITLPRRQTPPFVPPLPPPLAEPKIFPLKFEPRKLNPLQKLVASTLDMLEKSVVTKLEKKHKLNRTVDPEIQLEGNFAPVQECPVQHGLEIVGHIPSSLTGVYVRNGANPLFEPINGHHLFDGDGMIHAVKLDSVNNKASYSCRLTRTSRLVQEAVLGRPVFPKPIGELHGHLGLARLALFFARTSFGLVDATKGTGVANAGLIYFNGRLLAMSEDDLPYSVRITDNGDLETNGRYNFDGQIDDPLIAHPKVDPGELYTLSYNVLKKPFLKFFKFDMCGNKSRDISISLQHPTMIHDFAITENHVIIPDYQVVFKLSEMLMGGSPVVHDPKKVSRFGVLSKDDHDESRIRWIEVPNCFCMHLWNAWEELSENDDETLVIIGSCMSPPDSIFSGNGESLKSELSEIRLNLKTGKSTRQVIVSGMNLEAGQVNKTKLGRKTRYAFMAIADPWPKCSGLAKVDLVTGNVTKVLYGDKKFGGEPYFVPSTKEGKEDEGYLMSYVRDEIKEKSELVIVNASNMTQVASVKLPKRVPYGFHGTFVSSQDLCNQSSC; SEQ ID NO:3 (E. coli-preferred codon-optimized sequence of NtCCD11) SEQ ID NO:4(NtCCD11 A187V / D426G (E. coli preferred codon optimized sequence) SEQ ID NO:7: SEQ ID NO:8 pTEF sequence: AGAGACCGGGTTGGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTTCTGAGTATAAGAATCATTCAAAATGGTGAGTTTCAGAGGCAGCAGCAATTGCCACGGGCTTTGAGCACACGGCCGGGTGTGGTCCCATTCCCATCGACACAAGACGCCACGTCATCCGACCAGCACTTTTTGCAGTACTAACCGCAG; SEQ ID NO:9 tCYC1 sequence: TCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCTCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTTAATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGC。

Claims

1. A carotenoid lyase or a carotenoid lyase mutant, characterized in that, The amino acid sequence of the carotenoid lyase is shown in SEQ ID NO.1; the mutant is based on SEQ ID NO:1, with alanine at position 187 mutated to valine and aspartic acid at position 426 mutated to glycine.

2. The gene encoding the carotenoid lyase or a mutant of the carotenoid lyase as described in claim 1.

3. A recombinant vector containing the gene described in claim 2.

4. Recombinant microbial cells expressing the carotenoid lyase or a mutant of the carotenoid lyase as described in claim 1.

5. The use of the carotenoid lyase or carotenoid lyase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant microbial cell of claim 4 in lysing β-carotene or producing carotenoid-derived aroma compounds.

6. The application according to claim 5, characterized in that, The aroma compounds derived from the cleavage of carotenoids are β-ionone and β-cyclocitral.

7. The application of the carotenoid lyase or carotenoid lyase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant microbial cell of claim 4 in the preparation of cigarettes, the improvement of cigarette quality, and the preparation of fragrant and sweet flavorings and fragrances.

8. A method for preparing carotenoid cleavage aroma compounds, characterized in that, The carotenoid lyase or carotenoid lyase mutant described in claim 1 was added to a reaction system containing β-carotene emulsion and reacted at 40°C in the dark for at least 2 hours.

9. The method according to claim 8, characterized in that, The reaction system also includes 0.2 mM FeSO4 and 2 mM ascorbic acid.

10. A method for preparing carotenoid pyrolysis aroma compounds, characterized in that, The gene described in claim 2 was expressed in β-carotene-accumulating Yersinia lipolytica and obtained by fermentation for 72 hours.

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