Ferulic acid decarboxylase and mutant for biosynthesis of vanillin

Abstract

The invention relates to the technical field of biosynthesis, in particular to ferulic acid decarboxylase and a mutant for biosynthesizing vanillin, the amino acid sequence of the ferulic acid decarboxylase is as shown in SEQ ID NO.1, and the mutant contains one or more of the following mutation sites on the basis of the amino acid sequence of the ferulic acid decarboxylase as shown in SEQ ID NO.1: I143V, Y58W and K59V. The ferulic acid decarboxylase and the mutant provided by the invention have good catalytic activity, can keep stable and efficient catalytic activity in a high-concentration ferulic acid substrate environment, and provide powerful guarantee for high yield of vanillin.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, in particular to a ferulic acid decarboxylase and a mutant thereof for biosynthesis of vanillin. Background Art

[0002] Vanillin (4-hydroxy-3-methoxybenzaldehyde), one of the world's most important food flavorings, is widely and indispensable in a variety of industries, including food, cosmetics, and pharmaceuticals. Its unique aroma and flavor add significant value to products, and market demand for high-quality vanillin continues to rise.

[0003] Traditional methods of obtaining vanillin mainly include natural extraction from vanilla pods and chemical synthesis. However, the natural extraction method faces the dilemma of extremely limited supply of raw material vanilla pods. However, due to extremely low yields, long planting cycles and high costs, the acquisition cost is high and it can only serve a niche high-end market and cannot meet the huge global demand. Currently, the production of vanillin is still dominated by chemical synthesis, which mainly uses guaiacol, lignin or eugenol as raw materials. Although the chemical method is mature and the cost is relatively controllable, its inherent defects are becoming increasingly prominent: the production process often involves toxic and harmful chemical reagents, causing environmental pollution problems; its products cannot meet the regulatory definition requirements for "natural vanillin" and are difficult to adapt to market development trends. At the same time, the sources of some raw materials are limited, and sustainability faces challenges.

[0004] In recent years, with the significant increase in consumer demand for natural and healthy products, the market demand for vanillin with a "natural" label has surged. Therefore, bioconversion methods using microorganisms or their enzyme catalysts, using renewable biomass resources as raw materials, are widely considered the most promising sustainable solution. This approach offers mild reaction conditions and is environmentally friendly. The final product can be labeled "natural" as long as it complies with relevant regulations, offering significant market competitive advantages and environmental benefits.

[0005] Among the numerous biosynthetic routes for vanillin production, the one using ferulic acid as a precursor has attracted considerable attention due to its wide availability of raw materials. A key step in this pathway is the decarboxylation of ferulic acid to 4-vinylguaiacol (4-VG), catalyzed by ferulic acid decarboxylase. This reaction is the rate-limiting step in the entire biotransformation process, and its efficiency directly impacts the yield and production cost of the final vanillin. The application of ferulic acid decarboxylase in industrial-scale vanillin biosynthesis is generally plagued by a series of technical challenges. The primary issue is insufficient catalytic efficiency, manifested as a low substrate conversion rate, resulting in slow reaction progress and yields that fail to meet industrial production requirements. The enzyme's catalytic activity is easily inhibited by high concentrations of the substrate ferulic acid, the intermediate 4-VG, and the final product vanillin in the reaction system. This feedback inhibition further exacerbates the problem of insufficient conversion efficiency. Furthermore, ferulic acid decarboxylase exhibits generally poor stability in industrial production environments. Under complex conditions such as temperature fluctuations, pH changes, the presence of organic solvents, and high substrate or product concentrations, the enzyme rapidly inactivates, significantly limiting its application in industrial production.

[0006] Therefore, the development of a ferulic acid decarboxylase and mutants with higher catalytic activity, stability and adaptability to high-concentration substrates is of vital importance to improving the efficiency of vanillin biosynthesis, reducing production costs and promoting the development of the vanillin biosynthesis industry. It is also a key issue that needs to be urgently addressed in this field. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention provides a ferulic acid decarboxylase and mutant for the biosynthesis of vanillin, which solves the problems of low efficiency of ferulic acid decarboxylation reaction, susceptibility of the enzyme to substrate and product inhibition, and poor stability, significantly improves the efficiency of vanillin biosynthesis, and reduces the cost of industrial production.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a ferulic acid decarboxylase, wherein the amino acid sequence of the ferulic acid decarboxylase is the sequence shown in SEQ ID NO.1.

[0010] The present invention also provides a mutant of ferulic acid decarboxylase, which contains one or more of the following mutation sites: I143V, Y58W, and K59V based on the amino acid sequence of ferulic acid decarboxylase shown in SEQ ID NO.1.

[0011] As a preferred embodiment, the mutation site is:

[0012] I143V; or

[0013] I143V and Y58W; or

[0014] I143V, Y58W and K59V.

[0015] The invention also provides a nucleic acid molecule having at least one of the following:

[0016] (A1) is a nucleotide sequence encoding the above-mentioned ferulic acid decarboxylase;

[0017] (A2) is the nucleotide sequence encoding the above-mentioned ferulic acid decarboxylase mutant.

[0018] The invention also provides a recombinant expression vector having any of the following:

[0019] (B1), the nucleic acid molecule as described above;

[0020] (B1), the nucleic acid molecule as described above and the gene element for expressing carotenoid oxygenase.

[0021] The invention also provides a host cell, using Escherichia coli JM109 as a host, and having at least one of the following:

[0022] (C1), a nucleic acid molecule as described above;

[0023] (C2) The recombinant expression vector as described above.

[0024] The invention also provides the use of any of the following in the biocatalytic synthesis of vanillin:

[0025] (D1), ferulic acid decarboxylase as described above;

[0026] (D2), a mutant of the ferulic acid decarboxylase described above;

[0027] (D3) The host cell as described above.

[0028] The invention also provides a method for preparing vanillin, which uses the host cell as a biocatalyst and carries out conversion in a reaction system with ferulic acid as a substrate to obtain the product vanillin.

[0029] In some specific embodiments of the present invention, the preparation method comprises the following steps:

[0030] 1) culturing the host cell as described above, and isolating wet cells as a biocatalyst from the resulting culture;

[0031] 2) adding the wet cells obtained in step 1) into a reaction system containing ferulic acid for conversion to obtain the product vanillin.

[0032] In some specific embodiments of the present invention, the reaction system comprises: 0.05-0.1 g / mL wet bacteria, 85-115 mM Tris-HCl, and 95-160 mM ferulic acid.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The ferulic acid decarboxylase and mutants provided by the present invention have good catalytic activity and can significantly improve the efficiency of converting ferulic acid to 4-vinylguaiacol. Compared with the existing technology, the substrate conversion efficiency is significantly improved, the reaction cycle of vanillin biosynthesis is effectively shortened, the production efficiency is greatly improved, and the demand for high efficiency in industrial large-scale production is met. At the same time, the ferulic acid decarboxylase and mutants of the present invention exhibit excellent tolerance to substrate and product inhibition issues and can maintain stable and efficient catalytic activity in a high-concentration ferulic acid substrate environment. This overcomes the problem of reduced efficiency of traditional enzymes due to feedback inhibition in the reaction system, providing a strong guarantee for the high yield of vanillin. DETAILED DESCRIPTION

[0035] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0036] As used herein, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0037] Herein, "XaY" is used to represent the mutated amino acid in a mutant, where "a" represents the position of the amino acid in the wild-type form, "X" represents the type of amino acid at position a in the wild-type form, and "Y" represents the type of amino acid at position a in the wild-type form. XaY means that the amino acid at position a is replaced by "Y" instead of the wild-type amino acid X.

[0038] The invention provides ferulic acid decarboxylase. Ferulic acid decarboxylase Fdc (Ferulic Acid Decarboxylase) is isolated from Bacillus pumilus ATCC15884 strain and can specifically catalyze the decarboxylation of ferulic acid to generate 4-vinylguaiacol in a biosynthetic pathway, thereby providing a key intermediate for vanillin synthesis.

[0039] The amino acid sequence of the ferulic acid decarboxylase is the sequence shown in SEQ ID NO.1:

[0040]

[0041] The codon-optimized nucleic acid sequence of Escherichia coli is shown in SEQ ID NO: 2:

[0042]

[0043]

[0044] The present invention also provides a mutant of the ferulic acid decarboxylase, which is obtained by subjecting the ferulic acid decarboxylase shown in SEQ ID NO. 1 to point mutation, and contains one or more of the following mutation sites: I143V, Y58W, and K59V, i.e., the mutant's 143rd position Ile (isoleucine) is mutated to Val (valine); the mutant's 58th position Tyr (tyrosine) is mutated to Trp (tryptophan); and the mutant's 59th position Lys (lysine) is mutated to Val (valine).

[0045] The mutant is a single point mutation and a multi-point mutation applied for protection based on the amino acid sequence of ferulic acid decarboxylase as shown in SEQ ID NO.1, and contains one or more of the following mutation sites: I143V, Y58W, and K59V.

[0046] In a specific embodiment, the mutant of ferulic acid decarboxylase is based on the amino acid sequence of ferulic acid decarboxylase shown in SEQ ID NO.1, and contains one of the following mutation sites:

[0047] I143V; Y58W; K59V; I143V and Y58W; I143V and K59V; Y58W and K59V; I143V, Y58W and K59V.

[0048] As a preferred embodiment, it is a single point mutation, and the mutation site is: I143V;

[0049] As a preferred embodiment, it is a double-point mutation, with the mutation sites being: I143V and Y58W;

[0050] As a preferred embodiment, it is a triple-point mutation, and the mutation sites are: I143V, Y58W and K59V.

[0051] Other gene or protein sequence information involved in the present invention is as follows:

[0052] Carotene oxygenase CAO1 is derived from Neurospora crassa OR74A, and its amino acid sequence is shown in SEQ ID NO: 3:

[0053]

[0054] The codon-optimized nucleic acid sequence of Escherichia coli is shown in SEQ ID NO: 4:

[0055]

[0056]

[0057]

[0058] The decarboxylase used in the comparative example of the present invention is PAD (phenolic acid decarboxylase), which is derived from Heyndrickxia and has an NCBI accession number of WP017550974.1.

[0059] The codon-optimized nucleic acid sequence of Escherichia coli is shown in SEQ ID NO: 5:

[0060]

[0061] The culture medium used in the present invention is as follows:

[0062] TB medium: 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.32 g / L KH2PO4, and 12.54 g / L K2HPO4.

[0063] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0064] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0065] The HPLC detection conditions in the embodiment are:

[0066] Chromatographic column: ZORAB SB-C18, 250 mm × 4.6 mm, 5.0 μm; mobile phase composed of methanol (A) and 0.1% formic acid in water (B); flow rate 1 mL / min; 40% mobile phase B for 8 min, 40%-85% mobile phase B for 1 min, 85% mobile phase B for 5 min, 85%-40% mobile phase B for 1 min, 40% mobile phase B for 5 min.

[0067] The present invention will be further explained below in conjunction with specific embodiments.

[0068] Example 1 Construction of recombinant expression vector and recombinant strain of ferulic acid decarboxylase

[0069] 1. Construction of recombinant expression vector

[0070] The FDC gene was synthesized by General Biotechnology (Anhui) Co., Ltd. after codon optimization and cloned into the NdeI and HindIII sites of the pET-28a vector to obtain the expression vector plasmid pET28a-Fdc.

[0071] The CAO1 and FDC genes were synthesized by General Biotechnology (Anhui) Co., Ltd. after codon optimization and cloned into the NdeI and HindIII sites of the pET-28a vector. CAO 1 was amplified and recombined with the pET28a-Fdc vector to obtain the expression vector plasmid pET28a-CAO 1-Fdc.

[0072] 2. Construction of recombinant strains

[0073] The recombinant plasmid pET28a-Fdc was transformed into JM1 09 (DE3), and the competent cells were mixed with the plasmid. After electroporation, the cells were incubated in LB medium for 1 hour and then spread on a plate containing kanamycin (50 mg / L) resistance. After overnight culture, a single colony was picked to obtain the recombinant strain, which was designated as 4VG001.

[0074] The recombinant plasmid pET28a-CAO1-Fdc was transformed into JM109 (DE3), and the competent cells were mixed with the plasmid. After electroporation, the cells were incubated in LB medium for 1 hour and then spread on a plate containing kanamycin (50 mg / L) resistance. After overnight culture, a single colony was picked to obtain the recombinant strain, which was designated as VAN001.

[0075] Comparative Example 1 Construction of recombinant expression vector and recombinant strain of ferulic acid decarboxylase

[0076] 1. Construction of recombinant expression vector

[0077] The gene of ferulic acid decarboxylase PAD shown above was synthesized by General Bio (Anhui) Co., Ltd. after codon optimization and cloned into the NdeI and HindIII sites of the pET-28a vector to obtain the expression vector plasmid pET28a-PAD.

[0078] The CAO1 and PAD genes were synthesized by General Biotechnology (Anhui) Co., Ltd. after codon optimization and cloned into the NdeI and HindIII sites of the pET-28a vector. CAO 1 was amplified and recombined with the pET28a-PAD vector to obtain the expression vector plasmid pET28a-CAO 1-PAD.

[0079] 2. Construction of recombinant strains

[0080] The recombinant plasmid pET28a-PAD was transformed into JM109 (DE3), and the competent cells were mixed with the plasmid. After electroporation, the cells were incubated in LB medium for 1 hour and then spread on a plate containing kanamycin (50 mg / L) resistance. After overnight culture, a single colony was picked to obtain the recombinant strain, which was counted as 4VG000.

[0081] The recombinant plasmid pET28a-CAO 1-PAD was transformed into JM1 09 (DE3), and the competent cells were mixed with the plasmid. After electroporation, the cells were incubated in LB medium for 1 hour and then spread on a plate containing kanamycin (50 mg / L) resistance. After overnight culture, a single colony was picked to obtain the recombinant strain, which was counted as VAN000.

[0082] Example 2 Construction of Ferulic Acid Decarboxylase Mutant Gene, Recombinant Expression Vector and Recombinant Strain

[0083] 1. Construction of mutant genes

[0084] Take the I143V single point mutant as an example:

[0085] The required full plasmids were synthesized by Sangon Biotech Co., Ltd. using pET28a-Fdc and pET28a-CAO1-Fdc constructed in Example 1 as templates, respectively, to obtain plasmids pET28a-Fdc (I143V) and pET28a-CAO1-Fdc (I143V).

[0086] The construction methods of other plasmids were the same as those of pET28a-Fdc(I143V) and pET28a-CAO1-Fdc(I143V).

[0087] The plasmids were obtained as follows:

[0088] pET28a-CAO1-Fdc(Y58W), pET28a-CAO1-Fdc(Y58W);

[0089] pET28a-CAO1-Fdc(K59V), pET28a-CAO1-Fdc(K59V);

[0090] pET28a-CAO1-Fdc(I143V / Y58W), pET28a-CAO1-Fdc(I143V / Y58W);

[0091] pET28a-CAO1-Fdc(I143V / K59V), pET28a-CAO1-Fdc(I143V / K59V);

[0092] pET28a-CAO1-Fdc(Y58W / K59V), pET28a-CAO1-Fdc(Y58W / K59V);

[0093] pET28a-CAO1-Fdc(I143V / Y58W / K59V), pET28a-CAO1-Fdc(I143V / Y58W / K59V).

[0094] 2. Construction of recombinant strains

[0095] The recombinant plasmid pET28a-Fdc(I143V) was transformed into JM109(DE3). The competent cells were mixed with the plasmid and incubated in LB medium for 1 h after electroporation. The cells were then plated on plates containing kanamycin (50 mg / L) and cultured overnight. Single colonies were picked to obtain the recombinant strain, designated 4VG002.

[0096] The recombinant plasmid pET28a-CAO1-Fdc (I143V) was transformed into JM109 (DE3), and the competent cells were mixed with the plasmid. After electroporation, the cells were incubated in LB medium for 1 hour and then spread on a plate containing kanamycin (50 mg / L) resistance. After overnight culture, a single colony was picked to obtain the recombinant strain, which was designated as VAN002.

[0097] Using the same method, recombinant strains 4VG003-4VG008 and VAN003-VAN008 were obtained.

[0098] The basic information of the strain is shown in Table 1 below:

[0099] Table 1 Recombinant expression bacteria

[0100]

[0101]

[0102] Example 3 Biotransformation reaction experimental verification

[0103] The recombinant strains 4VG000-4VG008 constructed in Example 1, Example 2, and Comparative Example 1 were inoculated into 50 mL of TB culture medium containing 50 μg / mL kanamycin and cultured in a 250 mL shake flask at 200 rpm and 37° C. When the OD 600 reached 0.6, 0.2 mM IPTG and 1 mM FeSO 4 were added, and expression was induced at 22° C. for 16 h, followed by centrifugation at 8000 rpm for 10 min. The cells were collected, washed twice with 100 mM Tris-HCl (pH 7.0), and diluted with reaction buffer to a wet cell weight of 0.075 g / mL to obtain wet cells for biotransformation.

[0104] The catalytic reaction system (5 mL) consisted of 0.075 g / mL wet bacterial cells, 100 mM Tris-HCl (pH 7.0), and 103 mM ferulic acid. The reaction was incubated at 22°C for 18 hours. After 18 hours, a 1 mL sample of reaction buffer was removed from the biotransformation system and analyzed by HPLC. The conversion efficiency of 4VG was calculated. The conversion multiples of strains 4VG000-4VG008 were calculated based on the conversion efficiency of strain 4VG001 (calculated as 1).

[0105] The results are shown in Table 2:

[0106] Table 24 Catalytic conversion results of VG001-4VG008 bacteria

[0107]

[0108]

[0109] As can be seen from Table 2, the ferulic acid decarboxylase Fdc of the present invention and its mutants exhibit significant advantages in catalyzing the formation of the intermediate product 4-vinylguaiacol (4VG). This result also indirectly reflects that the enzyme of the present invention and its mutants have a higher tolerance to the substrate ferulic acid and the intermediate product 4VG. Conventional enzymes are prone to feedback inhibition, resulting in decreased activity, in environments with high substrate or intermediate concentrations. However, the enzyme of the present invention can still maintain efficient conversion during the catalytic process, continuously converting ferulic acid into 4VG, providing sufficient key intermediates for the subsequent synthesis of vanillin, thereby effectively increasing the final yield of vanillin.

[0110] Example 4 Whole-cell biotransformation reaction experimental verification

[0111] The recombinant strains VAN001-VAN008 constructed in Examples 1 and 2 were inoculated into 50 mL of TB culture medium containing 50 μg / mL kanamycin and cultured in a 250 mL shake flask at 200 rpm and 37°C. When the OD600 reached 0.6, 0.2 mM IPTG and 1 mM FeSO4 were added, and expression was induced at 22°C for 16 h. The cells were then centrifuged at 8000 rpm for 10 min, and the cells were harvested, washed twice with 100 mM Tris-HCl (pH 7.0), and diluted with reaction buffer to a wet weight of 0.075 g / mL. The wet cells were then used as biocatalysts in whole-cell catalytic reactions.

[0112] The catalytic reaction system (5 mL) consisted of 0.075 g / mL wet bacterial cells, 100 mM Tris-HCl (pH 7.0), and 103 mM ferulic acid. The reaction was incubated at 22°C for 18 hours. After 18 hours, a 1 mL sample of reaction buffer was removed from the bioconversion system and analyzed by HPLC. The conversion rate of vanillin was calculated. The conversion multiples of strains VAN001-VAN008 were calculated based on the conversion rate of strain VAN001 (calculated as 1).

[0113] The results are shown in Table 3:

[0114] Table 3 Catalytic conversion results of VAN000-VAN008 bacteria

[0115]

[0116]

[0117] As can be seen from Table 3, the ferulic acid decarboxylase Fdc and its mutants of the present invention exhibit significant advantages in catalyzing the synthesis of vanillin. The vanillin conversion rate of the strain expressing the decarboxylase PAD (VAN000) used in the comparative example was only 14.2%, while the conversion rate of the wild-type ferulic acid decarboxylase Fdc (VAN001) was 2.75 times that of the comparative example, highlighting the superiority of the Fdc enzyme source in catalytic activity.

[0118] On this basis, a series of mutants (VAN002-VAN008) obtained by modifying the wild-type Fdc enzyme further improved its catalytic efficiency. The vanillin conversion rate of the VAN008 mutant was 5.52 times higher than that of the control decarboxylase PAD and approximately 2 times higher than that of the wild-type Fdc enzyme. This fully demonstrates that the ferulic acid decarboxylase Fdc and its mutants of the present invention can effectively overcome the technical limitations of low catalytic efficiency of traditional enzymes, opening up a new path for the efficient biosynthesis of vanillin.

[0119] Example 5 Substrate concentration adaptability verification

[0120] This example uses the same experimental method as Example 4, except that the catalytic reaction systems are different. This example uses three different catalytic systems for verification.

[0121] Catalytic system 1: 0.075 g / mL wet bacteria, 100 mM Tris-HCl (pH 7.0), 51.5 mM ferulic acid, reaction at 22°C for 18 hours;

[0122] Catalytic system 2: 0.075 g / mL wet bacteria, 100 mM Tris-HCl (pH 7.0), 130 mM ferulic acid, reaction at 22°C for 18 h;

[0123] Catalytic system 3: 0.075 g / mL wet bacteria, 100 mM Tris-HCl (pH 7.0), 155 mM ferulic acid, reaction at 22°C for 18 hours.

[0124] Reactions were performed using the three catalytic systems described above. After completion, 1 mL of reaction buffer was sampled from the biotransformation system and analyzed by HPLC. The conversion rate was calculated. Based on the conversion rate of strain VAN001, the conversion rate multiples of strains VAN000-VAN008 were calculated. The conversion rate multiples are shown in Table 4.

[0125] Table 4 VAN000-VAN008 bacterial substrate adaptability results

[0126]

[0127]

[0128] As shown in Table 4, the ferulic acid decarboxylase Fdc and its mutants exhibit excellent substrate tolerance at varying substrate concentrations, overcoming the problem of decreased catalytic efficiency due to substrate inhibition in conventional enzymes. This property enables the enzyme to adapt to higher substrate concentrations in industrial production, reducing reaction volume and increasing product yield per unit volume, thereby significantly improving the economic efficiency and feasibility of vanillin biosynthesis.

[0129] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. The preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification.

Claims

1. Ferulic acid decarboxylase, characterized in that The amino acid sequence of the ferulic acid decarboxylase is the sequence shown in SEQ ID NO.

1.

2. The mutant of ferulic acid decarboxylase according to claim 1, wherein The mutant contains one or more of the following mutation sites: I143V, Y58W, and K59V based on the amino acid sequence of ferulic acid decarboxylase as shown in SEQ ID NO.

1.

3. The mutant of ferulic acid decarboxylase according to claim 2, wherein The mutation sites are: I143V; or I143V and Y58W; or I143V, Y58W and K59V.

4. A nucleic acid molecule, characterized in that Have at least one of the following: (A1) is a nucleotide sequence encoding the ferulic acid decarboxylase according to claim 1; (A2) is a nucleotide sequence encoding the ferulic acid decarboxylase mutant according to claim 2 or 3.

5. A recombinant expression vector, characterized in that: Have any of the following: (B1), the nucleic acid molecule according to claim 4; (B1), the nucleic acid molecule according to claim 4 and the gene element for expressing carotenoid oxygenase.

6. A host cell, characterized in that Escherichia coli JM109 is used as the host and at least one of the following is present: (C1), the nucleic acid molecule according to claim 4; (C2) The recombinant expression vector according to claim 5.

7. Application of any of the following in the biocatalytic synthesis of vanillin: (D1), the ferulic acid decarboxylase according to claim 1; (D2), according to claim 2 or 3, a mutant of ferulic acid decarboxylase; (D3) The host cell according to claim 6.

8. A method for preparing vanillin, characterized in that: The host cell according to claim 6 is used as a biocatalyst and conversion is carried out in a reaction system with ferulic acid as a substrate to obtain the product vanillin.

9. The method for preparing vanillin according to claim 8, characterized in that: The preparation method comprises the following steps: 1) culturing the host cell according to claim 6, and isolating wet cells as a biocatalyst from the resulting culture; 2) The wet cells obtained in step 1) are added to a reaction system containing ferulic acid for conversion to obtain the product vanillin.

10. The method for preparing vanillin according to claim 9, wherein: The reaction system is: 0.05-0.1 g / mL wet bacteria, 85-115 mM Tris-HCl, and 95-160 mM ferulic acid.

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