Carotenoid cleavage dioxygenase mutant and application thereof

Abstract

The invention relates to the technical field of carotenoid cleavage dioxygenase, and discloses a carotenoid cleavage dioxygenase mutant and application thereof. In order to improve the efficiency of converting carotenoid in tobacco into aroma molecule beta-ionone, specific mutation of characteristic sites is carried out on carotenoid cleavage dioxygenase (PhCCD1) from petunia, that is, serine (S) at the 428 site of the carotenoid cleavage dioxygenase is mutated into cysteine (C) or tyrosine (Y) to obtain a mutant S428C or S428Y, and the mutant S428C or S428Y is converted into beta-ionone. The yield of the beta-ionone converted from the carotenoid is greatly improved. Experiments prove that the yields of beta-ionone of S428C and S428Y respectively reach 54.2 mg / L and 50.6 mg / L, which are respectively increased by 69.7% and 58.5% compared with those of a wild strain WT.

Description

Technical Field

[0001] This invention relates to the field of carotenoid cleavage dioxygenase technology, and more particularly to a carotenoid cleavage dioxygenase mutant. Background Technology

[0002] Carotenoid degradation products are widely used in the food and flavoring industries due to their unique aroma and functional value. CCDs are non-heme iron-dependent enzymes that use carotenoids as substrates, and based on phylogenetic analysis of their amino acid sequences, they can be divided into six CCD family members. These include CCD1, CCD2, 9-cis-epoxy-carotenoid cleavage dioxygenase (NCED), CCD4, CCD7, and CCD8.

[0003] Existing research has shown that the substrates that the CCD family can interact with mainly include β-carotene, lutein, and lycopene, while the degradation products mainly include β-ionone, pseudoionone, 3-hydroxy-β-ionone, 6-methyl-5-hepten-2-one, geranylacetone, and citral. The specificity and activity of CCDs from different sources vary. These CCD oxidative degradation compounds are important components of aromas in the food, beverage, cosmetic, and tobacco industries. For example, β-ionone has a pleasant floral aroma and an extremely low olfactory threshold, as low as 0.007 μg / L in water.

[0004] Developing and screening CCDs with high specificity and catalytic activity is crucial for the biosynthesis of carotenoid-derived aroma components, including β-ionone. However, the low catalytic activity of natural CCDs limits their application in the oxidative cleavage of carotenoid substrates for aroma production.

[0005] In the prior art, Chinese patent application CN119101666A discloses a mutant of carotenoid cleavage dioxygenase, which obtains a mutant with enhanced enzyme activity by mutating positions 181 or 337 of the carotenoid cleavage dioxygenase. However, the catalytic activity of this enzyme is still not high. Summary of the Invention

[0006] To address the aforementioned technical problem of low catalytic activity of carotenoid cleavage dioxygenase, and more specifically, to improve the efficiency of converting carotenoids in tobacco into aroma molecules such as β-ionone, this invention provides a carotenoid cleavage dioxygenase mutant and its applications.

[0007] The specific technical solution of this invention is as follows: In a first aspect, a carotenoid cleavage dioxygenase mutant is characterized in that the amino acid sequence of the carotenoid cleavage dioxygenase mutant is as shown in SEQ ID NO.4 or as shown in SEQ ID NO.6.

[0008] To improve the efficiency of converting carotenoids in tobacco into aroma molecules such as β-ionone, this invention involves a specific mutation at a key site in the petunia hybrida carotenoid cleavagedioxygenase (PhCCD1), resulting in a significant increase in the yield of β-ionone converted from carotenoids. This specific mutation refers to changing the serine (S) at position 428 of the wild-type PhCCD1 to either cysteine ​​(C) or tyrosine (Y) to obtain mutants S428C or S428Y.

[0009] Specifically, experimental results showed that the β-ionone yields of S428C and S428Y reached 54.2 mg / L and 50.6 mg / L, respectively, which were 69.7% and 58.5% higher than those of the wild-type strain WT.

[0010] The amino acid sequence of wild-type PhCCD1 is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.1. The amino acid sequences of mutants S428C and S428Y are shown in SEQ ID NO.4 and SEQ ID NO.6, respectively.

[0011] Encoding genes are genes in DNA that encode protein sequences. They guide protein synthesis and are fundamental to protein synthesis in living organisms. Clearly, the gene encoding the aforementioned carotenoid cleavage dioxygenase mutant should also be included within the scope of protection of this invention. For those skilled in the art, given the disclosure of the amino acids in the aforementioned carotenoid cleavage dioxygenase mutant, the gene encoding this mutant can be obtained without any intellectual effort, using relevant knowledge or tools in the field.

[0012] Plasmids can be used as vectors to introduce exogenous DNA fragments into target cells for protein expression. Plasmids carrying genes encoding the aforementioned carotenoid cleavage dioxygenase mutant can serve as vectors for this mutant gene, introduced into target cells for expression. Clearly, plasmids carrying genes encoding the aforementioned carotenoid cleavage dioxygenase mutant should also be included within the scope of protection of this invention. Furthermore, the target cell, such as a genetically engineered bacterium, containing the aforementioned carotenoid cleavage dioxygenase mutant or its encoding gene, or containing plasmids encoding the aforementioned carotenoid cleavage dioxygenase mutant gene, should also be included within the scope of protection of this invention.

[0013] Preferably, the host bacterium of the genetically engineered bacteria is Escherichia coli.

[0014] On the other hand, the present invention provides an application of the above-mentioned carotenoid cleavage dioxygenase mutant in catalyzing carotenoid conversion.

[0015] Preferably, the method of application includes the following steps: In an enzyme-catalyzed system, using carotenoids as substrates, at least one of β-ionone, pseudoionone, 3-hydroxy-β-ionone, 6-methyl-5-hepten-2-one, geranylacetone, and citral is catalyzed to be generated. The enzyme catalytic system includes the carotenoid cleavage dioxygenase mutant as described in claim 1.

[0016] Preferably, the enzyme catalytic system uses a micelle system as the reaction system, wherein the reaction system is as follows: the carotenoid substrate is encapsulated with the surfactant Triton X-100 as an encapsulation medium and dispersed in the liquid phase.

[0017] Based on the above, the present invention also provides an application of the above-mentioned carotenoid cleavage dioxygenase mutant in catalyzing aroma production from tobacco substrates.

[0018] Compared with the prior art, the present invention has the following technical effects: To improve the efficiency of converting carotenoids in tobacco into the aroma molecule β-ionone, this invention involves a specific mutation at a key site in the carotenoid cleavage dioxygenase (PhCCD1) from petunia. Specifically, the serine (S) at position 428 is mutated to either cysteine ​​(C) or tyrosine (Y) to obtain mutants S428C or S428Y, which significantly increases the yield of β-ionone converted from carotenoids. Experimental results show that the β-ionone yields of S428C and S428Y reached 54.2 mg / L and 50.6 mg / L, respectively, representing increases of 69.7% and 58.5% compared to the wild-type strain WT, demonstrating a significant improvement in β-ionone production. Attached Figure Description

[0019] Figure 1 The fermentation product of engineered bacteria in mutant library 428-430 is β-ionone. Figure 2 To improve the secondary screening of mutant strains in the initial screening of the S428 mutant library; Figure 3 SDS-PAGE results of crude and purified enzymes prepared for mutants S428C and S428Y; Figure 4 The in vitro catalytic yield of carotenoid substrates by pure enzymes prepared for mutants S428C and S428Y; Figure 5 The reaction kinetics curves of the pure enzymes of mutants S428C and S428Y are shown. Figure 6 Results of β-carotene catalysis by purified enzymes of mutants S428C and S428Y; Figure 7 Results of pure enzyme catalysis of lutein by mutants S428C and S428Y; Figure 8 Aroma production results of crude enzyme-catalyzed tobacco extracts prepared for mutants S428C and S428Y. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0021] Example 1: Construction and fermentation of PhCCD1 mutant strain (1) Carotenoid cleavage dioxygenase (PhCCD1) from petunia and its 378-380, 404-406, and 428-430 sites (a total of 9 sites) were selected as mutation targets to improve the enzyme activity of PhCCD1. The nucleotide sequence of PhCCD1 is shown in SEQ ID NO.1, and the amino acid sequence of the carotenoid cleavage dioxygenase it encodes is shown in SEQ ID NO.2. The PhCCD1 gene was synthesized from the whole genome.

[0022] (2) Primer pairs were designed to perform saturation mutations at nine sites: 378-380, 404-406, and 428-430. NDT degenerate codons were used to replace the target amino acids, achieving coverage of up to 12 different types of amino acids at each site. Primer sequences are detailed in Table 1. The target DNA fragments were purified by PCR amplification and agarose gel electrophoresis followed by a single-round gel recovery.

[0023] Table 1

[0024] The relevant methods for this step (2) are as follows: PCR amplification method: The system consisted of 23.4 μL water, 0.3 μL upstream primer, 0.3 μL downstream primer, 25 μL KODdye mix, and 1 μL pβIon6.

[0025] Agarose gel electrophoresis and gel recovery method: Dissolve the agarose gel containing the target DNA in 500 μL Buffer PE (wash buffer) (55 ℃, 7 min), transfer it to a nucleic acid adsorption column (place the adsorption column in a collection tube), and centrifuge using a microcentrifuge (13000 rpm, 10 s); add 600 μL Buffer PW (wash buffer) to wash (13000 rpm, 10 s, repeated twice), spin dry (13000 rpm, 1 min), and then place the adsorption column in a clean centrifuge tube; add 40 μL of sterile water to elute the DNA sample on the silica gel column, let stand for 2 min, and centrifuge (13000 rpm, 2 min) to obtain the plasmid fragment.

[0026] (3) A plasmid fragment assembly system was constructed, and the assembly reaction was carried out by PCR. After 12 hours of overnight culture, nine mutant libraries were obtained, and six colonies were randomly selected for sequencing verification to ensure the mutation quality of the mutant libraries.

[0027] The relevant methods for this step (3) are as follows: Plasmid fragment assembly: The molar ratio of each plasmid fragment was 1:1. Take 2 μL of plasmid fragment and 2 μL of enzyme (2 × ClonExpress Mix, Novizan, C115-02) to construct a 4 μL system, assemble at 50 ℃ (PCR instrument) for 5 min, and then incubate overnight.

[0028] (4) After the mutation rate of the mutant library reached the target, the colonies on the plate were washed with LB liquid medium, and the plasmids were extracted using a small plasmid extraction kit (Tiangen, DP106-02). Then, the mutant plasmids at nine different sites were electroporated into β-carotene-producing engineered bacteria to construct a mutant library. The mutant library construction method is as follows: The β-carotene-producing engineered bacteria cultured overnight were transferred to 10 mL of LB liquid medium and cultured at 37 ℃ for 3 h. Then, the supernatant was removed by centrifugation. 1 mL of sterile water was taken and the bacterial cells were transferred to a 1.5 mL centrifuge tube and placed on ice for 10 min. Then, the supernatant was removed by centrifugation at 10000 rpm for 1 min, and 1 mL of sterile water was added to resuspend the cells. The cells were then centrifuged again at 10000 rpm for 1 min. After removing the supernatant, 100 μL of sterile water and 1 μL of mutant plasmid were added and mixed well. The mixture was placed on ice for 10 min. E. coli was electroporated using a BIO-RAD electroporator. Quickly add 600 μL of LB liquid medium, mix well, transfer to a test tube, and place on a shaker. Incubate at 30 ℃ and 250 rpm / min for 1 h to restore the bacterial culture. Take an appropriate amount of bacterial culture and spread it onto LB solid medium labeled with the corresponding antibiotic resistance.

[0029] (5) During the fermentation test, 30 colonies were randomly selected from nine mutant libraries for fermentation and detection and sequencing of β-ionone. The colony fermentation procedure was as follows: single colonies activated with LB solid medium were inoculated into LB test tubes and cultured overnight for 12 hours to prepare seed culture. Then, the seed culture was inoculated into the fermentation medium (fermentation medium components: 10 g / L glycerol, 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) at an inoculation rate of 2% (volume ratio) and cultured at 37 °C. When the OD of the bacterial culture was... 600 When the temperature reaches approximately 1, add 20% (v / v) dodecane and place at 30 °C for biphase fermentation.

[0030] The detection and analysis procedures were as follows: After fermentation of the genetically engineered bacteria for 24 h, 300 μL of the dodecane phase was taken into a 1.5 mL centrifuge tube and centrifuged at 13000 rpm for 5 min. Then, 10 μL of the upper organic phase was dissolved in 90 μL of n-hexane for sample preparation. The sample was detected using an Agilent GC-MS (7890B-5977B) column (30 m × 0.25 mm × 0.25 μm). The temperature program was as follows: initial temperature 50 °C, held for 1 min; increased to 100 °C at 5 °C / min, held for 1 min; then increased to 200 °C at 5 °C / min, held for 1 min; finally increased to 300 °C at 5 °C / min, held for 1 min. Inlet temperature: 230 °C; split ratio: 10:1; carrier gas: helium (purity > 99.999%), flow rate: 1.0 mL / min; injection volume: 2 μL; mass spectrometry scan range: 10–500 m / z. The fermentation products were qualitatively identified based on ion information from the ion spectral library and retention times of standards. The fermentation yield was quantified by preparing ion ketone standard solutions of different concentrations. The yield results of β-ionone from the engineered strains in mutant libraries 428-430 are shown below. Figure 1 The β-ionone yields of different mutant strains screened are shown in Table 2. Strains improved by the initial screening at the S428 site were selected for secondary screening; the β-ionone yield results are shown in Table 2. Figure 2 .

[0031] The results showed that two mutant strains with significantly increased yields were obtained by screening at the S428 site, namely S428C and S428Y, as detailed below. Figure 1 and Figure 2 The yields of β-ionone reached 54.2 mg / L and 50.6 mg / L, respectively, representing increases of 69.7% and 58.5% compared to the wild-type strain WT. In Table 2, the naming convention for mutant strains is as follows: the number represents the mutated site in PhCCD1, the letter before the number represents the amino acid at that site before the mutation, and the letter after the number represents the amino acid at that site after the mutation. For example, mutant S428C represents the mutation of serine (S) at position 428 of PhCCD1 to cysteine ​​(C).

[0032] Table 2

[0033] Table 2 shows that the yields of mutants S428C and S428Y obtained through mutation reached 54.2 mg / L and 50.6 mg / L, respectively, which were 69.7% and 58.5% higher than the wild-type strain WT, indicating a significant increase in yield. The yields of mutants S428V, S428T, and S428H were only slightly higher than the wild-type. The yields of mutants S428G, S428L, and S428I were decreased to varying degrees compared to the wild-type.

[0034] The nucleotide and amino acid sequences of the S428C mutant are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide and amino acid sequences of the S428Y mutant are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0035] Example 2: In vitro catalytic verification and enzyme catalytic reaction kinetic curve verification of highly active PhCCD1 mutant This embodiment aims to verify the in vitro catalytic performance of PhCCD1 mutants S428C and S428Y, and to analyze their enzyme catalytic reaction kinetic curves.

[0036] In this embodiment, a widely used micelle system was selected as the in vitro reaction medium, and the surfactant Triton X-100 was used as an encapsulating agent to uniformly disperse the carotenoid substrates in the aqueous phase. The catalytic activity of the highly active mutants S428C and S428Y, as well as the wild-type WT, on a series of carotenoid substrates was verified by analyzing protein expression levels and product yields.

[0037] Specifically, the micelle system was prepared as follows: 0, 40, 80, 160, and 320 μM trans-β-apo-8'-carotene solutions were prepared using dichloromethane. 200 μL of this solution was added to a 2 mL centrifuge tube, followed by 200 μL of ethanol and 1 μL of the surfactant Triton X-100. The solvent was evaporated to dryness using a rotary evaporator, and the resulting residue was stored at -20°C for use as an in vitro reaction medium in subsequent experiments.

[0038] The enzyme expression and crude enzyme extraction methods were as follows: plasmids containing highly active mutants S428C and S428Y, as well as wild-type WT, were transformed into *E. coli* BL21(DE3). The cells were cultured overnight at 37 °C in LB liquid medium, then transferred to 50 mL of LB medium and cultured at 37 °C until OD (exchange rate). 600 The concentration reached 0.8. At this point, 0.1 mM IPTG was added to induce protein expression, and the culture temperature was lowered to 30 °C to continue fermentation for 24 hours. After fermentation, (OD) 600Cells were collected by centrifugation at 4000 rpm for 10 minutes after reaching a pH of 4.0. The cells were then concentrated 10-fold with 1X PBS buffer, resuspended, and lysed. The supernatant was then obtained by centrifugation at 13000 rpm for 5 minutes.

[0039] Enzyme purification: The crude enzyme solution was purified using a GST tag purification kit (Sangon Biotech, C600327-0001). The column was equilibrated with 10 mL of Binding / Wash Buffer at a flow rate of 0.5–1 mL / min until the absorbance (A280) at 280 nm reached baseline. The crude enzyme solution was mixed with Binding / Wash Buffer and added to the resin at a flow rate of 0.5–1 mL / min. The resin was washed with 10 mL of Binding / Wash Buffer, and the wash was repeated until the absorbance of the eluent reached baseline by measuring the absorbance at 280 nm. GST-tagged proteins were eluted with 1 mL of Elution Buffer in fractions, and the washing step was repeated, with each eluent collected individually. The column was equilibrated with 3 mL of Binding / Wash Buffer and 5 column volumes of deionized water, and finally equilibrated with 5 mL of 20% ethanol. The column was then stored at 4 °C. The crude and purified enzyme concentrations of mutants S428C and S428Y, as well as the wild-type enzyme, in this embodiment are shown in Table 3. The SDS-PAGE results of the crude and purified enzymes prepared from mutants S428C and S428Y are shown in [Table 3]. Figure 3 The mutant enzymes S428C and S428Y are designated as S428-21C and S428-26Y, respectively.

[0040] Protein concentration determination: The purified enzyme solution or crude enzyme solution was mixed with 6× protein loading buffer, boiled at 98 °C for 5 minutes, and then centrifuged at 13000 rpm for 5 minutes. 10 μL of the supernatant was loaded and analyzed by SDS-PAGE at 150 V. Clear protein bands were visible after Coomassie brilliant blue staining and destaining.

[0041] Enzyme-catalyzed reaction: Different substrate concentrations and fixed-purity enzyme additions (35 μg) were prepared in 2 mL centrifuge tubes, and the volume was supplemented to 1 mL with 1×PBS buffer. After 30 min of in vitro catalytic reaction, 500 μL of ethyl acetate was added to terminate the reaction, and β-ionone was extracted in a metal shaker (30 ℃, 800 rpm). 100 μL of the upper organic phase was used as a sample for content determination using an Agilent gas chromatography-mass spectrometry (GC-MS) system. The reaction kinetic parameters of mutant S428C and S428Y and wild-type enzymes are shown in Table 4, and the enzyme reaction kinetic curves are shown in Table 5. Figure 5 The results of β-carotene catalysis by the mutant and wild-type pure enzymes are shown in [the table below]. Figure 6 The results of catalyzing lutein are shown in [the original text]. Figure 7 .

[0042] Table 3. Concentrations of crude and purified enzymes prepared from PhCCD1 mutants

[0043] The results of this embodiment show that, compared with wild-type WT, the enzyme expression capacity of the S428C mutant is increased, while the enzyme expression capacity of the S428Y mutant is decreased. See details below. Figure 3 and Table 3; according to Figure 4 The results show that, using 64 μM trans-β-apo-8'-carotene as a substrate, the catalytic activity of the S428C mutant was increased by 210.58%, and that of the S428Y mutant was increased by 126.68%; the enzyme kinetic parameter k... cat / K m The results of the value determination showed that the S428C mutant strain had a 2-fold increase in catalytic efficiency compared to the wild type, while the S428Y mutant strain had a 40% increase in catalytic efficiency compared to the wild type. (See details...) Figure 5 And Table 4.

[0044] Table 4. Kinetic parameters of the PhCCD1 mutant

[0045] Simultaneously, the experiment also evaluated the in vitro catalytic activity of the S428C and S428Y mutants for β-carotene and lutein. For example... Figure 6 and Figure 7 As shown, the results indicate that, compared with wild-type WT, both mutants have significantly improved catalytic activity for both substrates, with the S428C mutant exhibiting the most outstanding catalytic activity. This result is consistent with the in vivo fermentation experiment results described above.

[0046] Example 3: In vitro catalysis of tobacco aqueous extract by highly active PhCCD1 mutant Key neutral flavor components of tobacco include a range of secondary metabolites, including degradation products of carotenoids (such as beta-carotene).

[0047] This embodiment utilizes the highly active PhCCD1 mutants S428C and S428Y to prepare crude enzyme solution. A micellar system was chosen as the in vitro reaction medium. Waste tobacco leaf aqueous extract was used as the substrate, and other exogenous carotenoids were added to catalyze the production of aroma molecules such as β-ionone. This solution was then applied to the aroma production of waste tobacco leaf extract. The aroma production results are as follows: Figure 8 As shown.

[0048] The preparation method of the micelle system is as follows: a 320 μM solution of trans-β-apo-8'-carotene and β-carotene is prepared using dichloromethane. Then, 200 μL of this solution is added to a 2 ml centrifuge tube, followed by 200 μL of ethanol and 1 μL of surfactant TritonX-100. The solution is then evaporated to dryness using a rotary evaporator and stored at -20 °C for later use.

[0049] The enzyme expression and crude enzyme extraction methods were as follows: the plasmid with the best mutant activity was transformed into *E. coli* BL21(DE3). The mixture was cultured overnight at 37 °C in LB liquid medium, then transferred to 50 mL of LB medium and cultured at 37 °C until OD (excitement / depression) was achieved. 600 The OD value reached 0.6-1.0. At this point, 0.1 mM IPTG was added to induce protein expression, and the culture temperature was lowered to 30 °C to continue fermentation for 24 hours. After fermentation, the OD value was calculated. 600 (4) Collect cells by centrifugation at 4000 rpm for 10 minutes, concentrate the cells 10-fold with 1X PBS buffer, resuspend them, and then lyse the cells. Subsequently, centrifuge at 13000 rpm for 5 minutes to obtain the supernatant crude enzyme solution.

[0050] Enzymatic reaction: In a 2 mL centrifuge tube, prepare a mixture of 200 μL of waste tobacco leaf aqueous extract, substrate (if available, 32 μM), and crude enzyme (2.5 mg / mL, 500 μL), and bring the volume to 1 mL with 1×PBS buffer. After 15 hours of in vitro catalytic reaction, add 500 μL of ethyl acetate to terminate the reaction, and then extract aroma components by reacting in a metal shaker (30 ℃, 800 rpm). Prepare a 100 μL sample from the upper organic phase and analyze it using an Agilent gas chromatography-mass spectrometry (GC-MS) system.

[0051] The results are as follows Figure 8 As shown, the mutant can utilize waste tobacco leaf water extract to catalyze the production of neutral tobacco aroma components such as β-ionone and dihydroactinolone. When other carotenoids such as β-carotene and trans-β-apo-8'-carotene are added, the aroma molecule yield is higher, which is beneficial for the aroma production application of waste tobacco leaf extract.

[0052] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A carotenoid cleaving dioxygenase mutant, characterized in that: The amino acid sequence of the carotenoid cleavage dioxygenase mutant is shown in SEQ ID NO.4 or SEQ ID NO.

6.

2. A gene encoding the carotenoid cleavage dioxygenase mutant of claim 1.

3. A plasmid, characterized in that, The plasmid carries the gene described in claim 2.

4. A genetically engineered bacterium, wherein the genetically engineered bacterium contains the carotenoid cleavage dioxygenase mutant of claim 1, the gene of claim 2, or the plasmid of claim 3.

5. The genetically engineered bacterium according to claim 4, characterized in that, The host bacterium of the genetically engineered bacteria is Escherichia coli.

6. The application of a carotenoid cleavage dioxygenase mutant as described in claim 1, or the gene as described in claim 2, or the plasmid as described in claim 3, or the genetically engineered bacteria as described in any one of claims 4-5, in catalyzing carotenoid conversion.

7. The application according to claim 6, characterized in that, The method of application includes the following steps: In an enzyme-catalyzed system, using carotenoids as substrates, at least one of β-ionone, pseudoionone, 3-hydroxy-β-ionone, 6-methyl-5-hepten-2-one, geranylacetone, and citral is catalyzed to be generated. The enzyme catalytic system includes the carotenoid cleavage dioxygenase mutant as described in claim 1.

8. The application according to claim 7, characterized in that, The enzyme catalytic system uses a micelle system as the reaction system, which is as follows: the carotenoid substrate is encapsulated in a liquid phase using the surfactant Triton X-100 as the encapsulation medium.

9. The application of a carotenoid cleavage dioxygenase mutant as described in claim 1, or the gene as described in claim 2, or the plasmid as described in claim 3, or the genetically engineered bacteria as described in any one of claims 4-5, in catalyzing aroma production from tobacco substrates.

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