Carotenoid oxygenase CAO1 mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a carotenoid oxygenase CAO1 mutant and application thereof. The carotenoid oxygenase CAO1 mutant disclosed by the invention is characterized in that on the basis of an amino acid sequence of wild type carotenoid oxygenase CAO1 as shown in SEQ ID NO.1, the carotenoid oxygenase CAO1 mutant contains one or more of the following mutation sites: M426K, M426S, M426N, M426Q, M426R, A372L, V265R, T236V, L444D, W339E, S191T, A524G and V519G. The carotenoid oxygenase CAO1 mutant disclosed by the invention is obtained by mutating wild type carotenoid oxygenase CAO1, has higher catalytic activity, can be used for efficiently synthesizing vanillin by taking ferulic acid as a substrate, and solves the technical problems that the wild type carotenoid oxygenase CAO1 is limited in catalytic activity, relatively low in conversion efficiency and unfavorable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a carotenoid oxygenase CAO1 mutant and application thereof. Background Art

[0002] Vanillin, also known as vanillin, has the unique aroma of vanilla beans. It is a widely used flavoring compound and one of the most commonly used aromatic compounds in the food, fragrance, pharmaceutical and chemical industries. There are three main methods for the production of vanillin: plant raw material extraction, chemical synthesis and biosynthesis. Among them, the plant raw material extraction method, which directly extracts from natural plants such as vanilla beans, has a very low yield and high production cost, which cannot meet the demand; the chemical synthesis method usually uses guaiacol or lignin derivatives as raw materials. Although the yield is high, the method requires harsh reaction conditions and has defects such as strong reagent toxicity. In contrast, the biosynthesis method using renewable raw materials and engineered microbial fermentation enzymes provides a milder, more environmentally friendly and more sustainable process for the production of vanillin.

[0003] Currently, the primary natural substrates used for microbial conversion include renewable resources such as ferulic acid and eugenol. Ferulic acid is widely present in nature, inexpensive, and readily available, making it considered a preferred precursor for microbial production of vanillin. Vanillin synthesis is catalyzed by ferulic acid decarboxylase (Fdc) and carotenoid oxygenases (e.g., CAO1), using ferulic acid as a substrate. However, the existing wild-type CAO1 has low catalytic efficiency and low ferulic acid conversion rates, making current production processes potentially unsuitable for commercial-scale production. Summary of the Invention

[0004] In view of this, the present invention provides a carotenoid oxygenase CAO1 mutant and its application. Compared with the wild type, the mutant has higher catalytic activity and high catalytic efficiency, improves the conversion rate of ferulic acid, and is more suitable for industrial commercial-scale production.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a carotenoid oxygenase CAO1 mutant. The carotenoid oxygenase CAO1 mutant contains one or more of the following mutation sites on the basis of the amino acid sequence of the wild-type carotenoid oxygenase CAO1 as shown in SEQ ID NO.1: M426K, M426S, M426N, M426Q, M426R, A372L, V265R, T236V, L444D, W339E, S191T, A524G, and V519G.

[0007] In some specific embodiments of the present invention, the mutation site of the mutant comprises any of the following:

[0008] M426K; or

[0009] M426S; or

[0010] M426N; or

[0011] M426Q; or

[0012] M426R; or

[0013] A372L; or

[0014] V265R; or

[0015] T236V; or

[0016] L444D; or

[0017] W339E; or

[0018] S191T; or

[0019] A524G; or

[0020] V519G; or

[0021] M426K / A372L; or

[0022] M426K / V265R; or

[0023] V265R / A372L; or

[0024] M426K / A372L / V256R; or

[0025] M426K / A372L / S191T; or

[0026] M426K / A372L / S191T / A524G; or

[0027] M426K / A372L / S191T / V519G.

[0028] The present invention also provides a nucleic acid molecule having a nucleotide sequence encoding the mutant.

[0029] The present invention also provides an expression vector having any of the following:

[0030] (A1) the above-mentioned nucleic acid molecule;

[0031] (A2) The above-mentioned nucleic acid molecule and a gene element for expressing ferulic acid decarboxylase Fdc.

[0032] In some specific embodiments of the present invention, the backbone of the above expression vector is pET28a.

[0033] The present invention also provides a host cell, excluding cells with totipotency, having any of the following:

[0034] (B1) the above-mentioned nucleic acid molecule;

[0035] (B2) The above-mentioned expression vector.

[0036] In some specific embodiments of the present invention, the host cell is a bacterium;

[0037] Preferably, the bacteria include Escherichia coli;

[0038] Further preferably, the bacteria include E. coli JM109 (DE3).

[0039] The present invention also provides the use of the carotenoid oxygenase CAO1 mutant or the host cell in biocatalytic synthesis of vanillin.

[0040] The present invention also provides a method for preparing vanillin, which is characterized in that ferulic acid is used as a substrate, PEG200 is added to a catalytic reaction system containing a biocatalyst, and vanillin is produced.

[0041] In some embodiments of the present invention, the biocatalyst comprises wild-type carotenoid oxygenase CAO1 or a carotenoid oxygenase CAO1 mutant.

[0042] The addition of PEG200 can improve the oxygen transfer in the catalytic reaction system, thereby increasing the yield of vanillin.

[0043] In some specific embodiments of the present invention, preferably, the biocatalyst is the above-mentioned host cell; further, the biocatalyst is a wet cell obtained by culturing the above-mentioned host cell.

[0044] In some specific embodiments of the present invention, the preparation method comprises the following steps:

[0045] 1) culturing the host cell described above, and isolating wet cells as a biocatalyst from the resulting culture;

[0046] 2) The wet cells obtained in step 1) are added to a reaction system containing ferulic acid for conversion to obtain the product vanillin.

[0047] 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, 95-110 mM ferulic acid, and 5%-7.5% (V / V) PEG 200.

[0048] Further preferably, the reaction system comprises: 0.075 g / mL wet bacteria, 100 mM Tris-HCl, 103 mM ferulic acid, and 5% (V / V) PEG200.

[0049] In some specific embodiments of the present invention, the method for culturing host cells is as follows: the host cells are inoculated into 50 mL TB culture medium containing 50 ug / mL kanamycin, and cultured in a 250 mL shake flask at 200 rpm and 37°C. When OD600 reaches 0.6, 0.2 mM IPTG and 1 mM FeSO4 are added, and expression is induced at 22°C for 16 hours, and then placed at 8000 rpm for 10 minutes to collect the bacteria.

[0050] The present invention has the following beneficial effects:

[0051] The carotenoid oxygenase CAO1 mutant of the present invention has significantly improved catalytic efficiency compared to wild-type carotenoid oxygenase CAO1, with a conversion rate reaching 1.73 times that of wild-type carotenoid oxygenase CAO1. Furthermore, the mutant of the present invention can achieve efficient catalytic conversion at a higher substrate concentration (103 mM ferulic acid). Furthermore, the addition of PEG200 to the reaction system improves oxygen transfer and synergizes with the CAO1 mutant, further increasing the catalytic conversion efficiency and thereby increasing the yield of vanillin. Therefore, the present invention has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0053] Figure 1 Shown are the protein electrophoresis results of carotenoid oxygenase CAO1 and ferulic acid decarboxylase Fdc. DETAILED DESCRIPTION

[0054] The present invention discloses a carotenoid oxygenase CAO1 mutant and its applications. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify, alter, and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0055] 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).

[0056] Herein, mutation sites are represented by the format of "XaY", where a represents the position of the amino acid in SEQ ID NO: 1, X represents the wild-type amino acid species at position a in SEQ ID NO: 1, and Y represents the mutated amino acid species at position a in SEQ ID NO: 1. For example, in the present invention, "M426K" represents a mutation of the methionine M at position 426 corresponding to SEQ ID NO: 1 to lysine K; "A372L" represents a mutation of the alanine A at position 372 corresponding to SEQ ID NO: 1 to leucine L; "S191T" represents a mutation of the serine S at position 191 corresponding to SEQ ID NO: 1 to threonine T; and "M426K / A372L" represents a mutation of the methionine M at position 426 corresponding to SEQ ID NO: 1 to lysine K and a mutation of the alanine A at position 372 to leucine L.

[0057] The present invention provides a carotenoid oxygenase CAO1 mutant. The carotenoid oxygenase CAO1 mutant contains one or more of the following mutation sites on the basis of the amino acid sequence of the wild-type carotenoid oxygenase CAO1 as shown in SEQ ID NO.1: M426K, M426S, M426N, M426Q, M426R, A372L, V265R, T236V, L444D, W339E, S191T, A524G, and V519G.

[0058] The wild-type carotenoid oxygenase CAO1 of the present invention is derived from Neurospora crassa OR74A, and its amino acid sequence is shown in SEQ ID NO: 1:

[0059] * (SEQ ID NO: 1);

[0060] The codon-optimized nucleic acid sequence of Escherichia coli is shown in SEQ ID NO: 2:

[0061]

[0062] Other gene or protein sequence information involved in this application is as follows:

[0063] Ferulic acid decarboxylase Fdc is derived from Bacillus pumilus ATCC 15884, and its amino acid sequence is shown in SEQ ID NO: 3:

[0064] MDQFVGLHMIYTYENGWEYEIYIKNDHTIDYRIHSGMVGGRWVRDQEVNIVKLTKGVYKVSWTEPTGTDVSLNFMPEEKRMHGVIFFPKWVHERPDITVCYQNDYIDLMKESREKYETYPKYVVPEFADITYIHHAGVNDETIIAEAPYEGLTDEIRAGRK* (SEQ ID NO: 3);

[0065] The codon-optimized nucleic acid sequence of Escherichia coli is shown in SEQ ID NO: 4:

[0066] ATGGATCAGTTTGTTGGTCTGCACATGATCTATACCTATGAAAATGGTTGGGAATATGAAATCTATATCAAGAATGACCACACCATTGATTATCGCATTCATAGCGGTATGGTTGGCGGCCGCTGGGTTCGTGATCAGGAAGTGAATATTGTTAAACTGACCAAAGGCGTGTATAAAGTGAGCTGGACCGAACCGACCGGTACCGATGTGAGTCTGAATTTTATGCCGGAAGAAAAACGCATGCA TGGCGTGATTTTTCTTTCCGAAATGGGTTCATGAACGTCCGGATATTACCGTGTGTTATCAGAATGATTATATCGATCTGATGAAGGAAAGCCGCGAAAAATATGAAACCTATCCGAAATATGTGGTTCCGGAATTTGCCGATATTACCTATATTCATCATGCCGGCGTTAATGATGAAACATTATTGCAGAAGCACCGTATGAAGGTCTGACCGATGAAATTCGCGCAGGCCGTAAATAA (SEQ ID NO: 4).

[0067] The culture medium used in this application is as follows:

[0068] 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.

[0069] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0070] PEG200 in this application was purchased from Merck; ferulic acid was purchased from Merck.

[0071] 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.

[0072] The present invention will be further described below with reference to the embodiments.

[0073] Example 1 Construction of recombinant strains

[0074] 1. Construction of recombinant plasmid

[0075] The CAO1 and FDC genes were synthesized by General Biotech (Anhui) Co., Ltd. after codon optimization and cloned into the NdeI and HindIII sites of the pET-28a vector. CAO1 was amplified and recombined with the pET28a-Fdc vector to obtain the plasmid pET28a-CAO1-Fdc.

[0076] 2. Construction of mutants

[0077] Take the M426K single point mutant as an example:

[0078] Using pET28a-CAO1-Fdc as a template, the required full plasmid was synthesized by Sangon Biotech Co., Ltd. to obtain the plasmid pET28a-CAO1 (M426K)-Fdc.

[0079] The construction methods of the remaining plasmids were the same as those of pET28a-CAO1 (M426K)-Fdc, and the resulting plasmids were as follows:

[0080] pET28a-CAO1(M426K)-Fdc

[0081] pET28a-CA01(M426S)-Fdc

[0082] pET28a-CA01(M426N)-Fdc

[0083] pET28a-CA01(M426Q)-Fdc

[0084] pET28a-CA01(M426R)-Fdc

[0085] pET28a-CA01(A372L)-Fdc

[0086] pET28a-CA01(V265R)-Fdc

[0087] pET28a-CA01(T236V)-Fdc

[0088] pET28a-CA01(L444D)-Fdc

[0089] pET28a-CA01(W339E)-Fdc

[0090] pET28a-CAO1(S191T)-Fdc

[0091] pET28a-CA01(A524G)-Fdc

[0092] pET28a-CA01(V519G)-Fdc

[0093] pET28a-CAO1(M426K / A372L)-Fdc

[0094] pET28a-CAO1(M426K / V265R)-Fdc

[0095] pET28a-CA01(V265R / A372L)-Fdc

[0096] pET28a-CAO1(M426K / A372L / V256R)-Fdc

[0097] pET28a-CA01(M426K / A372L / S191T)-Fdc

[0098] pET28a-CA01(M426K / A372L / S191T / A524G)-Fdc

[0099] pET28a-CA01(M426K / A372L / S191T / V519G)-Fdc.

[0100] 3. Construction of recombinant expression bacteria

[0101] (1) Construction of recombinant strain V00

[0102] 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 one 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 V00.

[0103] (2) Construction of recombinant strains V01-V20

[0104] The construction and preparation methods of the recombinant strain containing the CAO1 mutant were the same as those of the recombinant strain V00, and the recombinant expression strains V01-V20 were obtained, as shown in Table 2 below.

[0105] Table 2 Recombinant expression bacteria

[0106] strain chassis Expression plasmid V00 JM109(DE3) pET28a-CAO1-Fdc V01 JM109(DE3) pET28a-CAO1(M426K)-Fdc V02 JM109(DE3) pET28a-CAO1(M426S)-Fdc V03 JM109(DE3) pET28a-CAO1(M426N)-Fdc V04 JM109(DE3) pET28a-CAO1(M426Q)-Fdc V05 JM109(DE3) pET28a-CAO1(M426R)-Fdc V06 JM109(DE3) pET28a-CAO1(A372L)-Fdc V07 JM109(DE3) pET28a-CAO1(V265R)-Fdc V08 JM109(DE3) pET28a-CAO1(T236V)-Fdc V09 JM109(DE3) pET28a-CAO1(L444D)-Fdc V10 JM109(DE3) pET28a-CAO1(W339E)-Fdc V11 JM109(DE3) pET28a-CAO1(S191T)-Fdc V12 JM109(DE3) pET28a-CAO1(A524G)-Fdc V13 JM109(DE3) pET28a-CAO1(V519G)-Fdc V14 JM109(DE3) pET28a-CAO1(M426K / A372L)-Fdc V15 JM109(DE3) pET28a-CAO1(M426K / V265R)-Fdc V16 JM109(DE3) pET28a-CAO1(V265R / A372L)-Fdc V17 JM109(DE3) pET28a-CAO1 (M426K / A372L / V256R)-Fdc V18 JM109(DE3) pET28a-CAO1 (M426K / A372L / S191T)-Fdc V19 JM109(DE3) pET28a-CAO1 (M426K / A372L / S191T / A524G)-Fdc V20 JM109(DE3) pET28a-CAO1 (M426K / A372L / S191T / V519G)-Fdc

[0107] Example 2 Whole-cell biotransformation reaction experimental verification

[0108] The V00-V20 cells obtained in 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 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 collected, washed twice with 100 mM Tris-HCl (pH 7.0), and diluted with reaction buffer to a wet weight of 0.075 g / mL. Wet cells were obtained and used as biocatalysts in whole-cell catalytic reactions.

[0109] 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 rate was calculated using the conversion rate of strain V00 (wild type) as the basis for the calculation of the conversion multiples for mutants V01-V20.

[0110] HPLC detection conditions are:

[0111] 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, and 40% mobile phase B for 5 min.

[0112] The results of the conversion rate fold change are shown in Table 3:

[0113] Table 3 Catalytic conversion results of V00-V20 bacteria

[0114] strain Conversion rate fold change V00 1 V01 1.17±0.02 V02 1.16±0.02 V03 1.19±0.05 V04 1.12±0.04 V05 1.11±0.03 V06 1.04±0.01 V07 1.21±0.03 V08 1.19±0.02 V09 1.15±0.03 V10 1.08±0.05 V11 1.15±0.03 V12 1.14±0.02 V13 1.17±0.03 V14 1.23±0.03 V15 1.24±0.02 V16 1.29±0.10 V17 1.31±0.05 V18 1.41±0.02（57.5%） V19 0.91±0.01 V20 1.11±0.02

[0115] As can be seen from Table 3, compared with the V00 strain (wild type), the conversion rate of the mutants of the present invention is higher. Among them, the conversion rate of the recombinant strain V18 (M426K / A372L / S191T) is 1.41 times that of the wild type, indicating that the catalytic efficiency of the CAO1 mutant is significantly improved, thereby increasing the conversion rate.

[0116] Example 3 Effect of surfactants on whole-cell biotransformation

[0117] 1. Effect of PEG200 on conversion rate (V00 wet cells)

[0118] Catalytic reaction system (5 mL): 0.075 g / mL V00 wet bacterial cells (wild-type CAO1), 100 mM Tris-HCl (pH 7.0), and 103 mM ferulic acid. Pluronic F-68, PEG 200, PEG 400, and Triton X-100 were added to the catalytic reaction system at 5% (v / v). 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 rate was calculated based on the conversion rate of the V00 strain (wild-type) in the reaction system without surfactant. The results are shown in Table 4.

[0119] Table 4 Effect of surfactant conversion system

[0120] strains surfactants Conversion rate fold change V00 Pluronic F-68 0.89±0.02 V00 none 1 V00 PEG200 1.16±0.07（45.5%） V00 PEG400 1.07±0.01 V00 TritonX-100 0.92±0.04

[0121] As can be seen from Table 4, based on the conversion rate without adding surfactant, the conversion rate of the reaction system with Pluronic F-68 and TritonX-100 is lower, and the conversion rate of the reaction system with PEG200 is higher. The conversion rate of the reaction system with PEG200 is 1.16 times that of the reaction system without adding surfactant, which proves that not all surfactants can improve the conversion rate. It shows that adding PEG 200 to the reaction system can improve oxygen transfer, increase catalytic conversion rate, and increase the yield of vanillin.

[0122] 2. Effect of PEG200 addition on the conversion system

[0123] Catalytic reaction system (5 mL): 0.075 g / mL V00 wet bacteria (wild-type CAO1), 100 mM Tris-HCl (pH 7.0), 103 mM ferulic acid. Different amounts of PEG200 (V / V) were added to the reaction system. The reaction was carried out at 22°C for 18 hours. After 18 hours, 1 mL of reaction buffer sample was taken from the biotransformation system and analyzed by HPLC.

[0124] Based on the conversion rate of the V00 strain (wild type) in the reaction system without adding PEG200, the conversion rate multiple of the V00 strain was calculated. The results are shown in Table 5:

[0125] Table 5 Effect of PEG200 addition amount

[0126] strains PEG 200 addition amount (V / V) Conversion rate fold change V00 20% 0.90±0.03 V00 15% 0.87±0.01 V00 10% 0.98±0.01 V00 7.5% 1.16±0.11 V00 5% 1.16±0.07（45.5%） V00 2.5% 0.58±0.12 V00 1% 0.58±0.00 V00 0 1

[0127] It can be seen from Table 5 that, based on the conversion rate without adding PEG200, the conversion rates when the addition amount of PEG200 is 10%, 15% and 20% are lower. The conversion rates when the addition amount is 1% and 2.5% are only 0.58 times the conversion rate without adding PEG200, while the conversion rates when the addition amount is 5% and 7.5% are 1.16 times the conversion rate without adding PEG200. It can be concluded that the effect of increasing the conversion rate when adding 7.5% is equivalent to that when adding 5%, and the addition amount of 5% is less than that of 7.5%. Therefore, the optimal addition amount of PEG 200 is 5%.

[0128] 3. Effect of PEG200 on mutant transformation system

[0129] Catalytic reaction system (5 mL): 0.075 g / mL V00-V20 wet cells, 100 mM Tris-HCl (pH 7.0), 103 mM ferulic acid, and 5% (v / v) PEG 200. 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 to calculate the conversion rate.

[0130] Based on the conversion rate of the V00 strain (wild type) in the reaction system with the addition of PEG 200, the conversion rate multiples of the V01-V20 strains were calculated. The results are shown in Table 6:

[0131] Table 6

[0132] strain Conversion rate fold change V00 1 V01 1.38±0.01 V02 1.32±0.00 V03 1.32±0.02 V04 1.32±0.02 V05 1.29±0.01 V06 1.33±0.02 V07 1.38±0.01 V08 1.25±0.03 V09 1.26±0.05 V10 1.25±0.03 V11 1.31±0.02 V12 1.27±0.03 V13 1.34±0.03 V14 1.51±0.02 V15 1.46±0.10 V16 1.50±0.03 V17 1.38±0.06 V18 1.73±0.02 (conversion rate 78.5%) V19 1.17±0.08 V20 1.17±0.02

[0133] As can be seen from Table 6, when PEG200 was added to the reaction system, the conversion rate of the mutant of the present invention was higher than that of the V00 bacteria (wild type), among which the conversion rate of the V18 bacteria (M426K / A372L / S191T) was 1.73 times that of the wild type, and the conversion rate of the recombinant strain V18 in the reaction system with the addition of PEG200 (78.5%) was higher than the conversion rate (57.5%) without the addition of PEG200, indicating that the addition of PEG200 to the reaction system can improve oxygen transfer, and PEG200 and the CAO1 mutant synergistically enhance the efficiency, further improving the catalytic conversion rate, thereby increasing the yield of vanillin.

[0134] In summary, the carotenoid oxygenase CAO1 mutant of the present invention has significantly improved catalytic efficiency compared to wild-type carotenoid oxygenase CAO1, with a conversion rate reaching 1.73 times that of wild-type carotenoid oxygenase CAO1. Furthermore, the mutant of the present invention can achieve efficient catalytic conversion at a higher substrate concentration (103 mM ferulic acid). Furthermore, the addition of PEG200 to the reaction system can improve oxygen transfer and synergize with CAO1mut to further enhance catalytic conversion efficiency and increase vanillin production. Therefore, the present invention has high application value.

[0135] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A carotenoid oxygenase CAO1 mutant, characterized in that: The carotenoid oxygenase CAO1 mutant contains one or more of the following mutation sites based on the amino acid sequence of the wild-type carotenoid oxygenase CAO1 as shown in SEQ ID NO.1: M426K, M426S, M426N, M426Q, M426R, A372L, V265R, T236V, L444D, W339E, S191T, A524G, V519G.

2. The carotenoid oxygenase CAO1 mutant according to claim 1, characterized in that: The mutation sites of the mutant include any one of the following: M426K, M426S, M426N, M426Q, M426R, A372L, V265R, T236V, L444D, W339E, S191T, A524G, V519G, M426K / A372L, M426K / V265R, V265R / A372L, M426K / A372L / V256R, M426K / A372L / S191T, M426K / A372L / S191T / A524G, M426K / A372L / S191T / V519G.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule has a nucleotide sequence encoding the mutant according to claim 1 or 2.

4. An expression vector, characterized in that Have any of the following: (A1) The nucleic acid molecule according to claim 3; (A2) The nucleic acid molecule according to claim 3 and a gene element for expressing ferulic acid decarboxylase Fdc.

5. A host cell, excluding totipotent cells, characterized in that Have any of the following: (B1) the nucleic acid molecule according to claim 3; (B2) The expression vector according to claim 4. 6 . Use of the carotenoid oxygenase CAO1 mutant according to claim 1 or the host cell according to claim 5 in biocatalytic synthesis of vanillin.

7. A method for preparing vanillin, characterized in that: With ferulic acid as substrate, PEG200 is added into the catalytic reaction system containing biocatalyst to produce vanillin.

8. The method for preparing vanillin according to claim 7, wherein: The biocatalyst in the reaction system is the host cell according to claim 5.

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 5, 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, 95-110 mM ferulic acid, and 5%-7.5% (V / V) PEG 200.

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