Enzyme Composition, Its Application and Method for Synthesizing Vanillin

Abstract

The present invention relates to the field of bioengineering, and particularly to an enzyme composition, its application, and a method for synthesizing vanillin. The present invention provides an enzyme composition, comprising: a hydroxylase and a methoxytransferase. The present invention selects Saccharomyces cerevisiae as a chassis to provide a new pathway for synthesizing vanillin using p-coumaric acid as a precursor, and provides a general strategy for realizing the high-value utilization of lignin through biotransformation.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to an enzyme composition, its application, and a method for synthesizing vanillin. Background Art

[0002] Lignin is the most abundant renewable aromatic resource in nature, existing in the cell walls of most terrestrial plants, accounting for about 15 - 40%. As a biopolymer composed of phenylpropyl groups, lignin has great potential in the sustainable production of aromatic products. Traditionally, most lignin has been used for power generation by combustion. However, with the emergence of biorefineries that convert cellulosic biomass into liquid fuels, the lignin production far exceeds the demand for powering the operation, so it is necessary to explore how to convert the relevant surplus lignin into more diverse and valuable products. Developing new biological funnel pathways to convert lignin into a single high - value product can improve the utilization efficiency of lignin, thus contributing to a viable bioeconomy. Despite these possibilities, the stabilization of lignin remains a major challenge, and due to the diversity of lignin - derived monomers generated during the depolymerization process, the production of high - value lignin products has not been fully developed yet.

[0003] Vanillin (also known as vanillic aldehyde) is one of the most widely used flavoring agents in the world. Its natural source is vanilla beans ( vanilla planifolia ), and the extraction rate of natural vanillin is very low (less than 1%), the production cost is very high, and the output is also limited. Natural extraction, chemical synthesis, and tissue culture techniques can no longer meet the annual demand for vanillin in the world market. Commercially available vanillin is chemically synthesized from petroleum or lignin, and this process is not environmentally friendly and lacks substrate selectivity. Consumers' preference and demand for natural flavors have led to an increase in the price of natural vanilla. In recent years, there have been studies on producing vanillin by constructing microbial cell factories using aromatic compound conversion methods, and the produced vanillin can be regarded as natural vanillin or bio - vanillin. However, vanillin has varying degrees of inhibitory effects on microbial growth and metabolism, and is easily endogenously metabolized by microorganisms into vanillic acid or vanillyl alcohol. The high price of aromatic precursors themselves, the compatibility problem between heterologous enzymes and chassis bacteria, and the low efficiency of enzymes are all the main factors restricting the sustainable synthesis of vanillin by biotransformation methods.

[0004] Saccharomyces cerevisiae is an important platform microorganism, and has many advantages as a cell factory for synthesizing various important chemicals, such as clear genetic background, mature gene editing tools, strong robustness, and potential for industrial application, etc. Currently, there have been studies on expressing 3DSD (3 - dehydroshikimate dehydratase, from Podospora pauciseta ), HsComt ( O -methyltransferase, from Homo sapiens ) and ACAR (aromatic acid reductase, fromNocardia sp.), protocatechuic acid is synthesized through the shikimic acid pathway, vanillic acid is synthesized through methylation, and then 45 mg / L vanillin is produced by reduction. This pathway can only use glucose as the raw material, and the vanillin yield is low. The aromatic acid reductase ACAR involved in the pathway is non-specific and will perform non-ideal reduction on the lignin-derived monomers p-coumaric acid and ferulic acid, producing by-products and thus reducing the conversion rate of the target product. In addition, by introducing TAL (tyrosine ammonia-lyase, from Actinomycete Saccharothrix espanaensis ), C3H (4-coumarate-3-hydroxylase, from Saccharothrix espanaensis ), COMT (caffeic acid O-methyltransferase, from Arabidopsis thaliana ), FCS (trans-feruloyl-CoA synthetase, from Streptomyces sp. strain V-1 ), and ECH (enoyl-CoA hydratase / aldolase, from Streptomyces sp. strain V-1 ), site-directed mutagenesis of ARO4 K229L and ARO7 G141S relieves the feedback inhibition of the intermediate tyrosine. Finally, using glucose supplemented with 400 mg / L ferulic acid as the substrate, 8.5 mg / L vanillin can be synthesized. This method uses pure ferulic acid as the precursor, and the yield and conversion rate of the target product vanillin are both very low. SUMMARY OF THE INVENTION

[0005] In view of this, the present invention provides an enzyme composition, its application, and a method for synthesizing vanillin. The present invention selects Saccharomyces cerevisiae as the chassis, provides a new pathway for synthesizing vanillin using p-coumaric acid as the precursor, and provides a general strategy for realizing the high-value utilization of lignin through biotransformation.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0007] The present invention provides an enzyme composition, including: hydroxylase and O-methyltransferase;

[0008] The hydroxylase includes: derived from Pseudomonas putida KT2440 PobA 、derived from Pseudomonas putida KT2440 PphpaB 、derived from Enterobacter cloacae of EnhpaB 、derived from Pseudomonas aeruginosa of PahpaB and / or derived from Salmonella enterica of SehpaC one or more of;

[0009] The O-methyltransferase includes: The O-methyltransferase includes: derived from Homo sapiens ofHsCOMT , derived from Arabidopsis thaliana of AtCOMT and / or derived from Nicotiana tabacum of NtCOMT one or more of

[0010] In some embodiments of the present invention, the above enzyme composition includes: hydroxylase and methoxylase;

[0011] The hydroxylase includes: derived from Pseudomonas aeruginosa of PahpaB and derived from Salmonella enterica of SehpaC ;

[0012] The methoxylase includes: derived from Arabidopsis thaliana of AtCOMT and / or derived from Nicotiana tabacum of NtCOMT .

[0013] In some embodiments of the present invention, the above enzyme composition includes: hydroxylase and methoxylase;

[0014] The hydroxylase includes: derived from Pseudomonas aeruginosa of PahpaB and derived from Salmonella enterica of SehpaC ;

[0015] The methoxylase includes: derived from Arabidopsis thaliana of AtCOMT .

[0016] In some embodiments of the present invention, in the above enzyme composition, the PobA has

[0017] (1), the amino acid sequence shown in SEQ ID NO: 1; or

[0018] (2), a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in (1); or

[0019] (3), a sequence having a homology of more than 90% with the amino acid sequence shown in (1) or (2); or

[0020] The PphpaB has:

[0021] (4), the amino acid sequence shown in SEQ ID NO: 2; or

[0022] (5) A sequence with one or more amino acids substituted, deleted, added, and / or replaced based on the amino acid sequence shown in (4); or

[0023] (6) A sequence with a homology of more than 90% to the amino acid sequence shown in (4) or (5); or

[0024] said EnhpaB having:

[0025] (7) An amino acid sequence as shown in SEQ ID NO:3; or

[0026] (8) A sequence with one or more amino acids substituted, deleted, added, and / or replaced based on the amino acid sequence shown in (7); or

[0027] (9) A sequence with a homology of more than 90% to the amino acid sequence shown in (7) or (8); or

[0028] said PahpaB having:

[0029] (10) An amino acid sequence as shown in SEQ ID NO:4; or

[0030] (11) A sequence with one or more amino acids substituted, deleted, added, and / or replaced based on the amino acid sequence shown in (10); or

[0031] (12) A sequence with a homology of more than 90% to the amino acid sequence shown in (10) or (11); or

[0032] said SehpaC having:

[0033] (13) An amino acid sequence as shown in SEQ ID NO:5; or

[0034] (14) A sequence with one or more amino acids substituted, deleted, added, and / or replaced based on the amino acid sequence shown in (13); or

[0035] (15) A sequence with a homology of more than 90% to the amino acid sequence shown in (13) or (14); and

[0036] said HsCOMT having:

[0037] (16) An amino acid sequence as shown in SEQ ID NO:6; or

[0038] (17) A sequence with one or more amino acids substituted, deleted, added, and / or replaced based on the amino acid sequence shown in (16); or

[0039] (18) A sequence with more than 90% homology to the amino acid sequence shown in (16) or (17); or

[0040] said AtCOMT having:

[0041] (19) The amino acid sequence shown in SEQ ID NO:7; or

[0042] (20) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence shown in (19); or

[0043] (21) A sequence with more than 90% homology to the amino acid sequence shown in (19) or (20); or

[0044] said NtCOMT having:

[0045] (22) The amino acid sequence shown in SEQ ID NO:8; or

[0046] (23) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence shown in (22); or

[0047] (24) A sequence with more than 90% homology to the amino acid sequence shown in (22) or (23).

[0048] In some embodiments of the present invention, the sequence of SEQ ID NO:1 is: MKTQVAIIGAGPSGLLLGQLLHKAGIDNIIVERQTAEYVLGRIRAGVLEQGTVDLLREAGVAERMDREGLVHEGVELLVGGRRQRLDLKALTGGKTVMVYGQTEVTRDLMQAREASGAPIIYSAANVQPHELKGEKPYLTFEKDGRVQRIDCDYIAGCDGFHGISRQSIPEGVLKQYERVYPFGWLGLLSDTPPVNHELIYAHHERGFALCSQRSQTRSRYYLQVPLQDRVEEWSDERFWDELKARLPAEVAADLVTGPALEKSIAPLRSLVVEPMQYGHLFLVGDAAHIVPPTGAKGLNLAASDVNYLYRILVKVYHEGRVDLLAQYSPLALRRVWKGERFSWFMTQLLHDFGSHKDAWDQKMQEADREYFLTSPAGLVNIAENYVGLPFEEVA.

[0049] In some embodiments of the present invention, the sequence of SEQ ID NO:2 above is: MKKPNPLLEDLKSVLPTIAANAMRAEQDRSVPAENIALLKSIGMHRAFLPKHFGGMEITLPEFAQCIALLAGACASTAWAMSLLCTHSHQMAMFSPKLQQEVWGSDPDATASSSIAPFGRTEEVEGGVSFSGEMGWSSGCDHAEWAILGFRRKNAEGAQDYCFAILPRSDYEIRDDWYAVGMRGSGSKTLIVRDAFVPEHRIQKAKDMMEGKSAGFGLYPDSKIFFAPYRPYFASGFSTVSLGVAERMLEVFREKTRNRVRAYTGAAVGAATPALMRLAESTHQVAAARALLEKSWDEIAEHSARHEYPSRGTLAFWRTNQGYAVKMCIQAVDRLMEAAGGGAWFESNELQRLFRDSHMTGAHAYTDYDVCAQILGRELMGLEPDPAMV。

[0050] In some embodiments of the present invention, the sequence of SEQ ID NO:3 above is: MKPEEFRADTKRPLTGEEYLKSLQDGREIYIYGERVKDVTTHPAFRNAAASIAQMYDALHKPDMQDTLCWGTDTGSGGYTHKFFRVAKSADDLRQQRDAIAEWSRLSYGWMGRTPDYKAAFGCALGANPAFYGQFEQNARNWYTRIQETGLYFNHAIVNPPIDRHKPADEVKDVYIKLEKETDAGIIVSGAKVVATNSALTHYNMIGFGSAQVMGENPDFALMFVAPMDAEGVKLISRASYEMVAGATGSPYDYPLSSRFDENDAILVMDNVLIPWENVLIYRDFDRCRRWTMEGGFARMYPLQACVRLAVKLDFITALLKKSLECTGTLEFRGVQADLGEVVAWRNMFWALSDSMCSEATPWVNGAYLPDHAALQTYRVMAPMAYAKIKNIIERNVTSGLIYLPSSARDLNNPQIDQYLAKYVRGSNGMDHVERIKILKLMWDAIGSEFGGRHELYEINYSGSQDEIRLQCLRQAQSSGNMDKMMAMVDRCMSEYDQHGWTVPHLHNNSDINMLDKLLK。

[0051] In some embodiments of the present invention, the sequence of SEQ ID NO:4 above is: MKPEDFRASATRPFTGEEYLASLRDDREIYIYGDRVKDVTSHPAFRNAAASMARLYDALHDPQSKEKLCWETDTGNGGYTHKFFRYARSADELRQQRDAIAEWSRLTYGWMGRTPDYKAAFGSALGANPGFYGRFEDNAKTWYKRIQEACLYLNHAIVNPPIDRDKPVDQVKDVFISVDEEVDGGIVVSGAKVVATNSALTHYNFVGQGSAQLLGDNTDFALMFIAPMNTPGMKLICRPSYELVAGIAGSPFDYPLSSRFDENDAILVMDKVFIPWENVLIYRDFERCKQWFPQGGFGRLFPMQGCTRLAVKLDFITGALYKALQCTGSLEFRGVQAQVGEVVAWRNLFWSLTDAMYGNASEWHGGAFLPSAEALQAYRVLAPQAYPEIKKTIEQVVASGLIYLPSGVRDLHNPQLDKYLSTYCRGSGGMGHRERIKILKLLWDAIGSEFGGRHELYEINYAGSQDEIRMQALRQAIGSGAMKGMLGMVEQCMGDYDENGWTVPHLHNPDDINVLDRIRQ。

[0052] In some embodiments of the present invention, the sequence of SEQ ID NO:5 above is: MQVDEQRLHFRDAMASLAAAVNIVTTAGHAGRCGITATAVCSVTDTPPSVMVCINANSAMNPVFQGNGRLCINVLNHEQELMARHFAGMTGMAMEERFHQPCWQNGPLGQPVLNGALAGLEGEISEVQTIGTHLVYLVAIKNIILSQDGHGLIYFKRRFHPVRLEMEAPV。

[0053] In some embodiments of the present invention, the sequence of SEQ ID NO:6 is as follows: MPEAPPLLLAAVLLGLVLLVVLLLLLRHWGWGLCLIGWNEFILQPIHNLLMGDTKEQRILNHVLQHAEPGNAQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEHQPSVLLELGAYCGYSAVRMARLLSPGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVGASQDIIPQLKKKYDVDTLDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVICPGAPDFLAHVRGSSCFECTHYQSFLEYREVVDGLEKAIYKGPGSEAGP。

[0054] In some embodiments of the present invention, the sequence of SEQ ID NO:7 is as follows: MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPMSPTEIASKLPTKNPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVERIYGLGPVCKYLTKNEDGVSIAALCLMNQDKVLMESWYHLKDAILDGGIPFNKAYGMSAFEYHGTDPRFNKVFNNGMSNHSTITMKKILETYKGFEGLTSLVDVGGGIGATLKMIVSKYPNLKGINFDLPHVIEDAPSHPGIEHVGGDMFVSVPKGDAIFMKWICHDWSDEHCVKFLKNCYESLPEDGKVILAECILPETPDSSLSTKQVVHVDCIMLAHNPGGKERTEKEFEALAKASGFKGIKVVCDAFGVNLIELLKKL。

[0055] In some embodiments of the present invention, the sequence of SEQ ID NO:8 is: MESSTKSQIPTQSEEERNCTYAMQLLSSSVLPFVLHSTIQLEVFEILAKSNDTKLSASQIVSQIPNCKNPDAATMLDRMLYVLASYSLFTCSIVEDEENNGGQKRVYGLSQVGKFFVRDEDGASMGPLLALLQDKVFINSWFELKDAVLEGGVPFDRVHGVVHAFEYPKSDPKFNDVFNKAMINHTTVVMKKILENYKGFENLKTLVDVGGGLGVNLKMITSKYPTIKGTNFDLPHVVQHAPSYPGVEHVGGDMFESVPEGDAIFMKWILHDWSDSHNLKLLKNCYKALPDNGKVIVVEAILPVKPDIDTAVVGVSQCDLIMMAQNPGGKERSEEEFRALATEAGFKGVNLICCVCNFWVMEFCK。

[0056] The present invention also provides a nucleic acid molecule encoding the above enzyme composition, including:

[0057] a nucleic acid molecule encoding the PobA ; or a nucleic acid molecule encoding the PphpaB ; or a nucleic acid molecule encoding the EnhpaB ; or a nucleic acid molecule encoding the PahpaB ; or a nucleic acid molecule encoding the SehpaC ; and a nucleic acid molecule encoding the HsCOMT ; or a nucleic acid molecule encoding the AtCOMT ; or a nucleic acid molecule encoding the NtCOMT 。

[0058] In some embodiments of the present invention, among the above nucleic acid molecules, the nucleic acid molecule of the PobA has:

[0059] (25), a nucleotide sequence as shown in SEQ ID NO:9; or

[0060] (26), a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (25), and having the same or similar function as the nucleotide sequence as shown in (25); or

[0061] (27), a nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (25) or (26); or

[0062] the nucleic acid molecule of the PphpaB has:

[0063] (28) The nucleotide sequence as shown in SEQ ID NO:10; or

[0064] (29) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (28), and having the same or similar function as the nucleotide sequence as shown in (28); or

[0065] (30) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (28) or (29); or

[0066] The EnhpaB nucleic acid molecule has:

[0067] (31) The nucleotide sequence as shown in SEQ ID NO:11; or

[0068] (32) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (31), and having the same or similar function as the nucleotide sequence as shown in (31); or

[0069] (33) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (31) or (32); or

[0070] The PahpaB nucleic acid molecule has:

[0071] (34) The nucleotide sequence as shown in SEQ ID NO:12; or

[0072] (35) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (34), and having the same or similar function as the nucleotide sequence as shown in (34); or

[0073] (36) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (34) or (35); or

[0074] The SehpaC nucleic acid molecule has:

[0075] (37) The nucleotide sequence as shown in SEQ ID NO:13; or

[0076] (38) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (37), and having the same or similar function as the nucleotide sequence as shown in (37); or

[0077] (39) A nucleotide sequence having at least 80% identity to the nucleotide sequence shown in (37) or (38); and

[0078] The HsCOMT nucleic acid molecule has:

[0079] (40) A nucleotide sequence as shown in SEQ ID NO:14; or

[0080] (41) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (40), and having the same or similar function as the nucleotide sequence shown in (40); or

[0081] (42) A nucleotide sequence having at least 80% identity to the nucleotide sequence shown in (40) or (41); or

[0082] The AtCOMT nucleic acid molecule has:

[0083] (43) A nucleotide sequence as shown in SEQ ID NO:15; or

[0084] (44) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (43), and having the same or similar function as the nucleotide sequence shown in (43); or

[0085] (45) A nucleotide sequence having at least 80% identity to the nucleotide sequence shown in (43) or (44); or

[0086] The NtCOMT nucleic acid molecule has:

[0087] (46) A nucleotide sequence as shown in SEQ ID NO:16; or

[0088] (47) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (46), and having the same or similar function as the nucleotide sequence shown in (46); or

[0089] (48) A nucleotide sequence having at least 80% identity to the nucleotide sequence shown in (46) or (47).

[0090]

[0091]

[0092]

[0093]

[0094] In some embodiments of the present invention, the sequence of SEQ ID NO:13 above is: ATGCAAGTTGATGAACAAAGATTGCATTTTAGAGATGCAATGGCTTCTTTAGCTGCAGCTGTTAATATTGTTACTACAGCAGGTCATGCTGGTAGATGTGGTATTACTGCAACAGCTGTTTGTTCTGTTACTGATACACCACCATCAGTTATGGTTTGTATCAACGCAAACTCAGCTATGAACCCAGTTTTCCAGGGTAACGGTAGATTGTGTATCAACGTTTTGAACCATGAACAAGAATTGATGGCAAGACATTTTGCTGGTATGACTGGTATGGCTATGGAAGAAAGATTTCATCAACCATGTTGGCAAAATGGTCCATTGGGTCAACCAGTTTTAAATGGTGCATTGGCTGGTTTAGAAGGTGAAATTTCTGAAGTTCAAACTATCGGTACACATTTGGTTTATTTGGTTGCAATTAAAAATATCATCTTGTCACAAGATGGTCATGGTTTGATCTATTTCAAGAGAAGATTTCATCCAGTTAGATTAGAAATGGAAGCTCCAGTTTAA。

[0095] In some embodiments of the present invention, the sequence of SEQ ID NO:14 is as follows: ATGCCAGAAGCTCCACCATTATTATTAGCCGCAGTTTTGTTGGGCTTGGTTTTATTGGTCGTCTTGTTGTTGTTGTTGAGACATTGGGGTTGGGGTCTTTGTCTTATTGGTTGGAACGAATTCATCTTGCAGCCAATTCACAACTTGTTGATGGGCGATACTAAGGAACAGAGAATCTTGAACCACGTTTTACAACACGCAGAACCAGGTAACGCTCAATCCGTTTTGGAAGCTATCGATACTTATTGCGAACAGAAGGAGTGGGCTATGAACGTTGGCGATAAGAAGGGTAAAATCGTGGACGCTGTTATTCAAGAACATCAACCATCCGTCTTGTTGGAATTAGGAGCTTATTGCGGTTACAGCGCAGTTAGAATGGCTAGATTGTTGTCTCCAGGAGCTAGATTGATCACCATTGAAATCAACCCAGATTGCGCAGCTATTACTCAAAGAATGGTCGATTTCGCAGGCGTTAAAGATAAGGTTACCTTGGTTGTTGGAGCTTCTCAAGACATCATCCCACAGTTGAAGAAGAAATACGACGTTGATACCTTGGATATGGTGTTCTTGGATCATTGGAAGGACAGATACTTGCCAGATACTTTGTTGTTGGAGGAGTGCGGTTTACTAAGAAAAGGTACCGTTTTGTTGGCCGATAACGTTATTTGCCCAGGAGCTCCAGATTTCTTAGCTCACGTTAGAGGTTCTTCTTGCTTTGAATGCACTCATTACCAGTCCTTCTTGGAATACAGAGAAGTTGTTGACGGTTTGGAAAAGGCTATATATAAAGGTCCAGGTTCAGAAGCAGGTCCATAA。

[0096]

[0097]

[0098] The present invention also provides an expression cassette, comprising the above enzyme composition and / or the above nucleic acid molecule.

[0099] The present invention also provides an expression module, comprising a first expression module, a second expression module and an endogenous expression module;

[0100] The first expression module comprises: a linker protein and any two of the above enzyme composition and / or the above nucleic acid molecule; PobA the PphpaB the EnhpaB the PahpaB and / or the SehpaC ;

[0101] The second expression module comprises: one or more of the above enzyme composition and / or the above nucleic acid molecule; HsCOMT the AtCOMT and / or the NtCOMT ;

[0102] The endogenous expression module comprises: MET13 atsc , SAH1 and MET6.

[0103] In some embodiments of the present invention, MET13 in the above expression module atsc is methylenetetrahydrofolate reductase, MET6 is homocysteine methyltransferase and SAH1 is S-adenosylhomocysteine hydrolase.

[0104] In some embodiments of the present invention, the linker protein in the above expression module comprises: GGGGS.

[0105] In some embodiments of the present invention, the sequence of the linker protein in the above expression module is as shown in SEQ ID NO:17: GGTGGTGGTGGTTCT.

[0106] In some embodiments of the present invention, the first expression module in the above expression module comprises: a linker protein and the PahpaB and the SehpaC。

[0107]

[0108] In some embodiments of the present invention, in the above expression module, the copy number of the connexin is not less than 3; the copy number of the second expression module is not less than 2; and the endogenous expression module is overexpressed.

[0109] In some embodiments of the present invention, in the above expression module, an endogenous promoter and an endogenous terminator are further included.

[0110] In some embodiments of the present invention, in the above expression module, the endogenous promoter includes: P TPI1 , P FBA1 , P PGK1 , P TEF2 , P TDH3 , P GPM1 and / or one or more of P PDC1 .

[0111] In some embodiments of the present invention, in the above expression module, the endogenous terminator includes: T GPM1 , T ADH2 , T TDH1 , T PGI1 , T HXT7 , T CHO1 , T TEF1 , T CYC1 and / or one or more of T ADH3 .

[0112] In some embodiments of the present invention, in the above expression module, a connecting homologous arm is further included; the connecting homologous arm includes one or more of HO-HAs, delta15-HAs and / or delta22-HAs.

[0113] In some embodiments of the present invention, in the above expression module, a gRNA plasmid is further included.

[0114] The present invention also provides a strain, into which the above expression cassette and / or the above expression module are introduced into a chassis strain.

[0115] In some embodiments of the present invention, the above chassis strain includes: SyBE_Sc05220018 (ZR05) and / or SyBE_Sc05220038 (VAN01).

[0116] The present invention also provides the use of the above enzyme composition, the above nucleic acid molecule, the above expression cassette, the above expression module and / or the above strain in the synthesis of vanillin.

[0117] The present invention also provides a method for synthesizing vanillin. Take the above strain, after culturing, mix it with lignin or its stock solution, and after fermentation, obtain the vanillin.

[0118] In some embodiments of the present invention, in the above method, the lignin includes: p - coumaric acid and ferulic acid.

[0119] In some embodiments of the present invention, in the above method, the preparation method of the stock solution includes the following steps: mixing straw with an alkali solution, performing an oil bath, then cooling, and separating the solid and liquid to obtain the stock solution.

[0120] In some embodiments of the present invention, in the above method, the alkali solution includes NaOH (0.1 g / g straw).

[0121] In some embodiments of the present invention, in the above method, the final loading of the alkali solution is 100 mg / g.

[0122] In some embodiments of the present invention, in the above method, the addition amount of the straw is 10% wt.

[0123] In some embodiments of the present invention, in the above method, the temperature of the oil bath is 130 °C and the time is 30 min.

[0124] In some embodiments of the present invention, in the above method, the pH value of the stock solution is 6.

[0125] In some embodiments of the present invention, in the above method, the fermentation uses a fermentation system; the fermentation system includes: 0.4X the stock solution, 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract powder.

[0126] In some embodiments of the present invention, in the above method, the fermentation time is 96 h and the temperature is 30 °C

[0127] The present invention provides an enzyme composition, including: hydroxylase and methoxytransferase;

[0128] The hydroxylase includes: derived from Pseudomonas putida KT2440 PobA and derived from Pseudomonas putida KT2440 PphpaB and derived from Enterobacter cloacae of EnhpaB and derived from Pseudomonas aeruginosa of PahpaB and / or derived from Salmonella enterica of SehpaC one or more of;

[0129] The methoxytransferase includes: derived from Homo sapiens of HsCOMT and derived fromArabidopsis thaliana from the AtCOMT and / or Nicotiana tabacum from the NtCOMT one or more of them.

[0130] The beneficial effects of the present invention include:

[0131] (1) Compared with the existing lignin utilization methods, the treatment method in this study is mild, with less pollution, low cost, and no special equipment is required;

[0132] (2) Compared with the existing vanillin synthesis methods, starting from corn stover, the raw materials are cheap, easily available and sustainable, replacing expensive pure monomers as precursors;

[0133] (3) As a generally recognized safe strain, the Saccharomyces cerevisiae strain has better biosafety compared with other lignin-degrading bacteria, and it is easier to achieve subsequent optimization processes;

[0134] (4) Fermentation is carried out by directly adding the original lignin hydrolysis solution to the culture medium, without the need for prior monomer separation and inhibitor detoxification processes.

[0135] (5) The vanillin yield obtained by converting lignin hydrolysis solution in this study is 1.35 mmol / L, which is the highest yield of vanillin synthesized by Saccharomyces cerevisiae using lignin at present.

[0136] (6) Compared with other chassis microorganisms, Saccharomyces cerevisiae has biosafety and good robustness, with a clear genetic background and mature molecular operation techniques, which is convenient for introducing exogenous pathways and metabolic regulation;

[0137] (7) Saccharomyces cerevisiae is easy to be cultured at high density, which can effectively alleviate the problems of substrate and product inhibition, and improve the expression level and catalytic efficiency of low-efficiency enzymes; (the low-efficiency enzymes include caffeic acid O-methyltransferase COMT and hydroxylase hpaBC, and the corresponding measures are to strengthen the SAM cycle and increase the copy number and enzyme fusion respectively)

[0138] (8) Compared with the published vanillin biosynthesis methods, the "biological funnel pathway" proposed in this study greatly broadens the substrate spectrum, providing more reference significance and research targets for vanillin biosynthesis;

[0139] (9) The treatment process of lignin is simple and mild, without using polluting substances and expensive reagents, reducing costs and reducing the environmental pressure of agricultural waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0141] Figure 1 Demonstrate the fermentation verification of the p - coumaric acid - p - hydroxybenzaldehyde pathway;

[0142] Figure 2 Demonstrate the screening and fermentation verification of the p - hydroxybenzaldehyde - protocatechuic aldehyde pathway;

[0143] Figure 3 Demonstrate the screening and fermentation verification of the protocatechuic aldehyde - vanillin and caffeic acid - ferulic acid pathways;

[0144] Figure 4 Demonstrate the pathways for the conversion of ferulic acid and p - coumaric acid into vanillin;

[0145] Figure 5 Demonstrate the fermentation verification of the conversion of lignin - derived monomers into vanillin; wherein: A shows the fermentation result of ferulic acid - vanillin; B shows the fermentation result of p - coumaric acid - vanillin;

[0146] Figure 6 Demonstrate the result of enzyme fusion - enhanced hydroxylation;

[0147] Figure 7 Demonstrate two pathways for p - coumaric acid - vanillin;

[0148] Figure 8 Demonstrate the result of SAM - regulated enhanced methoxylation process;

[0149] Figure 9 Demonstrate the fermentation result of p - coumaric acid & ferulic acid - vanillin;

[0150] Figure 10 Demonstrate the fermentation result of APL - vanillin. Specific embodiments

[0151] The present invention discloses an enzyme composition, its application, and a method for synthesizing vanillin.

[0152] It should be understood that the expression "one or more of..." individually includes each of the recited objects following the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0153] The terms "comprising", "having", or "containing", including the use of their grammatical synonyms, should generally be understood as open - ended and non - restrictive, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0154] It should be understood that as long as the present invention remains operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0155] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. Any language in this specification should not be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0156] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0157] In Examples 1 to 5 and Verification Examples 1 to 2 of the present invention,

[0158] (1) Obtaining a Saccharomyces cerevisiae strain capable of stably accumulating vanillin

[0159] Provided by the research group of Yuan Yingjin, and the strain number is SyBE_Sc05220018 (the strain ZR05 in Patent CN 113201465 A)

[0160] (2) Obtaining a Saccharomyces cerevisiae strain capable of synthesizing and stably accumulating vanillin using ferulic acid as a precursor

[0161] Provided by the research group of Yuan Yingjin, and the strain number is SyBE_Sc05220038 (the strain VAN01 in Patent CN 113201465 A). This strain can only use ferulic acid monomer to synthesize vanillin and cannot use p-coumaric acid (the content in lignin is 3-4 times that of ferulic acid), which is not conducive to the effective utilization of resources.

[0162] In Examples 1 to 5 and Verification Examples 1 to 2 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0163] The present invention will be further elaborated below in conjunction with the examples:

[0164] Example 1 Verification of a strain capable of converting p-coumaric acid into p-hydroxybenzaldehyde

[0165] Contained in strain VAN01 (named VA01 in the present invention) Petroselinum crispum derived from 4CL and Pseudomonas putida derived from KT2440 EchGenes that can convert p - coumaric acid into p - hydroxybenzaldehyde. Through fermentation verification, 1.22 mmol of p - coumaric acid was converted into 0.66 mmol of p - hydroxybenzaldehyde after 24 h of fermentation. ZR05 that did not introduce this pathway could not convert p - coumaric acid into p - hydroxybenzaldehyde (as shown in Table 1 and Figure 1 as follows).

[0166] Table 1 Fermentation results of the p - coumaric acid - p - hydroxybenzaldehyde pathway

[0167] Strain p-Hydroxybenzaldehyde (mmol / L) ZR05 0 VA01 0.66±0.00

[0168] Example 2 Construction and verification of a strain capable of converting p - hydroxybenzaldehyde into protocatechualdehyde

[0169] Using the ZR05 strain that can stably accumulate vanillin as the chassis, hydroxylase from different sources was expressed through a free plasmid, and the best active combination was screened to achieve the hydroxylation of p - hydroxybenzaldehyde and thus convert it into protocatechualdehyde. The hydroxylase involved in the screening mainly included Pseudomonas putida from KT2440 PobA , Pseudomonas putida from KT2440 PphpaB , Enterobacter cloacae from EnhpaB , Pseudomonas aeruginosa from PahpaB and Salmonella enterica from SehpaC . The nucleotide sequences after codon optimization are as shown in SEQ ID NO:9 - SEQ ID NO:13.

[0170] Experimental method:

[0171] Yeast endogenous promoter P PGK1 and terminator T HXT7 were amplified by PCR and overlapped with the PahpaB gene to obtain the module P PGK1 - PahpaB -T HXT7 . The pRS415 plasmid was digested with XhoI and BamI restriction endonucleases, and the linearized vector PRS415 and the module P PGK1 - PahpaB -T HXT7 were subjected to Gibson assembly and heat - shocked and transformed into competent Escherichia coli DH5α cells. Amp resistance screening was carried out, and the transformants were picked for preliminary verification by colony PCR. The strains with correct preliminary verification were inoculated into LB liquid medium for activation, and the plasmids were extracted and sequenced for verification. The transformants with correct verification were preserved. The correctly verified plasmid was named pRS415 - P PGK1 - PahpaB -T HXT7 .

[0172] The plasmid pRS416-P was obtained by the same experimental method as described above. TEF2 - SehpaC -T ADH2 , pRS415-P PGK1 - PphpaB -T HXT7 , pRS415-P PGK1 - EnhpaB -T HXT7 , pRS415-P PGK1 - PobA -T HXT7 .

[0173] The plasmids (1) pRS415-P PGK1 - PobA -T HXT7 (2) pRS415-P PGK1 - PahpaB -T HXT7 and pRS416-P TEF2 - SehpaC -T ADH2 (3) pRS415-P PGK1 - PphpaB -T HXT7 and pRS416-P TEF2 - SehpaC -T ADH2 (4) pRS415-P PGK1 - EnhpaB -T HXT7 and pRS416-P TEF2 - SehpaC -T ADH2 were transformed into the ZR05 strain to obtain the strains VA02, VA03, VA04, and VA05.

[0174] Experimental results:

[0175] Through fermentation, using p-hydroxybenzaldehyde as the substrate, the ability of the strains VA02, VA03, VA04, and VA05 to convert p-hydroxybenzaldehyde into protocatechuic aldehyde was verified. As can be seen from the results, the highest yield of protocatechuic aldehyde obtained by fermenting p-hydroxybenzaldehyde with the strain VA03 for 96 h was 0.87 mmol (as Figure 2 shown in Table 2).

[0176] Therefore, select Pseudomonas aeruginosa sourced from PahpaB and Salmonella enterica sourced from SehpaC to complete the hydroxylation process in the pathway.

[0177] Table 2 Screening and fermentation results of the p-hydroxybenzaldehyde-protocatechuic aldehyde pathway

[0178] Strain Protocatechuic aldehyde (mmol / L) VA02 0 VA03 0.87±0.01 VA04 0.29±0.00 VA05 0.33±0.05

[0179] Example 3 Construction and verification of a strain capable of converting protocatechuic aldehyde into vanillin

[0180] Using the ZR05 strain that can stably accumulate vanillin as the chassis, and expressing through a free plasmid Homo sapiens derived from HsCOMT , Arabidopsis thaliana derived from AtCOMT or Nicotiana tabacum derived from NtCOMT , screen the enzymes for the methoxylation process to convert protocatechuic aldehyde into vanillin. The codon-optimized nucleotide sequences are shown in SEQ ID NO:14~SEQ ID NO:16.

[0181] Experimental method:

[0182] Construct plasmids pRS413-P TDH3 - HsCOMT -T TDH1 , pRS413-P TDH3 - NtCOMT -T TDH1 and pRS413-P TDH3 - AtCOMT -T TDH1 and introduce them into yeast ZR05 to obtain strains VA06, VA07 and VA08.

[0183] Considering that caffeic acid will appear in the subsequent fermentation system, when selecting in this step, the catalytic conditions for both protocatechuic aldehyde and caffeic acid as substrates are considered. Through fermentation, using protocatechuic aldehyde and caffeic acid as substrates, verify the ability of the above strains to convert protocatechuic aldehyde into vanillin and caffeic acid into ferulic acid. After 72 h of fermentation, VA06, VA07 and VA08 produced 0.08 mmol / L, 1.22 mmol and 1.42 mmol of vanillin and 0.03 mmol / L, 0.11 mmol and 0.35 mmol of ferulic acid respectively (as Figure 3 and Table 3 show). Therefore, AtCOMT is preliminarily selected to catalyze this step of the reaction.

[0184] Table 3 Screening and fermentation results of the protocatechuic aldehyde-vanillin and caffeic acid-ferulic acid pathways

[0185] Strain Vanillin (mmol / L) Ferulic acid (mmol / L) VA06 0.08±0.00 0.03±0.01 VA07 1.42±0.01 0.11±0.00 VA08 1.22±0.02 0.35±0.01

[0186] Construction of Saccharomyces cerevisiae strain VA09 for the conversion and synthesis of vanillin using ferulic acid and p-coumaric acid as precursors

[0187] After verification in Examples 1 and 3, the roles of the selected enzymes in this pathway were confirmed, so the above genes were integrated. Strain VAN01 already contains Petroselinum crispum from 4CL and Pseudomonas putida from KT2440 Ech genes. These two genes can catalyze the conversion of ferulic acid to vanillin and, as verified by experiments, can also convert p-coumaric acid to p-hydroxybenzaldehyde (as shown in Figure 1 ). On this basis, further expression of Pseudomonas aeruginosa from PahpaB , Salmonella enterica from SehpaC , Arabidopsis thaliana from AtCOMT or Nicotiana tabacum from NtCOMT was achieved to construct the p-coumaric acid - p-hydroxybenzaldehyde - protocatechuic aldehyde - vanillin pathway. The resulting strain was able to convert ferulic acid and p-coumaric acid to vanillin ( Figure 4 ).

[0188] Experimental method:

[0189] The yeast promoter and terminator were amplified by PCR and ligated with each gene by the Overlap PCR method to form module T PGI1 -P PGK1 - PahpaB -T HXT7 , T TDH1 -P TEF2 - SehpaC -T ADH2 and T HXT7 -P TDH3 - AtCOMT -T TDH1 , and sequencing verification was performed; approximately 400 bp sequences on the left and right of the Saccharomyces cerevisiae HO were obtained by PCR and named HO-L and HO-R, respectively. They were overlapped with the terminator T PGI1 and T ADH2 sequences to obtain HO-L-T PGI1 and HO-R-T ADH2 as the linking homologous arms (HO-HAs). A gRNA plasmid (containing the gene encoding the cas9 protein) was constructed according to the HO sequence. The gRNA plasmid, HO-HAs, and module T PGI1 -P PGK1 - PahpaB -T HXT7 , TTDH1 -P TEF2 - SehpaC -T ADH2 and T HXT7 -P TDH3 - AtCOMT -T TDH1 It was introduced into Saccharomyces cerevisiae SyBE_Sc05220038 by the lithium acetate transformation method. The SC screening plate was coated, and the obtained transformants were subjected to streak purification culture, and then the yeast genome was extracted for PCR verification and sequencing. The correctly verified transformants were named VA09.

[0190] Experimental results:

[0191] Through fermentation, using p - coumaric acid and ferulic acid as substrates, the ability of strain VA09 to produce vanillin was verified, and the results are as Figure 5 , shown in Table 4 and Table 5 ( Figure 5 A is 2.49 mmol / L vanillin produced using ferulic acid as the substrate, Figure 5 B is 0.37 mmol / L vanillin produced using p - coumaric acid as the substrate).

[0192] Table 4 Fermentation results of ferulic acid - vanillin

[0193]

[0194] Table 5 Fermentation results of p - coumaric acid - vanillin

[0195]

[0196] Example 5 Construction of Saccharomyces cerevisiae strains VA10 and VA11 with enhanced hydroxylation and methoxylation steps

[0197] From the fermentation results of the above - mentioned strain VA09, it can be seen that when ferulic acid is used as the substrate, the yield and conversion rate of vanillin synthesis are relatively high. However, when p - coumaric acid is used as the substrate, the conversion rate is low, and there is an accumulation of two intermediate products, 0.06 mmol / L p - hydroxybenzaldehyde and 0.06 mmol / L protocatechualdehyde. Therefore, it is necessary to further strengthen the two catalytic processes of hydroxylation and methoxylation. On the basis of strain VA09, by further expressing the fusion of SehpaC -(GGGGS)3- PahpaB and adding one AtCOMT copy to over - express MET13 atsc , SAH1 and MET6 to strengthen the SAM cycle process and strengthen the hydroxylation and methoxylation processes in the vanillin synthesis pathway.

[0198] Experimental method:

[0199] The fusion protein linker sequence GGGGS is encoded by GGTGGTGGTGGTTCT (as shown in SEQ ID NO:17). Using the above module T PGI1 -P PGK1 - PahpaB -T HXT7 and T TDH1 -P TEF2 - SehpaC -T ADH2 as templates, T PGI1 -P PGK1 and PahpaB -T HXT7 were obtained by PCR amplification; T PGI1 -P PGK1 , gene SehpaC , three sets of linker sequences (GGGGS)3 (i.e., GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT) and PahpaB -T HXT7 were ligated into a fragment, named SehpaC -(GGGGS)3- PahpaB . Approximately 400 bp sequences on both the left and right sides of Saccharomyces cerevisiae delta15 were obtained by PCR and named delta15-L and delta15-R, respectively. They were overlapped with the terminator T PGI1 and T ADH1 sequences to obtain delta15-L-T PGI1 and delta15-R-T ADH1 as ligation homologous arms (delta15-HAs). A gRNA plasmid was constructed based on the delta15 sequence. The delta15 gRNA plasmid, delta15-HAs, SehpaC -(GGGGS)3- PahpaB and T HXT7 -P TDH3 - AtCOMT -T TDH1 were co-transformed into VA09 by the lithium acetate transformation method. After being verified successfully by the above method, it was named VA10.

[0200] Modules T CHO1 -P TDH3 -SAH1-T PGI1 , T PGI1 -P GPM1 -MET6-T TEF1 , T TEF1 -T CYC1 and T CYC1 -P PDC1 -MET13atsc -T TDH3 and delta22-L-T CHO1 and delta22-R-T ADH3 (delta22-HAs), the delta22-gRNA plasmid, was transformed into VA10 with lithium acetate and verified to obtain strain VA11.

[0201] Experimental results:

[0202] First, using p-coumaric acid as a substrate, the ability of strain VA10 to convert p-coumaric acid to vanillin was verified. The results are as Figure 6 shown in and Table 6. Compared with VA09, the vanillin production (0.31 mmol / L) did not increase, but new substance accumulation was found in the system, namely caffeic acid (0.32 mmol / L). The appearance of caffeic acid indicates the existence of another synthetic pathway (as Figure 7 shown), that is, the enhanced hydroxylation process diverted the substrate from the p-coumaric acid - p-hydroxybenzaldehyde process, and also indicates the necessity of further enhancing the O-methyltransferase process.

[0203] Table 6 Results of enzyme fusion enhanced hydroxylation fermentation

[0204]

[0205] Therefore, the O-methyltransferase process was further enhanced by strengthening the SAM cycle. The obtained strain VA11 could synthesize 0.54 mmol / L vanillin using p-coumaric acid as a substrate, with almost no accumulation of p-hydroxybenzaldehyde, protocatechuic aldehyde, and caffeic acid (as Figure 8 shown in and Table 7), indicating that the O-methyltransferase process was effectively enhanced.

[0206] Table 7 Results of SAM regulation enhanced O-methyltransferase process

[0207]

[0208] Verification example 1

[0209] Simulating the situation of lignin-released monomers after alkali pretreatment of corn stover, using 1.31 mmol / L ferulic acid and 5.18 mmol / L p-coumaric acid as substrates, the ability of VA11 to synthesize vanillin was verified. 2.23 mmol / L vanillin was obtained by fermentation, and almost no remaining of the two substrates (as Figure 9 shown in and Table 8). Although there are still intermediate products remaining, the simultaneous conversion of the two substrates has been achieved and the yield is the highest value reported for batch fermentation using Saccharomyces cerevisiae as a chassis so far. Therefore, strain VA11 was used to transform lignin to synthesize vanillin.

[0210] Table 8 Fermentation results of p-coumaric acid & ferulic acid - vanillin

[0211]

[0212] Verification Example 2 Pretreatment of Corn Stover and Fermentation of Hydrolysate

[0213] The corn stover was first washed with water, ground after natural air drying, sieved through a 20 - 80 mesh sieve, and the water content was measured. A pretreatment system was prepared according to 100 mg NaOH / g dry weight of the stover and a 10%wt loading. It was placed in an oil bath at 130°C for 30 min, cooled to room temperature with running water, and the solid - liquid separation was carried out to obtain the alkali - pretreated stock solution (APL). The pH was adjusted to 6 and sterilized at 115°C for 10 min. A 20 - mL fermentation system was prepared: 8 mL APL (i.e., 0.4×), 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract powder. The strain AV11 was secondarily activated, and the initial OD600 for fermentation inoculation was 1. It was cultured at 30°C and 220 rpm for 96 h.

[0214] Experimental Results:

[0215] The monomer concentrations in APL after pretreatment were: p - coumaric acid 2.03 g / L and ferulic acid 0.37 g / L. In the initial fermentation system, p - coumaric acid was 820 mg / L (4.99 mmol / L) and ferulic acid was 150 mg / L (0.77 mmol / L). After fermentation with the VA11 strain, 1.35 mmol / L vanillin was obtained, and there was basically no remaining p - coumaric acid and ferulic acid (as Figure 10 shown in Table 9). This result proves the successful realization of the goal of engineering Saccharomyces cerevisiae to convert the lignin component in corn stover into vanillin.

[0216] Table 9 APL - Vanillin Fermentation Results

[0217]

[0218] The above - mentioned is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An enzyme composition, characterized in that, Comprising: Hydroxylase and O-methyltransferase; The hydroxylase is derived from Pseudomonas aeruginosa of PahpaB and derived from Salmonella enterica of SehpaC ; The methoxytransferase is: derived from Arabidopsis thaliana of AtCOMT ; The said PahpaB sequence: the amino acid sequence shown in SEQ ID NO:4; The said SehpaC sequence: the amino acid sequence as shown in SEQ ID NO:5; The AtCOMT sequence: the amino acid sequence as shown in SEQ ID NO:

7.

2. A nucleic acid molecule encoding the enzyme composition according to claim 1, characterized in that, Comprising: The nucleic acid molecule encoding the PahpaB , the nucleic acid molecule encoding the AtCOMT , and the nucleic acid molecule encoding the SehpaC .

3. The nucleic acid molecule according to claim 2, characterized in that, The said PahpaB nucleic acid molecule is: The nucleotide sequence shown in SEQ ID NO:12; The said SehpaC nucleic acid molecule is: The nucleotide sequence shown in SEQ ID NO:13 The said AtCOMT nucleic acid molecule is: The nucleotide sequence shown in SEQ ID NO:

15.

4. Expression cassette, characterized in that, Comprising the nucleic acid molecule according to claim 2 or 3.

5. Expression module, characterized in that, Comprising a first expression module, a second expression module and an endogenous expression module; The first expression module expresses connexin and the PahpaB and the SehpaC ; The second expression module expresses the AtCOMT ; The endogenous expression module includes: MET13 atsc , SAH1 and MET6.

6. The expression module according to claim 5, wherein, The copy number of the said linker protein is not less than 3; the copy number of the second expression module is not less than 2; the endogenous expression module is overexpressed.

7. A strain, characterized in that, Introducing the expression cassette according to claim 4 and / or the expression module according to claim 5 or 6 into a chassis strain; the chassis strain is Saccharomyces cerevisiae.

8. Use of the enzyme composition according to claim 1, the nucleic acid molecule according to claim 2 or 3, the expression cassette according to claim 4, the expression module according to claim 5 or 6 and / or the strain according to claim 7 in the synthesis of vanillin.

9. A method for synthesizing vanillin, characterized in that, Taking the strain according to claim 7, culturing it, mixing it with lignin or the original solution of lignin hydrolysis solution, and fermenting it to obtain the said vanillin.

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