Caffeic acid o-methyltransferase mutants and uses thereof

Abstract

The application provides a caffeic acid O-methyltransferase (COMT) mutant and application thereof. The inventors find a site closely related to catalytic activity in an amino acid sequence of COMT, and on this basis, disclose a COMT mutant and application thereof in catalyzing O-methylation reaction. The mutant can participate in a vanillin biosynthesis pathway and improve the yield of vanillin.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and plant biology, and more specifically, this invention relates to caffeic acid O-methyltransferase mutants and their applications. Background Technology

[0002] O-methyltransferases (OMTs) are a class of enzymes that use a methyl donor, such as S-adenosylmethionine (SAM), to replace the hydroxyl group of the substrate with a methoxy group. There are many types of OMTs, typically distinguished by the size of the protein molecular weight and whether magnesium is required for the catalytic reaction. 2+ They can be divided into two categories: Category I OMTs have a molecular weight of 26-30 kDa, and the reaction process requires Mg. 2+ Furthermore, their substrate profiles are relatively simple. For example, caffeoyl-CoA O-methyltransferase mainly participates in the synthesis of plant lignin, converting caffeoyl-CoA to feruloyl-CoA. Class II OMTs proteins have a molecular weight of 38-43 kDa, are mostly dimers, and do not require the participation of metal cations in the reaction. Their substrates are diverse and varied. For example, Arabidopsis thaliana caffeic acid O-methyltransferase (AtCOMT) belongs to the class II O-methyltransferases. This enzyme mainly participates in lignin synthesis, O-methylating caffeic acid to ferulic acid. It can catalyze a series of compounds containing phenylpropane-like structures and does not require metal cations as coenzymes. Comparative analysis with isozymes from different species revealed the following mechanism of AtCOMT-catalyzed substrate O-methylation: First, H267 abstracts a proton from the phenolic hydroxyl group, deprotonating it to form a phenolic anionic group. Subsequently, the newly generated anionic group nucleophilically attacks the active methyl group of the coenzyme SAM. Through a bimolecular nucleophilic substitution reaction (SN2), the substrate anionic group replaces the methyl group of SAM, ultimately forming a methoxy group, completing the O-methylation reaction. AtCOMT has a broad substrate spectrum, involving more than ten substrates including caffeic acid, N-acetyl-5-hydroxytryptamine (NAS), resveratrol, erythromycin, guaiacol, methyl salicylate, and p-hydroxybenzaldehyde, but its protein crystal structure has not yet been resolved. Summary of the Invention

[0003] The purpose of this invention is to provide a caffeic acid O-methyltransferase mutant and its application.

[0004] In a first aspect of the invention, a caffeic acid O-methyltransferase (COMT) mutant is provided, the mutant comprising: (1) a protein whose amino acid sequence corresponds to the 310th position of SEQ ID NO:2; or (2) a conserved variant of the protein (1), wherein the amino acid corresponding to the 310th position of SEQ ID NO:2 is the same as the amino acid mutated at the corresponding position of the protein (1).

[0005] In one or more embodiments, the conserved variant protein includes a protein selected from: (a) a protein formed by substituting, deleting or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequence of protein (1), and having the function of protein (1); (b) a protein having more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology to the amino acid sequence of protein (1), and having the function of protein (1); (c) an active fragment of protein (1) containing a structure in the spatial structure of caffeic acid O-methyltransferase that interacts with a methyl donor and a methyl acceptor, and having the function of protein (1); or (d) a protein formed by adding a tag sequence, a signal sequence or a secretion signal sequence to both ends of any of the proteins (1) or (2).

[0006] In one or more embodiments, the methyl donor includes S-adenosylmethionine.

[0007] In one or more embodiments, the methyl receptor comprises: a compound having a phenolic hydroxyl group; preferably, the methyl receptor comprises (but is not limited to): ferulic acid, N-acetyl-5-hydroxytryptamine (NAS), resveratrol, erythromycin, guaiacol, methyl salicylate, p-hydroxybenzaldehyde, phenethyl alcohol, UV-O, 2-decaisoprene-3-methyl-5-hydroxy-6-methoxy-hydroquinone.

[0008] In one or more embodiments, the 310th position is mutated to Gly(G); preferably, the amino acid sequence of the mutant is shown in SEQ ID NO:4.

[0009] In another aspect of the invention, an isolated polynucleotide is provided that encodes the caffeic acid O-methyltransferase mutant.

[0010] In another aspect of the invention, a carrier or a host cell containing the carrier is provided, the carrier containing the aforementioned polynucleotide.

[0011] In one or more embodiments, the cell includes a ferulic acid synthesis pathway gene, thereby enabling the cell to synthesize ferulic acid; preferably, the ferulic acid synthesis pathway gene includes a gene encoding the following enzymes: tyrosine deaminase (TAL), p-coumaric acid 3-hydroxylase (SAM5), and the aforementioned caffeic acid O-methyltransferase (COMT) mutant; preferably, the tyrosine deaminase is derived from Rhodotorula glutinis, and the p-coumaric acid 3-hydroxylase is derived from Saccharothrix espanaensis.

[0012] In one or more embodiments, the cell further includes a vanillin synthesis pathway gene, thereby enabling the cell to synthesize vanillin; preferably, the vanillin synthesis pathway gene includes: a gene encoding ferulic acid decarboxylase and dioxygenase; preferably, the gene encoding ferulic acid decarboxylase is the fdc gene; preferably, the gene encoding dioxygenase is the cso2 gene; preferably, the fdc gene is from Bacillus pumilus, and the cso2 gene is from Caulobacter segnis; preferably, the cso2 gene is a mutant gene that encodes an enzyme with mutations A49P and Q390A (cso2(A49P-Q390A)).

[0013] In one or more embodiments, the host cell includes: prokaryotic cells or eukaryotic cells; preferably, the prokaryotic host cell includes Escherichia coli, Bacillus subtilis, etc.; the eukaryotic host cell includes: fungal cells, yeast cells, insect cells, mammalian cells, etc.

[0014] In one or more embodiments, the host cell is Escherichia coli.

[0015] In another aspect of the present invention, a method for producing the caffeic acid O-methyltransferase mutant is provided, comprising the steps of: (1) culturing the host cells to obtain a culture; and (2) isolating the caffeic acid O-methyltransferase mutant from the culture.

[0016] In another aspect of the present invention, a method for improving the catalytic activity of caffeic acid O-methyltransferase is provided, comprising: mutating the amino acid corresponding to position 310 of SEQ ID NO:2 in its amino acid sequence; preferably, the position 310 is mutated to Gly; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO:4.

[0017] In one or more embodiments, the improvement in catalytic activity is a statistically significant improvement, such as an increase of 20% or more, 40% or more, 60% or more, 70% or more, or higher.

[0018] In another aspect of the invention, the application of any of the caffeic acid O-methyltransferase mutants described above is provided for catalyzing substrate O-methylation reactions; or for preparing reagents (including catalytic reagents) or culture media for catalyzing substrate O-methylation reactions.

[0019] In one or more embodiments, the substrate of the caffeic acid O-methyltransferase mutant includes a compound containing a phenolic hydroxyl group.

[0020] In one or more embodiments, the substrate includes (but is not limited to): caffeic acid, N-acetyl-5-hydroxytryptamine (NAS), resveratrol, erythromycin, guaiacol, methyl salicylate, p-hydroxybenzaldehyde, phenethyl alcohol, UV-O, 2-decaisoprene-3-methyl-5-hydroxy-6-methoxy-hydroquinone.

[0021] In one or more embodiments, during catalysis, a methyl group is obtained from a methyl donor; preferably, the methyl donor comprises S-adenosylmethionine.

[0022] In one or more embodiments, the substrate includes ferulic acid, which, as an intermediate, can further form vanillin in cells.

[0023] In one or more embodiments, the cells include genes for the ferulic acid synthesis pathway and / or genes for the vanillin synthesis pathway.

[0024] In one or more embodiments, the ferulic acid synthesis pathway gene includes a gene encoding the following enzymes: tyrosine deaminase, p-coumaric acid 3-hydroxylase, and the caffeic acid O-methyltransferase mutant; preferably, the tyrosine deaminase is derived from Rhodotorula glutinis, and the p-coumaric acid 3-hydroxylase is derived from Saccharothrix espanaensis;

[0025] In one or more embodiments, the vanillin synthesis pathway gene includes: genes encoding ferulic acid decarboxylase and dioxygenase; preferably, the gene encoding ferulic acid decarboxylase is the fdc gene; preferably, the gene encoding dioxygenase is the cso2 gene; preferably, the fdc gene includes the fdc gene from Bacillus pumilus, and the cso2 gene includes the cso2 gene from Caulobacter segnis; preferably, the cso2 gene is a mutant gene that encodes an enzyme with mutations A49P and Q390A (cso2(A49P-Q390A)).

[0026] In another aspect of the invention, a composition for catalyzing O-methylation reactions is provided, the composition comprising: any of the preceding caffeic acid O-methyltransferase mutants; or the aforementioned host cell.

[0027] In one or more embodiments, the composition further includes a pharmaceutically or industrially acceptable carrier.

[0028] In another aspect of the invention, a kit for O-methylation of a substrate is provided, comprising: any of the preceding caffeic acid O-methyltransferase mutants; any of the preceding host cells; or the preceding compositions.

[0029] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0030] Figure 1 AtCOMT agarose gel electrophoresis images and sequencing alignment results.

[0031] Figure 2 a. Schematic diagram of vanillin biosynthesis pathway.

[0032] Figure 2 b. The fermentation products involved by the AtCOMT mutant.

[0033] Figure 2 c. HPLC analysis of vanillin synthesis using an artificially constructed recombinant Escherichia coli system. Detailed Implementation

[0034] Through in-depth research, the inventors identified a site in the amino acid sequence of caffeic acid O-methyltransferase (COMT) that is closely related to its catalytic activity. Based on this, they revealed a COMT mutant and its application in catalyzing O-methylation reactions. This mutant can participate in the vanillin biosynthesis pathway and increase vanillin yield.

[0035] mutant

[0036] As used herein, unless otherwise stated, the terms "caffeic acid O-methyltransferase mutant," "COMT mutant," "mutant caffeic acid O-methyltransferase," and "mutant COMT" are used interchangeably and refer to a polypeptide corresponding to wild-type caffeic acid O-methyltransferase formed by a mutation at position 310 of its corresponding amino acid sequence. Preferably, such mutation is Q310G.

[0037] If it is necessary to represent the wild-type COMT, it can be a protein with an amino acid sequence such as SEQ ID NO:2, or it can be a homologous variant or active fragment of the protein. Preferably, the wild-type COMT is derived from Arabidopsis thaliana; however, it should be understood that the present invention also covers COMT homologs derived from other species that are homologous and functionally identical.

[0038] As used herein, "isolated COMT" means that the COMT mutant is substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify the COMT mutant using standard protein purification techniques. Substantially pure protein produces a single master band on a non-reducing polyacrylamide gel.

[0039] The proteins of this invention can be recombinant proteins, natural proteins, or synthetic proteins, with recombinant proteins being preferred. The proteins of this invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology.

[0040] This invention also includes fragments, derivatives, and analogs of the COMT mutant. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the natural COMT mutant of this invention. The protein fragments, derivatives, or analogs of this invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing an additional amino acid sequence to this protein sequence (such as a leader sequence or secretory sequence or a sequence used to purify this protein or a proteogenic sequence, or a fusion protein). These fragments, derivatives, and analogs are within the scope well known to those skilled in the art as defined herein. However, the amino acid sequences of the COMT mutant and its fragments, derivatives, and analogs certainly contain the mutations described above in this invention; preferably, the mutation corresponds to amino acid position 310 in SEQ ID NO:2.

[0041] In this invention, the term "COMT mutant" also includes (but is not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-20, more preferably 1-10, and even more preferably 1-8, 1-5, 1-3, or 1-2), and the addition or deletion of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition or deletion of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the COMT mutant. However, these variant forms certainly contain the mutations described above in this invention; preferably, the mutation corresponds to amino acid position 310 in SEQ ID NO:2.

[0042] In this invention, the term "COMT mutant" also includes (but is not limited to): derived proteins that retain their protein activity and have at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, such as at least 98% or 99%, sequence identity with the amino acid sequence of the said COMT mutant. Similarly, these derived proteins certainly contain the mutation described above in this invention, preferably a mutation corresponding to amino acid position 310 in SEQ ID NO:2.

[0043] The present invention also provides a multinucleotide sequence encoding the COMT mutant of the present invention or a conserved variant thereof.

[0044] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0045] The polynucleotide encoding the mature protein of the mutant includes: a coding sequence that encodes only the mature protein; a coding sequence for the mature protein and various additional coding sequences; a coding sequence for the mature protein (and optional additional coding sequences) and a non-coding sequence.

[0046] "A polynucleotide encoding a protein" can be a polynucleotide that includes the protein itself, or it can include polynucleotides that also include additional coding and / or non-coding sequences.

[0047] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or COMT mutant coding sequences, and methods for generating the proteins of the present invention via recombinant technology.

[0048] In this invention, the COMT mutant polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0049] Methods well known to those skilled in the art can be used to construct expression vectors containing a COMT mutant-encoding DNA sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0050] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0051] In this invention, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include: *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, and *Agrobacterium*; eukaryotic cells such as yeast and plant cells. In a specific embodiment of this invention, *Escherichia coli* is used as the host cell.

[0052] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0053] Recombinant COMT mutants can be expressed or produced using the polynucleotide sequences of this invention through conventional recombinant DNA techniques. Generally, the following steps are involved:

[0054] (1). Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the COMT mutant of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0055] (2) Host cells cultured in a suitable culture medium;

[0056] (3) Isolate and purify proteins from culture media or cells.

[0057] In a specific embodiment of the present invention, the COMT mutant is expressed and purified in *E. coli*: the wild-type COMT gene is amplified from the cloning vector and subcloned into a prokaryotic expression vector by PCR. Using the COMT gene expression vector as a template, a mutation is introduced through PCR primers, and after PCR amplification, a mutant protein expression vector is constructed. The correctly sequenced mutant is heterologously expressed in *E. coli*. After purification, the protein is subjected to in vitro enzyme activity testing or applied in other areas.

[0058] As a preferred embodiment of the present invention, the specific steps of the expression method are as follows:

[0059] Construction of engineered bacteria: The coding genome of the COMT mutant was inserted into a plasmid for the synthesis of ferulic acid and a plasmid for the key functional gene for the synthesis of ferulic acid to vanillin, and co-transformed into Escherichia coli BL21(DE3) to obtain engineered Escherichia coli containing the coding gene of the COMT mutant;

[0060] Fermentation with engineered bacteria: Using 1 g / L L-tyrosine as a precursor, the constructed *E. coli* engineered bacteria were cultured in TB medium at 37℃ and 250 rpm until OD = 0.6. IPTG (final concentration 0.1 mM) was then added and fermentation was induced at 22℃ for 5 days to obtain the fermentation broth. 500 μL of the fermentation broth was extracted twice with an equal volume of ethyl acetate. The supernatant was transferred to a clean EP tube, concentrated under vacuum until dry, and reconstituted with 100 μL of methanol for HPLC analysis.

[0061] Application of mutants

[0062] Based on the new findings of this invention, applications of the COMT mutant are provided for catalyzing O-methylation reactions of substrates; or for preparing reagents (including catalytic reagents) or culture media for catalyzing O-methylation reactions of substrates.

[0063] Given that COMT is a methyltransferase capable of catalyzing O-methylation of substrates and has a relatively broad range of substrates (compounds containing phenolic hydroxyl groups); for example, said substrates include (but are not limited to): ferulic acid, N-acetyl-5-hydroxytryptamine (NAS), resveratrol, erythromycin, guaiacol, methyl salicylate, p-hydroxybenzaldehyde, etc.; it is understood that mutant COMT also has the same function as wild-type COMT, and also has a relatively broad range of substrates and catalyzes O-methylation of them.

[0064] As a preferred embodiment of the present invention, the COMT mutant can serve as a methyltransferase in the vanillin synthesis pathway.

[0065] Vanillin, 3-methoxy-4-hydroxybenzaldehyde, also known as vanillin, is a widely used edible flavoring agent. It was successfully synthesized by Dr. M. Harman and Dr. G. Teymann in 1874, becoming the first synthetic flavoring agent. Its molecular formula is C8H8O3, molecular weight is 152.15, density is 1.056, melting point is 81-82℃, boiling point is 285℃, it has a slightly sweet taste, and is soluble in hot water, ethanol, ether, glycerol, and alcohol. It is not easily soluble in cold water and vegetable oils. Its aqueous solution reacts with ferric chloride to turn blue-purple. It is easily oxidized in air and discolors easily in alkaline substances. Natural vanillin is found in tobacco leaves, asparagus, coffee, and vanilla fragrans (Salisb.) Ames. It is one of the most widely used food flavoring agents globally and is known as the "King of Food Flavors." Vanillin is commonly used in infant formula and cereal products, and can also be used as a plant growth promoter, bactericide, and defoamer for lubricants. It is an important intermediate in the synthesis of pharmaceuticals and other fragrances. Currently, vanillin is mainly prepared through chemical synthesis, but the resulting products have a relatively simple aroma, lacking the complex aroma of natural vanillin, and are prone to causing environmental pollution. In natural vanillin pods, vanillin is the most important aroma component. Because vanilla pod plants have very high requirements for soil and climate factors, and the natural fermentation process is complex, the natural source of vanillin is very limited. Vanillin products extracted from natural plants account for less than 1% of global production. Fresh vanillin pods do not have that sweet aroma because the vanillin they contain is not free, but exists in the form of glycosides, and the aroma cannot be released at this time. After a long process of sun-drying and fermentation, vanilla pods finally acquire their aroma.

[0066] With the development of metabolic engineering and synthetic biology, many researchers are constructing artificial biological systems to understand and develop plant natural products. Simulating and optimizing natural synthetic pathways within microorganisms has become increasingly important and widespread. In a preferred embodiment of this invention, the inventors assembled the vanillin pathway in *E. coli* to simulate the natural synthetic pathway in plants, and optimized the fermentation pathway using a series of metabolic engineering techniques, making heterologous and efficient vanillin synthesis in *E. coli* possible. The enzyme activity detection method for mutant COMT can utilize techniques known in the art. For example, the same enzyme reaction system can be used to test the in vitro enzyme activity of different mutant proteins. After the reaction, product formation is detected by HPLC, and the changes in catalytic activity of the mutant are analyzed by comparing the relative conversion rates of the mutant and wild-type to the substrate.

[0067] In a preferred embodiment of the present invention, the caffeic acid O-methyltransferase AtCOMT from Arabidopsis thaliana (L.) Heynh is used as the parent, and the glutamine Gln(Q) site at the critical amino acid residue 310 is mutated to glycine Gly(G) to improve the catalytic activity of caffeic acid compounds. The modified caffeic acid O-methyltransferase COMT mutant is applied to an artificially constructed recombinant Escherichia coli system. This recombinant microorganism can produce vanillin, and its in vivo fermentation efficiency for vanillin production is 1.6 times that of the wild type, exhibiting better catalytic activity and making it more suitable for the industrial production of vanillin.

[0068] The mutant COMT of the present invention, or host cells expressing the mutant COMT, can be formulated in pharmaceutically or industrially acceptable vectors to obtain compositions suitable for catalytic reactions or for storage. As a preferred embodiment of the present invention, the host cells further include other ferulic acid synthesis pathway genes and / or vanillin synthesis pathway genes; preferably, the ferulic acid synthesis pathway genes include: Rgtal, sam5, and AtCOMT; preferably, the vanillin synthesis pathway genes include: fdc and cso2(A49P-Q390A).

[0069] The mutant COMT of the present invention or host cells containing it (preferably also containing other ferulic acid synthesis pathway genes and / or vanillin synthesis pathway genes) can also be included in the kit for use or sale. Generally, the kit also includes instructions for use.

[0070] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0071] Materials and Instruments

[0072] Arabidopsis cDNA was obtained from Arabidopsis plants. Oligonucleotide primers, antibiotics, and ethidium bromide (EB) nucleic acid dye were purchased from Sangon Biotech Ltd. Polymerase chain reaction (PCR) gel recovery kits and plasmid extraction kits were products of the United States; PrimeSTAR Max DNA Polymerase, a high-fidelity PCR enzyme, was a product of Takara Bio Inc., Japan. The II One Step Cloning Kit is a Vazyme product. Escherichia coli DH10B and BL21(DE3) strains and pET21a and pET28a vectors are used for gene cloning, protein expression, and fermentation assays.

[0073] Vanillin, a standard compound, was purchased from Shanghai Runjie Chemical Reagent Co., Ltd.; ferulic acid, a standard compound, was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; caffeic acid, a standard compound, was purchased from Ailan Shanghai Chemical Technology Co., Ltd.; and p-coumaric acid, a standard compound, was purchased from Shanghai Yuanye Biotechnology Co., Ltd. L-tyrosine was purchased from Sangon Biotech Co., Ltd. Other reagents were domestically produced analytical grade or chromatographic grade reagents, purchased from Sinopharm Chemical Reagent Co., Ltd. Chromatographic separation was performed using a C18 column (Silgreen HPLC column 250×4.6mm).

[0074] PCR was performed using an Arktik Thermal Cycler (Thermo Fisher Scientific); isothermal incubation was performed using a ZXGP-A2050 incubator (Zhicheng) and a ZWY-211G isothermal shaker (Zhicheng); centrifugation was performed using a 5418R high-speed refrigerated centrifuge and a 5418 mini centrifuge (Eppendorf). Vacuum concentration was performed using a Concentrator Plus concentrator (Eppendorf); OD 600Detection was performed using a UV-1200 UV-Vis spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.). The rotary evaporation system consisted of an IKA RV 10-digital rotary evaporator (IKA), an MZ 2C NT chemical diaphragm pump, and a CVC3000 vacuum controller (vacuubrand). High-performance liquid chromatography (HPLC) was performed using a Dionex UltiMate 3000 HPLC system (ThermoFisher Scientific).

[0075] The culture media and solutions used in subsequent examples are shown in Table 1.

[0076] Table 1

[0077]

[0078] The AtCOMT nucleotide sequence involved in subsequent embodiments is as shown in SEQ ID NO:1:

[0079] AtCOMT

[0080]

[0081] The AtCOMT amino acid sequence involved in subsequent embodiments is as shown in SEQ ID NO:2:

[0082] AtCOMT

[0083] MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPMSPTEIASKLPTKNPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVERIYGLGPVCKYLTKNEDGVSIAALCLMNQDKVLMESWYHLKDAILDGGIPFNKAYGMSAFEYHGTDPRFNKVFNNGMSNHS TITMKKILETYKGFEGLTSLVDVGGGIGATLKMIVSKYPNLKGINFDLPHVIEDAPSHPGIEHVGGDMFVSVPKGDAIFMKWICHDWSDEHCVKFLKNCYESLPEDGKVILAECILPETPDSSLSTKQVVHVDCIMLAHNPGGKERTEKEFEALAKASGFKGIKVVCDAFGVNLIELLKKL*

[0084] The AtCOMT(Q310G) mutant nucleotide sequence involved in the following examples is shown in SEQ ID NO:3:

[0085] AtCOMT(Q310G)

[0086] atgggttcaacggcagagacacaattaactccggtgcaagtcaccgacgacgaagctgccctcttcgccatgcaactagccagtgcttccgttcttccgatggctttaaaatccgccttagagcttgaccttcttgagattatggccaagaatggttctcccatgtctcctaccgagatcgcttctaaacttccgaccaaaaatcctgaagctccggtcatgctcgaccgtatcctccgtcttcttacgtcttactccgtcttaacctgctccaaccgtaaactttccggtgatggcgttgaacggatttacgggcttggtccggtttgcaagtatttgaccaagaacgaagatggtgtttccattgctgctctttgtcttatgaaccaagacaaggttctcatggaaagctggtaccatttgaaggatgcaattcttgatggtgggattccattcaacaaggcttatggaatgagcgcgttcgagtaccacgggactgaccctagattcaacaaggtctttaacaatggaatgtctaaccattccacaatcaccatgaagaagattcttgagacctataagggttttgaaggattgacttctttggttgatgttggtggtggcattggtgctacactcaaaatgattgtctccaagtaccctaatcttaaaggcatcaactttgatctcccacatgtcatcgaagatgctccttctcatcctggtattgagcatgttggaggagatatgtttgtaagtgtccctaaaggtgatgccatattcatgaagtggatatgtcatgactggagtgacgaacattgcgtgaaattcttgaagaactgctacgagtcacttccagaggatggaaaagtgatattagcagagtgtatacttccagagacaccagactcaagcctctcaaccaaa ggagtagtccatgtcgattgcattatgttggctcacaatcccggaggcaaagaacgaaccgagaaagagtttgaggcattagccaaagcatcaggcttcaagggcatcaaagttgtctgcgacgcttttggtgttaaccttattgagttatcaagaagctctaa

[0087] The amino acid sequence of the AtCOMT mutant (Q310G) involved in the following examples is shown in SEQ ID NO:4:

[0088] AtCOMT(Q310G)

[0089] MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPMSPTEIASKLPTKNPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVERIYGLGPVCKYLTKNEDGVSIAALCLMNQDKVLMESWYHLKDAILDGGIPFNK AYGMSAFEYHGTDPRFNKVFNNGMSNHSTITMKKILETYKGFEGLTSLVDVGGGIGATLKMIVSKYPNLKGINFDLPHVIEDAPSHPGIEHVGGDMFVSVPKGDAIFMKWICHDWSDEHCVKFLKNCYESLPEDGKVILAECILPETPDSSLSTK G VVHVDCIMLAHNPGGKERTEKEFEALAKASGFKGIKVVCDAFGVNLIELLKKL*

[0090] The RgTAL nucleotide sequence involved in the following examples is as shown in SEQ ID NO:5:

[0091] >RgTAL

[0092]

[0093] The RgTAL amino acid sequence involved in the following examples is shown in SEQ ID NO: 6:

[0094] >RgTAL

[0095] MAPRPTSQNQYRTCPTTQVTQVDIVEKMLAAPTDSTLELDGYSLNLGDVVSAARKGRPVRVKDSDEIRSKIDKSVEFLRSQLSMSVYGVTTGFGGSADTRTEDAISLQKALLEHQLCGVLPSSFDSFRLGRGLENSLPLEVVRGAMTIRVNSLTRGHSAVRLVVLEALTNFLNHGITPIVPLRGTISASGDLSPLSYIAAAISGHPDSKVHVVHEGKEKILYAREAMALFNLEPVVLGPKEGLGLVNGTAVSASMATLALHDAHMLSLLSQSLTAMTVEAMVGHAGSFHPFLHDVTRPHPTQIEVAGNIRKLLEGSRFAVHHEEEVKVKDDEGILRQDRYPLRTSPQWLGPLVSDLIHAHAVLTIEAGQSTTDNPLIDVENKTSHHGGNFQAAAVANTMEKTRLGLAQIGKLNFTQLTEMLNAGMNRGLPSCLAAEDPSLSYHCKGLDIAAAAYTSELGHLANPVTTHVQPAEMANQAVNSLALISARRTTESNDVLSLLLATHLYCVLQAIDLRAIEFEFKKQFGPAIVSLIDQHFGSAMTGSNLRDELVEKVNKTLAKRLEQTNSYDLVPRWHDAFSFAAGTVVEVLSSTSLSLAAVNAWKVAAAESAISLTRQVRETFWSAASTSSPALSYLSPRTQILYAFVREELGVKARRGDVFLGKQEVTIGSNVSKIYEAIKSGRINNVLLKMLA*

[0096] The sam5 nucleotide sequence involved in the following examples is shown in SEQ ID NO: 7:

[0097] >sam5

[0098]

[0099] The sam5 amino acid sequence involved in the following examples is as shown in SEQ ID NO:8:

[0100] >sam5

[0101] MTITSPAPAGRLNNVRPMTGEEYLESLRDGREVYIYGERVDDVTTHLAFRNSVRSIARLYDVLHDPASEGVLRVPTDTGNGGFTHPFFKTARSSEDLVAAREAIVGWQRLVYGWMGRTPDYKAAFFGT LDANAEFYGPFEANARRWYRDAQERVLYFNHAIVHPPVDRDRPADRTADICVHVEEETDSGLIVSGAKVVATGSAMTNANLIAHYGLPVRDKKFGLVFTVPMNSPGLKLICRTSYELMVATQGSPFDY PLSSRLDENDSIMIFDRVLVPWENVFMYDAGAANSFATGSGFLERFTFHGCTRLAVKLDFIAGCVMKAVEVTGTTHFRGVQAQVGEVLNWRDVFWGLSDAMAKSPNSWVGGSVQPNLNYGLAYRTFMG VGYPRIKEIIQQTLGSGLIYLNSSAADWKNPDVRPYLDRYLRGSRGIQAIDRVKLLKLLWDAVGTEFAGRHELYERNYGGDHEGIRVQTLQAYQANGQAAALKGFAEQCMSEYDLDGWTRPDLINPGT*

[0102] The fdc nucleotide sequence involved in the following examples is as shown in SEQ ID NO:9:

[0103] >fdc

[0104] atggatcagtttgttggtctgcacatgatctatacctatgaaaatggttgggaatatgaaatctatatcaagaatgaccacaccattgattatcgcattcatagcggtatggttggcggccgctgggttcgtgatcaggaagtgaatattgttaaactgaccaaaggcgtgtataaagtgagctggaccgaaccgaccggtaccgatgtgagtctgaattttatgccggaagaaaaacgcatgcatggcgtgattttctttccgaaatgggttcatgaacgtccggatattaccgtgtgttatcagaatgattatatcgatctgatgaaggaaagccgcgaaaaatatgaaacctatccgaaatatgtggttccggaatttgccgatattacctatattcatcatgccggcgttaatgatgaaaccattattgcagaagcaccgtatgaaggtctgaccgatgaaattcgcgcaggccgtaaataa

[0105] The fdc amino acid sequence involved in the following examples is set forth in SEQ ID NO: 10:

[0106] >fdc

[0107] MDQFVGLHMIYTYENGWEYEIYIKNDHTIDYRIHSGMVGGRWVRDQEVNIVKLTKGVYKVSWTEPTGTDVSLNFMPEEKRMHGVIFFPKWVHERPDITVCYQNDYIDLMKESREKYETYPKYVVPEFADITYIHHAGVNDETIIAEAPYEGLTDEIRAGRK*

[0108] The cso2 nucleotide sequence involved in the following examples is set forth in SEQ ID NO: 11:

[0109] >cso2

[0110]

[0111] The cso2 amino acid sequence involved in the following examples is set forth in SEQ ID NO: 12:

[0112] >cso2

[0113] MTARFPNTREFTGALYRPSRFEGEVFDLEVDGQLPTDIDGTFFSVAPDAAFPPMREDDIFFNGDGAVSAFRFGGGHVDFQRRYVRTQRLEAQRAARRSLHGVYRNPSTNDPSVLGLNNSTANTNVLEHAGVLLAMKEDSLPYALDPLTLETKGLWNFGGQLTDAPFTAHPKIDPLTGDMIAFGYEARGDGSRDIVYYEFDEHGAKTREIWVQAPVSAMVHDFAVTERFVVFPIIPLSVDVERLRQGGRHFQWQPDLPQYFGVMRRDGDGGDLHWFTAPNGFQGHTLNAFDDGEKVYADMTSTNGNVFYFFPPADGFVPSPETLVSQLVRWTFDLSVKGGRLDMSPLTPFPAEFPRIDDRVALRPHRHGWMMAMDPTKPYAEDRVGPRPFAFFNQLAHLNIATGKIQTWFADEASCFQEPVFVPRTGSSREGDGYLLSLVNRLDERTTDMVVLDALRLGEGPVATVKLPLRMRMALHGNWSRAVSPSSIKAV*

[0114] The cso2(A49P-Q390A) nucleotide sequence involved in the following examples is set forth in SEQ ID NO: 13:

[0115] >cso2(A49P-Q390A)

[0116] atgaccgctcgttttccaaacacccgtgaatttactggcgcactgtatcgcccatctcgctttgaaggtgaagttttcgacctggaagtggatggtcagctgccgactgacattgatggtaccttcttctccgtagctccagat ccg gcg ttcttcaaccagctggctcatctgaacatcgctaccggcaaaatccagacttggttcgccgacgaagcgtcctgttttcaggaacctgtgttcgtcccgcgtaccggctcttcccgtgaaggtgacggttatctgctgtctctggttaaccgcctggacgaacgcaccacggacatggttgttctggatgcactgcgtctgggtgaaggtccggttgctaccgttaaactgccgctgcgtatgcgtatggctctgcacggcaactggtcccgtgcggtttctccgagctccatcaaagcggtgtaa

[0117] The amino acid sequence of cso2(A49P-Q390A) involved in the following examples is shown in SEQ ID NO:14:

[0118] >cso2(A49P-Q390A)

[0119] MTARFPNTREFTGALYRPSRFEGEVFDLEVDGQLPTDIDGTFFSVAPD P AFPPMREDDIFFNGDGAVSAFRFGGGHVDFQRRYVRTQRLEAQRAARRSLHGVYRNPSTNDPSVLGLNNSTANTNVLEHAGVLLAMKEDSLPYALDPLTLETKGLWNFGGQLTDAPFTAHPKIDPLTGDMIAFGYEARGDGSRDIVYYEFDEHGAKTREIWVQAPVSAMVHDFAVTERFVVFPIIPLSVDVERLRQGGRHFQWQPDLPQYFGVMRRDGDGGDLHWFTAPNGFQGHTLNAFDDGEKVYADMTSTNGNVFYFFPPADGFVPSPETLVSQLVRWTFDLSVKGGRLDMSPLTPFPAEFPRIDDRVALRPHRHGWMMAMDPTKPYAEDRVGPRPF A FFNQLAHLNIATGKIQTWFADEASCFQEPVFVPRTGSSREGDGYLLSLVNRLDERTTDMVVLDALRLGEGPVATVKLPLRMRMALHGNWSRAVSPSSIKAV*

[0120] Example 1: Cloning of the AtCOMT gene

[0121] Primers AtCOMT-F / R were designed based on the AtCOMT DNA sequence (SEQ ID NO:1 and its upstream and downstream sequences in Arabidopsis thaliana) (as shown in Table 2). PCR amplification was performed using AtCOMT-F / R primers and Arabidopsis thaliana cDNA as a template (reaction system and conditions are shown in Table 3), followed by 1.0% agarose gel electrophoresis (results are shown in Table 3). Figure 1 This yields the strip F-AtCOMT, which is the same size as the target.

[0122] Then, using infu-AtCOMT-F / R as primers and F-AtCOMT as template, PCR amplification was performed (reaction system and conditions are the same as in Table 3), followed by 1.0% agarose gel electrophoresis (results are shown in Table 3). Figure 1 F1-infu-AtCOMT, containing the pET28a homologous arm, was obtained, matching the target size. F1-infu-AtCOMT was then subjected to infusion homologous recombination with the double-digested (NcoI & BamHI) pET28a linear vector (see Table 4 for the infusion homologous recombination reaction system and conditions). This was transformed into DH10B competent cells, and single clones were selected for sequencing. Clone 1 was found to be correct and named pZYL20-pET28a-AtCOMT. Its sequence matched the NCBI sequence, thus yielding the full-length AtCOMT gene.

[0123] Table 2. Primers used for AtCOMT gene cloning

[0124]

[0125] Table 3. PCR reaction system and conditions

[0126]

[0127] Table 4. Ligation System and Conditions for Seamless DNA Cloning Technology

[0128]

[0129] Example 2: AtCOMT site-directed mutagenesis

[0130] The mutant AtCOMT(Q310G) was constructed using site-directed mutagenesis technology. First, site-directed mutagenesis primers were designed (as shown in Table 5). Using the pZYL20-pET28a-AtCOMT plasmid as a template, PCR amplification was performed (reaction system and conditions as shown in Table 3), followed by 1.0% agarose gel electrophoresis (results are shown in Table 5). Figure 1The resulting band F128 was consistent with the target size. The gel-recovered product was transformed into DH10B competent cells, and single clones were selected for sequencing, namely pZYL128: pET28a-AtCOMT(Q310G).

[0131] Table 5. Primers used for AtCOMT site-directed mutagenesis

[0132]

[0133] Example 3: Assembly of AtCOMT (Q310G) into the vanillin synthesis pathway

[0134] The inventors analyzed the synthetic pathway of vanillin, which requires five enzymatic reactions: TAL, Sam5, COMT, FDC, and CSO2. Considering the selection markers for E. coli, the first three genes were constructed into the vector pET21d containing ampicillin, namely pZYL51: pET21d-Rgtal-sam5-AtCOMT; then the remaining two genes were constructed into the vector pET28a containing kanamycin, namely pZYL33-pET28a-fdc-cso2(A49P-Q390A).

[0135] Construction of pZYL51: pET21d-Rgtal-sam5-AtCOMT: This plasmid includes three enzymes: TAL, Sam5, and COMT, namely the TAL gene (encoding tyrosine ammonia-lyase, RgTAL, SEQ ID NO:5) from *Rhodotorula glutinis*, the sam5 gene (encoding ρ-coumaric acid 3-hydroxylase, SeSAM5, SEQ ID NO:7) from *Saccharomyces cerevisiae*, and the COMT gene (encoding caffeic acid O-methyltransferase, AtCOMT, SEQ ID NO:1) from *Arabidopsis thaliana*. Gene synthesis and codon optimization were performed, and the above three genes were constructed into the plasmids pET21d-Rgtal, pET21a-sam5, and pET21a-AtCOMT. The entire pathway was assembled sequentially using XbaI / SpeI based on the BioBrick assembly method. The XbaI / HindIII excised DNA fragment from pET21a-sam5 was inserted between the SpeI / HindIII sites of pET21d-Rgtal to obtain plasmid pET21d-tal-sam5. Then, the XbaI / HindIII excised DNA fragment from pET21a-AtCOMT was sequentially inserted between the SpeI / HindIII sites of pET21d-Rgtal-sam5 to generate plasmid pZYL51:pET21d-Rgtal-sam5-AtCOMT.

[0136] A single mutation was introduced into the AtCOMT gene of the pZYL51:pET21d-Rgtal-sam5-AtCOMT plasmid, following the same plasmid construction method as in Example 1. First, site-directed mutagenesis primers were designed (as shown in Table 6). Using the pZYL51 plasmid as a template and 168-F / R as primers, PCR amplification was performed (reaction system and conditions as in Table 2), followed by 1.0% agarose gel electrophoresis. Band F168 (containing AtCOMT (Q310G), SEQ ID NO:3) was obtained, consistent with the target size. 10 μL of F168 was then directly transformed into DH10B competent cells. Single clones were selected and sequenced to obtain pZYL168: pET-21d-Rgtal-sam5-Atcomt(Q310G). This plasmid can be used to synthesize ferulic acid. It was then co-transformed with pZYL33: pET28a-fdc-cso2(A49P-Q390A) into E. coli BL21(DE3) for vanillin synthesis.

[0137] Establishment of pZYL33: pET28a-fdc-cso2 (A49P-Q390A): This plasmid includes two enzymes, FDC and CSO2: the Fdc (ferulic acid decarboxylase, FDC, SEQ ID NO: 9) gene from *Bacillus pumilus* and the Cso2 (carotenoid lysinogenase, CSO2, SEQ ID NO: 11) gene from *Sigmoidobacterium*. Gene synthesis and codon optimization were performed, and the two genes were constructed into the operon on the pET28a plasmid backbone, named pZYL34-pET28a-fdc-cso2. A double mutation was introduced into the cso2 gene of the pZYL34-pET28a-fdc-cso2 plasmid to achieve vanillin production from ferulic acid. The plasmid construction method is consistent with that in Example 1. First, site-directed mutagenesis primers were designed (as shown in Table 6). Using 34-F / R primers and pZYL34 plasmid as a template, PCR amplification was performed (reaction system and conditions as in Table 2), followed by 1.0% agarose gel electrophoresis, yielding band F35 (containing CSO2(A49P)), consistent with the target size. Then, using F35 as a template and 33-F / R primers, PCR amplification was performed (reaction system and conditions as in Table 2), followed by 1.0% agarose gel electrophoresis, yielding band F33 (containing CSO2(A49P-Q390A), SEQ ID NO: 13), consistent with the target size. 10 μL of the PCR product F33 was directly transformed into DH10B competent cells. Single clones were selected and sequenced to obtain pZYL33: pET28a-fdc-cso2(A49P-Q390A), which was used for vanillin synthesis.

[0138] pZYL168-pET-21d-Rgtal-sam5-Atcomt (Q310G) and pZYL33-pET28a-fdc-cso2 (A49P-Q390A) were co-transformed into Escherichia coli BL21 (DE3) for vanillin synthesis.

[0139] Table 6. Primers used for site-directed mutagenesis

[0140]

[0141] The strains and plasmids used in this embodiment are shown in Table 7.

[0142] Table 7. Strains and plasmids used in this example

[0143] Strains: SYL100 BL21(DE3)-pZYL168-pZYL33-pGro7 Car+Kan+Cm Plasmids: pZYL168 pZYL168-pET21d-Rgtal-sam5-Atcomt(Q310G) Car Plasmids: pZYL33 pZYL33-pET28a-fdc-cso2(A49P-Q390A) Kan

[0144] Example 4: Synthesis of vanillin using an artificially constructed recombinant Escherichia coli system

[0145] pZYL168 (pET-21d-Rgtal-sam5-Atcomt(Q310G)), pZYL33 (pET28a-fdc-cso2(A49P-Q390A)), and pGro7 (as a molecular chaperone, purchased from Shanghai Sangon Biotech) were co-transformed into E. coli BL21(DE3) and plated on Car+Kan+Cm antibiotic-resistant plates. The plates were incubated overnight at 37°C with the plates inverted. The next day, single colonies were picked from the plates and inoculated into 2 mL LB (glass tubes) containing Car+Kan+Cm antibiotics, and incubated overnight for 10 h. A 1% overnight culture was then inoculated into TB medium containing Car+Kan+Cm antibiotics, with an initial addition of 1 g / L L-tyrosine (…). Figure 2 a) Incubate at 37℃ and 250 rpm until OD600 ∈ (0.6-1.0), then incubate on ice for 10 min. Induce fermentation by adding IPTG to a final concentration of 0.1 mM and arabinose to a final concentration of 10 mM, and ferment at 22℃ for 5 days.

[0146] 1) Samples were taken and detected at OD600nm, and the results were recorded.

[0147] 2) Extract 500 μL of fermentation broth twice with an equal volume (500 μL) of ethyl acetate, and evaporate to dryness under vacuum. Redissolve in 100 μL of methanol, centrifuge at 12000 rpm for 1 min, and transfer the supernatant to a liner-lined HPLC vial for HPLC analysis.

[0148] HPLC detection conditions: The fermentation broth product was analyzed using a C18 column (Silgreen HPLC column 250×4.6mm). The HPLC conditions were: column temperature 30℃, flow rate 1 mL / min, and mobile phase B consisting of water containing 0.1% glacial acetic acid. Mobile phase C consisted of methanol containing 0.1% glacial acetic acid. The flow rates were: 0-2 min 80% B + 20% C, 2-17 min 50% B + 50% C, 17-22 min 100% C, 22-23 min 80% B + 20% C, and 25 min 80% B + 20% C.

[0149] According to HPLC results, the vanillin yield of WT and Atcomt (Q310G) mutant fermentation samples was only 198.65 mg / L, while the vanillin yield of Atcomt (Q310G) reached 327.72 mg / L, significantly increasing the vanillin yield by 65%. Figure 2 bc).

[0150] Furthermore, the inventors discovered that as vanillin concentration increased, the accumulation of intermediate products also gradually increased, indicating that the improvement of Atcomt(Q310G) protein activity can open up the metabolic flux of the entire vanillin synthesis pathway, which is unexpected.

[0151] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. sequence list <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> Caffeic acid O-methyltransferase mutants and their applications <130> 21A686 <160> 26 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1092 <212> DNA <213> Arabidopsis thaliana <400> 1 atgggttcaa cggcagagac acaattaact ccggtgcaag tcaccgacga cgaagctgcc 60 ctcttcgcca tgcaactagc cagtgcttcc gttcttccga tggctttaaa atccgcctta 120 gagcttgacc ttcttgagat tatggccaag aatggttctc ccatgtctcc taccgagatc 180 gcttctaaac ttccgaccaa aaatcctgaa gctccggtca tgctcgaccg tatcctccgt 240 cttcttacgt cttactccgt cttaacctgc tccaaccgta aactttccgg tgatggcgtt 300 gaacggattt acgggcttgg tccggtttgc aagtatttga ccaagaacga agatggtgtt 360 tccattgctg ctctttgtct tatgaaccaa gacaaggttc tcatggaaag ctggtaccat 420 ttgaaggatg caattcttga tggtgggatt ccattcaaca aggcttatgg aatgagcgcg 480 ttcgagtacc acgggactga ccctagattc aacaaggtct ttaacaatgg aatgtctaac 540 cattccacaa tcaccatgaa gaagattctt gagacctata agggttttga aggattgact 600 tctttggttg atgttggtgg tggcattggt gctacactca aaatgattgt ctccaagtac 660 cctaatctta aaggcatcaa ctttgatctc ccacatgtca tcgaagatgc tccttctcat 720 cctggtattg agcatgttgg aggagatatg tttgtaagtg tccctaaagg tgatgccata 780 ttcatgaagt ggatatgtca tgactggagt gacgaacatt gcgtgaaatt cttgaagaac 840 tgctacgagt cacttccaga ggatggaaaa gtgatattag cagagtgtat acttccagag 900 acaccagact caagcctctc aaccaaacaa gtagtccatg tcgattgcat tatgttggct 960 cacaatcccg gaggcaaaga acgaaccgag aaagagtttg aggcattagc caaagcatca 1020 ggcttcaagg gcatcaaagt tgtctgcgac gctttttggtg ttaaccttat tgagttactc 1080 aagaagctct aa 1092 <210> 2 <211> 363 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Gly Ser Thr Ala Glu Thr Gln Leu Thr Pro Val Gln Val Thr Asp 1 5 10 15 Asp Glu Ala Ala Leu Phe Ala Met Gln Leu Ala Ser Ala Ser Val Leu 20 25 30 Pro Met Ala Leu Lys Ser Ala Leu Glu Leu Asp Leu Leu Glu Ile Met 35 40 45 Ala Lys Asn Gly Ser Pro Met Ser Pro Thr Glu Ile Ala Ser Lys Leu 50 55 60 Pro Thr Lys Asn Pro Glu Ala Pro Val Met Leu Asp Arg Ile Leu Arg 65 70 75 80 Leu Leu Thr Ser Tyr Ser Val Leu Thr Cys Ser Asn Arg Lys Leu Ser 85 90 95 Gly Asp Gly Val Glu Arg Ile Tyr Gly Leu Gly Pro Val Cys Lys Tyr 100 105 110 Leu Thr Lys Asn Glu Asp Gly Val Ser Ile Ala Ala Leu Cys Leu Met 115 120 125 Asn Gln Asp Lys Val Leu Met Glu Ser Trp Tyr His Leu Lys Asp Ala 130 135 140 Ile Leu Asp Gly Gly Ile Pro Phe Asn Lys Ala Tyr Gly Met Ser Ala 145 150 155 160 Phe Glu Tyr His Gly Thr Asp Pro Arg Phe Asn Lys Val Phe Asn Asn 165 170 175 Gly Met Ser Asn His Ser Thr Ile Thr Met Lys Lys Ile Leu Glu Thr 180 185 190 Tyr Lys Gly Phe Glu Gly Leu Thr Ser Leu Val Asp Val Gly Gly Gly 195 200 205 Ile Gly Ala Thr Leu Lys Met Ile Val Ser Lys Tyr Pro Asn Leu Lys 210 215 220 Gly Ile Asn Phe Asp Leu Pro His Val Ile Glu Asp Ala Pro Ser His 225 230 235 240 Pro Gly Ile Glu His Val Gly Gly Asp Met Phe Val Ser Val Pro Lys 245 250 255 Gly Asp Ala Ile Phe Met Lys Trp Ile Cys His Asp Trp Ser Asp Glu 260 265 270 His Cys Val Lys Phe Leu Lys Asn Cys Tyr Glu Ser Leu Pro Glu Asp 275 280 285 Gly Lys Val Ile Leu Ala Glu Cys Ile Leu Pro Glu Thr Pro Asp Ser 290 295 300 Ser Leu Ser Thr Lys Gln Val Val His Val Asp Cys Ile Met Leu Ala 305 310 315 320 His Asn Pro Gly Gly Lys Glu Arg Thr Glu Lys Glu Phe Glu Ala Leu 325 330 335 Ala Lys Ala Ser Gly Phe Lys Gly Ile Lys Val Val Cys Asp Ala Phe 340 345 350 Gly Val Asn Leu Ile Glu Leu Leu Lys Lys Leu 355 360 <210> 3 <211> 1092 <212> DNA <213> Artificial Sequence <400> 3 atgggttcaa cggcagagac acaattaact ccggtgcaag tcaccgacga cgaagctgcc 60 ctcttcgcca tgcaactagc cagtgcttcc gttcttccga tggctttaaa atccgcctta 120 gagcttgacc ttcttgagat tatggccaag aatggttctc ccatgtctcc taccgagatc 180 gcttctaaac ttccgaccaa aaatcctgaa gctccggtca tgctcgaccg tatcctccgt 240 cttcttacgt cttactccgt cttaacctgc tccaaccgta aactttccgg tgatggcgtt 300 gaacggattt acgggcttgg tccggtttgc aagtatttga ccaagaacga agatggtgtt 360 tccattgctg ctctttgtct tatgaaccaa gacaaggttc tcatggaaag ctggtaccat 420 ttgaaggatg caattcttga tggtgggatt ccattcaaca aggcttatgg aatgagcgcg 480 ttcgagtacc acgggactga ccctagattc aacaaggtct ttaacaatgg aatgtctaac 540 cattccacaa tcaccatgaa gaagattctt gagacctata agggttttga aggattgact 600 tctttggttg atgttggtgg tggcattggt gctacactca aaatgattgt ctccaagtac 660 cctaatctta aaggcatcaa ctttgatctc ccacatgtca tcgaagatgc tccttctcat cctggtattg agcatgttgg agggatatg tttgtaagtg tccctaaagg tgatgccata 780 ttcatgaagt ggatatgtca tgactggagt gacgaacatt gcgtgaatt cttgaagaac tgctacgagt cacttccaga ggatggaaaa gtgatattag cagagtgtat acttccagag acaccagact caagcctctc aaccaaagga gtagtccatg tcgattgcat tatgttggct cacaatcccg gaggcaaaga aacgaaccgag aaagagtttg aggcattagc caaagcatca ggcttcaagg gcatcaaagt tgtctgcgac gcttttggtg ttaaccttat tgagttactc 1080 aagaagctct aa <210> 4 <211> 363 <212> PRT <213> Artificial Sequence <400> 4 Met Gly Ser Thr Ala Glu Thr Gln Leu Thr Pro Val Gln Val Thr Asp 1 5 10 15 Asp Glu Met Gln Free Mp3 Download 20 25 30 Pro Met Ala Leu Lys Ser Ala Leu Glu Leu Asp Leu Leu Glu Ile Met 35 40 45 Ala Lys Asn Gly Ser Pro Met Ser Pro Thr Glu Ile Ala Ser Lys Leu 50 55 60 Pro Thr Lys Asn Pro Glu Ala Pro Val Met Leu Asp Arg Ile Leu Arg 65 70 75 80 Leu Leu Thr Ser Tyr Ser Val Leu Thr Cys Ser Asn Arg Lys Leu Ser 85 90 95 Gly Asp Gly Val Glu Arg Ile Tyr Gly Leu Gly Pro Val Cys Lys Tyr 100 105 110 Leu Thr Lys Asn Glu Asp Gly Val Ser Ile Ala Ala Leu Cys Leu Met 115 120 125 Asn Gln Asp Lys Val Leu Met Glu Ser Trp Tyr His Leu Lys Asp Ala 130 135 140 Ile Leu Asp Gly Gly Ile Pro Phe Asn Lys Ala Tyr Gly Met Ser Ala 145 150 155 160 Phe Glu Tyr His Gly Thr Asp Pro Arg Phe Asn Lys Val Phe Asn Asn 165 170 175 Gly Met Ser Asn His Ser Thr Ile Thr Met Lys Lys Ile Leu Glu Thr 180 185 190 Tyr Lys Gly Phe Glu Gly Leu Thr Ser Leu Val Asp Val Gly Gly Gly 195 200 205 Ile Gly Ala Thr Leu Lys Met Ile Val Ser Lys Tyr Pro Asn Leu Lys 210 215 220 Gly Ile Asn Phe Asp Leu Pro His Val Ile Glu Asp Ala Pro Ser His 225 230 235 240 Pro Gly Ile Glu His Val Gly Gly Asp Met Phe Val Ser Val Pro Lys 245 250 255 Gly Asp Ala Ile Phe Met Lys Trp Ile Cys His Asp Trp Ser Asp Glu 260 265 270 His Cys Val Lys Phe Leu Lys Asn Cys Tyr Glu Ser Leu Pro Glu Asp 275 280 285 Gly Lys Val Ile Leu Ala Glu Cys Ile Leu Pro Glu Thr Pro Asp Ser 290 295 300 Ser Leu Ser Thr Lys Gly Val Val His Val Asp Cys Ile Met Leu Ala 305 310 315 320 His Asn Pro Gly Gly Lys Glu Arg Thr Glu Lys Glu Phe Glu Ala Leu 325 330 335 Ala Lys Ala Ser Gly Phe Lys Gly Ile Lys Val Val Cys Asp Ala Phe 340 345 350 Gly Val Asn Leu Ile Glu Leu Leu Lys Lys Leu 355 360 <210> 5 <211> 2082 <212> DNA <213> Rhodotorula glutinis <400> 5 atggccccgc gtccgacctc acagaatcag tatcgtacct gtccgaccac ccaggtgacc 60 caggtggata ttgtggaaaa aatgctggca gccccgaccg attcaacctt agaactggat 120 ggctatagtc tgaatctggg cgatgttgtg agcgccgcac gtaaaggtcg tccggttcgt 180 gttaaagata gcgatgaaat tcgctctaaa attgataaat cagtggaatt tctgcgctca 240 cagctgtcta tgagcgtgta tggcgtgacc accggctttg gcggtagtgc agatacccgt 300 accgaagatg ccatttcttt acagaaagca ttattagaac atcagttatg tggcgtgctg 360 ccgagtagct ttgatagctt tcgcctgggt cgcggcttag aaaatagtct gccgttagaa 420 gtggttcgcg gtgccatgac cattcgtgtt aatagtttaa cccgtggtca tagcgccgtg 480 cgcttagttg tgttagaagc cctgaccaat tttctgaatc atggtattac cccgattgtt 540 ccgttacgcg gcaccattag tgcaagcggc gatctgagtc cgctgagcta tattgcagca 600 gccatttcag gtcatccgga ttctaaagtt catgttgtgc atgaaggcaa agaaaaaatt 660 ctgtatgcac gcgaagcaat ggccctgttt aatctggaac ccgttgtttt aggcccgaaa 720 gaaggcctgg gcttagttaa tgggaccgca gtgagcgcct ctatggccac cctggcctta 780 catgatgcac acatgctgtc actgctgtct cagagcttaa ccgcaatgac cgtggaagca 840 atggtgggtc atgcaggctc tttcatccg ttttacatg atgtgacccg tccgcatccg 900 acccagattg aagttgccgg tatattcgt aaactgctgg aaggctctcg ctttgcagtt 960 catcatgaag aagaagttaa agtgaaagat gatgaaggta ttctgcgtca ggatcgctat 1020 ccgttacgta cctctccgca gtggttaggc ccgctggtga gcgatctgat tcatgcacat 1080 gccgttttaa ccattgaagc cggccagtca accaccgata atccgctgat tgatgtggaa 1140 aataaaacct cacatcatgg cggtaatttt caggccgcag ccgttgccaa tactatggaa 1200 aaaacccgcc tgggcttagc acagattggc aaactgaatt ttacccagtt aaccgaaatg 1260 ctgaatgccg gtatgaatcg cggcttaccg tcttgtctgg ccgccgaaga tccgagctta 1320 tcatatcatt gtaaaggttt agatattgcc gcagcagcct ataccagcga actgggtcat 1380 ttagccaatc cggtgaccac ccatgttcag ccggcagaaa tggccaatca ggcagtgaat 1440 agcttagccc tgattagtgc acgtcgtacc accgaatcta atgatgtgct gtcactgtta 1500 ttagccaccc atctgtattg tgtgttacag gcaattgatc tgcgcgcaat tgaatttgaa 1560 tttaaaaaac agtttggtcc ggccattgtg tcactgattg atcagcattt tggtagcgca 1620 atgactggca gtaatttacg cgatgaactg gttgaaaaag tgaataaaac cttagccaaa 1680 cgcctggaac agaccaatag ctatgatctg gttccgcgtt ggcatgatgc cttttctttt 1740 gcagcaggca ccgtggtgga agtgctgtct tcaacctcac tgagtttagc agccgttaat 1800 gcatggaaag ttgccgcagc agaaagcgcc atttctttaa cccgtcaggt gcgcgaaacc 1860 ttttggagtg ccgcctctac ctcaagtccg gccctgagct atctgtctcc gcgtacccag 1920 attctgtatg cctttgttcg cgaagaactg ggcgttaaag cacgtcgcgg cgatgtgttt 1980 ctgggcaaac aggaagtgac cattggtagt aatgtgagta aaatttatga agcaattaaa 2040 tcaggtcgta ttaataatgt gttactgaaa atgctggcat aa 2082 <210> 6 <211> 693 <212> PRT <213> Rhodotorula glutinis <400> 6 Met Ala Pro Arg Pro Thr Ser Gln Asn Gln Tyr Arg Thr Cys Pro Thr 1 5 10 15 Thr Gln Val Thr Gln Val Asp Ile Val Glu Lys Met Leu Ala Ala Pro 20 25 30 Thr Asp Ser Thr Leu Glu Leu Asp Gly Tyr Ser Leu Asn Leu Gly Asp 35 40 45 Val Val Ser Ala Ala Arg Lys Gly Arg Pro Val Arg Val Lys Asp Ser 50 55 60 Asp Glu Ile Arg Ser Lys Ile Asp Lys Ser Val Glu Phe Leu Arg Ser 65 70 75 80 Gln Leu Ser Met Ser Val Tyr Gly Val Thr Thr Gly Phe Gly Gly Ser 85 90 95 Ala Asp Thr Arg Thr Glu Asp Ala Ile Ser Leu Gln Lys Ala Leu Leu 100 105 110 Glu His Gln Leu Cys Gly Val Leu Pro Ser Ser Phe Asp Ser Phe Arg 115 120 125 Leu Gly Arg Gly Leu Glu Asn Ser Leu Pro Leu Glu Val Val Arg Gly 130 135 140 Ala Met Thr Ile Arg Val Asn Ser Leu Thr Arg Gly His Ser Ala Val 145 150 155 160 Arg Leu Val Val Leu Glu Ala Leu Thr Asn Phe Leu Asn His Gly Ile 165 170 175 Thr Pro Ile Val Pro Leu Arg Gly Thr Ile Ser Ala Ser Gly Asp Leu 180 185 190 Ser Pro Leu Ser Tyr Ile Ala Ala Ala Ile Ser Gly His Pro Asp Ser 195 200 205 Lys Val His Val Val His Glu Gly Lys Glu Lys Ile Leu Tyr Ala Arg 210 215 220 Glu Ala Met Ala Leu Phe Asn Leu Glu Pro Val Val Leu Gly Pro Lys 225 230 235 240 Glu Gly Leu Gly Leu Val Asn Gly Thr Ala Val Ser Ala Ser Met Ala 245 250 255 Thr Leu Ala Leu His Asp Ala His Met Leu Ser Leu Leu Ser Gln Ser 260 265 270 Leu Thr Ala Met Thr Val Glu Ala Met Val Gly His Ala Gly Ser Phe 275 280 285 His Pro Phe Leu His Asp Val Thr Arg Pro His Pro Thr Gln Ile Glu 290 295 300 Val Ala Gly Asn Ile Arg Lys Leu Leu Glu Gly Ser Arg Phe Ala Val 305 310 315 320 His His Glu Glu Glu Val Lys Val Lys Asp Asp Glu Gly Ile Leu Arg 325 330 335 Gln Asp Arg Tyr Pro Leu Arg Thr Ser Pro Gln Trp Leu Gly Pro Leu 340 345 350 Val Ser Asp Leu Ile His Ala His Ala Val Leu Thr Ile Glu Ala Gly 355 360 365 Gln Ser Thr Thr Asp Asn Pro Leu Ile Asp Val Glu Asn Lys Thr Ser 370 375 380 His His Gly Gly Asn Phe Gln Ala Ala Ala Val Ala Asn Thr Met Glu 385 390 395 400 Lys Thr Arg Leu Gly Leu Ala Gln Ile Gly Lys Leu Asn Phe Thr Gln 405 410 415 Leu Thr Glu Met Leu Asn Ala Gly Met Asn Arg Gly Leu Pro Ser Cys 420 425 430 Leu Ala Ala Glu Asp Pro Ser Leu Ser Tyr His Cys Lys Gly Leu Asp 435 440 445 Ile Ala Ala Ala Ala Tyr Thr Ser Glu Leu Gly His Leu Ala Asn Pro 450 455 460 Val Thr Thr His Val Gln Pro Ala Glu Met Ala Asn Gln Ala Val Asn 465 470 475 480 Ser Leu Ala Leu Ile Ser Ala Arg Arg Thr Thr Glu Ser Asn Asp Val 485 490 495 Leu Ser Leu Leu Leu Ala Thr His Leu Tyr Cys Val Leu Gln Ala Ile 500 505 510 Asp Leu Arg Ala Ile Glu Phe Glu Phe Lys Lys Gln Phe Gly Pro Ala 515 520 525 Ile Val Ser Leu Ile Asp Gln His Phe Gly Ser Ala Met Thr Gly Ser 530 535 540 Asn Leu Arg Asp Glu Leu Val Glu Lys Val Asn Lys Thr Leu Ala Lys 545 550 555 560 Arg Leu Glu Gln Thr Asn Ser Tyr Asp Leu Val Pro Arg Trp His Asp 565 570 575 Ala Phe Ser Phe Ala Ala Gly Thr Val Val Glu Val Leu Ser Ser Thr 580 585 590 Ser Leu Ser Leu Ala Ala Val Asn Ala Trp Lys Val Ala Ala Ala Glu 595 600 605 Ser Ala Ile Ser Leu Thr Arg Gln Val Arg Glu Thr Phe Trp Ser Ala 610 615 620 Ala Ser Thr Ser Ser Pro Ala Leu Ser Tyr Leu Ser Pro Arg Thr Gln 625 630 635 640 Ile Leu Tyr Ala Phe Val Arg Glu Glu Leu Gly Val Lys Ala Arg Arg 645 650 655 Gly Asp Val Phe Leu Gly Lys Gln Glu Val Thr Ile Gly Ser Asn Val 660 665 670 Ser Lys Ile Tyr Glu Ala Ile Lys Ser Gly Arg Ile Asn Asn Val Leu 675 680 685 Leu Lys Met Leu Ala 690 <210> 7 <211> 1539 <212> DNA <213> Saccharothrix espanaensis <400> 7 atgaccatca cctctccggc gccggcgggt cgtctgaaca acgttcgtcc gatgaccggt 60 gaagatacc tggaatctct gcgtgacggt cgtgaagttt acatctacgg tgaacgtgtt 120 gacgacgtta ccacccacct ggcgttccgt aactctgttc gttctatcgc gcgtctgtac 180 gacgttctgc acgacccggc gtctgaaggt gttctgcgtg ttccgaccga caccggtaac 240 ggtggtttca cccacccgtt cttcaaaacc gcgcgttctt ctgaagacct ggttgcggcg 300 cgtgaagcga tcgttggttg gcagcgtctg gtttacggtt ggatgggtcg taccccggac 360 tacaaagcgg cgttcttcgg taccctggac gcgaacgcgg aattctacgg tccgttcgaa 420 gcgaacgcgc gtcgttggta ccgtgacgcg caggaacgtg ttctgtactt caaccacgcg 480 atcgttcacc cgccggttga ccgtgaccgt ccggcggacc gtaccgcgga catctgcgtt 540 cacgttgaag aagaaaccga ctctggtctg atcgtttctg gtgcgaaagt tgttgcgacc 600 ggttctgcga tgaccaacgc gaacctgatc gcgcactacg gtctgccggt tcgtgacaaa 660 aaattcggtc tggttttcac cgttccgatg aactctccgg gtctgaaact gatctgccgt 720 acctcttacg aactgatggt tgcgacccag ggttctccgt tcgactaccc gctgtcttct 780 cgtctggacg aaaacgactc tatcatgatc ttcgaccgtg ttctggttcc gtgggaaaac 840 gttttcatgt acgacgcggg tgcggcgaac tctttcgcga ccggttctgg tttcctggaa 900 cgtttcacct tccacggttg cacccgtctg gcggttaaac tggacttcat cgcgggttgc 960 gttatgaaag cggttgaagt taccggtacc acccacttcc gtggtgttca ggcgcaggtt 1020 ggtgaagttc tgaactggcg tgacgttttc tggggtctgt ctgacgcgat ggcgaaatct 1080 ccgaactctt gggttggtgg ttctgttcag ccgaacctga actacggtct ggcgtaccgt 1140 accttcatgg gtgttggtta cccgcgtatc aaagaaatca tccagcagac cctgggttct 1200 ggtctgatct acctgaactc ttctgcggcg gactggaaaa acccggacgt tcgtccgtac 1260 ctggaccgtt acctgcgtgg ttctcgtggt atccaggcga tcgaccgtgt taaactgctg 1320 aaactgctgt gggacgcggt tggtaccgaa ttcgcgggtc gtcacgaact gtacgaacgt 1380 aactacggtg gtgaccacga aggtatccgt gttcagaccc tgcaggcgta ccaggcgaac 1440 ggtcaggcgg cggcgctgaa aggtttcgcg gaacagtgca tgtctgaata cgacctggac 1500 ggttggaccc gtccggacct gatcaacccg ggtacctaa 1539 <210> 8 <211> 512 <212> PRT <213> Saccharothrix espanaensis <400> 8 Met Thr Ile Thr Ser Pro Ala Pro Ala Gly Arg Leu Asn Asn Val Arg 1 5 10 15 Pro Met Thr Gly Glu Glu Tyr Leu Glu Ser Leu Arg Asp Gly Arg Glu 20 25 30 Val Tyr Ile Tyr Gly Glu Arg Val Asp Asp Val Thr Thr His Leu Ala 35 40 45 Phe Arg Asn Ser Val Arg Ser Ile Ala Arg Leu Tyr Asp Val Leu His 50 55 60 Asp Pro Ala Ser Glu Gly Val Leu Arg Val Pro Thr Asp Thr Gly Asn 65 70 75 80 Gly Gly Phe Thr His Pro Phe Phe Lys Thr Ala Arg Ser Ser Glu Asp 85 90 95 Leu Val Ala Ala Arg Glu Ala Ile Val Gly Trp Gln Arg Leu Val Tyr 100 105 110 Gly Trp Met Gly Arg Thr Pro Asp Tyr Lys Ala Ala Phe Phe Gly Thr 115 120 125 Leu Asp Ala Asn Ala Glu Phe Tyr Gly Pro Phe Glu Ala Asn Ala Arg 130 135 140 Arg Trp Tyr Arg Asp Ala Gln Glu Arg Val Leu Tyr Phe Asn His Ala 145 150 155 160 Ile Val His Pro Pro Val Asp Arg Asp Arg Pro Ala Asp Arg Thr Ala 165 170 175 Asp Ile Cys Val His Val Glu Glu Glu Thr Asp Ser Gly Leu Ile Val 180 185 190 Ser Gly Ala Lys Val Val Ala Thr Gly Ser Ala Met Thr Asn Ala Asn 195 200 205 Leu Ile Ala His Tyr Gly Leu Pro Val Arg Asp Lys Lys Phe Gly Leu 210 215 220 Val Phe Thr Val Pro Met Asn Ser Pro Gly Leu Lys Leu Ile Cys Arg 225 230 235 240 Thr Ser Tyr Glu Leu Met Val Ala Thr Gln Gly Ser Pro Phe Asp Tyr 245 250 255 Pro Leu Ser Ser Arg Leu Asp Glu Asn Asp Ser Ile Met Ile Phe Asp 260 265 270 Arg Val Leu Val Pro Trp Glu Asn Val Phe Met Tyr Asp Ala Gly Ala 275 280 285 Ala Asn Ser Phe Ala Thr Gly Ser Gly Phe Leu Glu Arg Phe Thr Phe 290 295 300 His Gly Cys Thr Arg Leu Ala Val Lys Leu Asp Phe Ile Ala Gly Cys 305 310 315 320 Val Met Lys Ala Val Glu Val Thr Gly Thr Thr His Phe Arg Gly Val 325 330 335 Gln Ala Gln Val Gly Glu Val Leu Asn Trp Arg Asp Val Phe Trp Gly 340 345 350 Leu Ser Asp Ala Met Ala Lys Ser Pro Asn Ser Trp Val Gly Gly Ser 355 360 365 Val Gln Pro Asn Leu Asn Tyr Gly Leu Ala Tyr Arg Thr Phe Met Gly 370 375 380 Val Gly Tyr Pro Arg Ile Lys Glu Ile Ile Gln Gln Thr Leu Gly Ser 385 390 395 400 Gly Leu Ile Tyr Leu Asn Ser Ser Ala Ala Asp Trp Lys Asn Pro Asp 405 410 415 Val Arg Pro Tyr Leu Asp Arg Tyr Leu Arg Gly Ser Arg Gly Ile Gln 420 425 430 Ala Ile Asp Arg Val Lys Leu Leu Lys Leu Leu Trp Asp Ala Val Gly 435 440 445 Thr Glu Phe Ala Gly Arg His Glu Leu Tyr Glu Arg Asn Tyr Gly Gly 450 455 460 Asp His Glu Gly Ile Arg Val Gln Thr Leu Gln Ala Tyr Gln Ala Asn 465 470 475 480 Gly Gln Ala Ala Ala Leu Lys Gly Phe Ala Glu Gln Cys Met Ser Glu 485 490 495 Tyr Asp Leu Asp Gly Trp Thr Arg Pro Asp Leu Ile Asn Pro Gly Thr 500 505 510 <210> 9 <211> 486 <212> DNA <213> Bacillus pumilus <400> 9 atggatcagt ttgttggtct gcacatgatc tatacctatg aaaatggttg ggaatatgaa 60 atctatatca agaatgacca caccattgat tatcgcattc atagcggtat ggttggcggc 120 cgctgggttc gtgatcagga agtgaatatt gttaaactga ccaaaggcgt gtataaagtg 180 agctggaccg aaccgaccgg taccgatgtg agtctgaatt ttatgccgga agaaaaacgc 240 atgcatggcg tgattttctt tccgaaatgg gttcatgaac gtccggatat taccgtgtgt 300 tatcagaatg attatatcga tctgatgaag gaaagccgcg aaaaatatga aacctatccg 360 aaatatgtgg ttccggaatt tgccgatatt acctatattc atcatgccgg cgttaatgat 420 gaaaccatta ttgcagaagc accgtatgaa ggtctgaccg atgaaattcg cgcaggccgt 480 aaataa 486 <210> 10 <211> 161 <212> PRT <213> Bacillus pumilus <400> 10 Met Asp Gln Phe Val Gly Leu His Met Ile Tyr Thr Tyr Glu Asn Gly 1 5 10 15 Trp Glu Tyr Glu Ile Tyr Ile Lys Asn Asp His Thr Ile Asp Tyr Arg 20 25 30 Ile His Ser Gly Met Val Gly Gly Arg Trp Val Arg Asp Gln Glu Val 35 40 45 Asn Ile Val Lys Leu Thr Lys Gly Val Tyr Lys Val Ser Trp Thr Glu 50 55 60 Pro Thr Gly Thr Asp Val Ser Leu Asn Phe Met Pro Glu Glu Lys Arg 65 70 75 80 Met His Gly Val Ile Phe Phe Pro Lys Trp Val His Glu Arg Pro Asp 85 90 95 Ile Thr Val Cys Tyr Gln Asn Asp Tyr Ile Asp Leu Met Lys Glu Ser 100 105 110 Arg Glu Lys Tyr Glu Thr Tyr Pro Lys Tyr Val Val Pro Glu Phe Ala 115 120 125 Asp Ile Thr Tyr Ile His His Ala Gly Val Asn Asp Glu Thr Ile Ile 130 135 140 Ala Glu Ala Pro Tyr Glu Gly Leu Thr Asp Glu Ile Arg Ala Gly Arg 145 150 155 160 Lys <210> 11 <211> 1476 <212> DNA <213> Caulobacter segnis <400> 11 atgaccgctc gttttccaaa cacccgtgaa tttactggcg cactgtatcg cccatctcgc 60 tttgaaggtg aagttttcga cctggaagtg gatggtcagc tgccgactga cattgatggt 120 accttcttct ccgtagctcc agatgctgct ttcccgccga tgcgtgaaga tgacattttt 180 ttcaacggcg acggtgccgt gtctgcattc cgttttggtg gtggtcatgt ggatttccag 240 cgccgttacg tacgcactca gcgtctggaa gctcagcgtg cagctcgtcg ctccctgcat 300 ggtgtttacc gcaacccgag caccaatgac ccgtctgttc tgggcctgaa caactctacc 360 gcgaatacca acgttctgga acatgcgggt gtcctgctgg ccatgaaaga ggattctctg 420 ccatacgccc tggaccctct gactctggaa accaaaggcc tgtggaattt cggcggccaa 480 ctgaccgacg ctccgtttac tgctcatcca aagattgacc cgctgactgg tgacatgatc 540 gcctttggtt acgaggcgcg tggtgacggc tctcgtgata ttgtttatta cgagttcgat 600 gaacatggtg ctaaaacccg tgagatttgg gttcaggctc cggtgtctgc tatggttcac 660 gattttgccg tcactgagcg ttttgtggtc ttcccgatta tccctctgtc cgttgatgtt 720 gaacgcctgc gtcaaggtgg tcgtcatttc cagtggcaac cggatctgcc gcagtacttc 780 ggtgtgatgc gtcgtgacgg tgatggcggt gatctgcact ggttcaccgc tccgaacggt 840 ttccagggcc acacgctgaa cgctttcgac gacggtgaaa aagtttatgc ggacatgacc 900 agcactaacg gtaacgtatt ctacttcttt ccgccggctg atggttttgt tccgagcccg 960 gaaaccctgg ttagccagct ggtacgttgg actttcgatc tgagcgtgaa gggtggtcgt 1020 ctggacatgt ctccgctgac ccctttcccg gcggaatttc cgcgtattga cgaccgcgta 1080 gctctgcgtc cgcatcgtca cggttggatg atggcaatgg acccgactaa accgtacgcc 1140 gaagaccgtg ttggtccacg tccgttcgcg ttcttcaacc agctggctca tctgaacatc 1200 gctaccggca aaatccagac ttggttcgcc gacgaagcgt cctgttttca ggaacctgtg 1260 ttcgtcccgc gtaccggctc ttcccgtgaa ggtgacggtt atctgctgtc tctggttaac 1320 cgcctggacg aacgcaccac ggacatggtt gttctggatg cactgcgtct gggtgaaggt 1380 ccggttgcta ccgttaaact gccgctgcgt atgcgtatgg ctctgcacgg caactggtcc 1440 cgtgcggttt ctccgagctc catcaaagcg gtgtaa 1476 <210> 12 <211> 491 <212> PRT <213> Caulobacter segnis <400> 12 Met Thr Ala Arg Phe Pro Asn Thr Arg Glu Phe Thr Gly Ala Leu Tyr 1 5 10 15 Arg Pro Ser Arg Phe Glu Gly Glu Val Phe Asp Leu Glu Val Asp Gly 20 25 30 Gln Leu Pro Thr Asp Ile Asp Gly Thr Phe Phe Ser Val Ala Pro Asp 35 40 45 Ala Ala Phe Pro Pro Met Arg Glu Asp Asp Ile Phe Phe Asn Gly Asp 50 55 60 Gly Ala Val Ser Ala Phe Arg Phe Gly Gly Gly His Val Asp Phe Gln 65 70 75 80 Arg Arg Tyr Val Arg Thr Gln Arg Leu Glu Ala Gln Arg Ala Ala Arg 85 90 95 Arg Ser Leu His Gly Val Tyr Arg Asn Pro Ser Thr Asn Asp Pro Ser 100 105 110 Val Leu Gly Leu Asn Asn Ser Thr Ala Asn Thr Asn Val Leu Glu His 115 120 125 Ala Gly Val Leu Leu Ala Met Lys Glu Asp Ser Leu Pro Tyr Ala Leu 130 135 140 Asp Pro Leu Thr Leu Glu Thr Lys Gly Leu Trp Asn Phe Gly Gly Gln 145 150 155 160 Leu Thr Asp Ala Pro Phe Thr Ala His Pro Lys Ile Asp Pro Leu Thr 165 170 175 Gly Asp Met Ile Ala Phe Gly Tyr Glu Ala Arg Gly Asp Gly Ser Arg 180 185 190 Asp Ile Val Tyr Tyr Glu Phe Asp Glu His Gly Ala Lys Thr Arg Glu 195 200 205 Ile Trp Val Gln Ala Pro Val Ser Ala Met Val His Asp Phe Ala Val 210 215 220 Thr Glu Arg Phe Val Val Phe Pro Ile Ile Pro Leu Ser Val Asp Val 225 230 235 240 Glu Arg Leu Arg Gln Gly Gly Arg His Phe Gln Trp Gln Pro Asp Leu 245 250 255 Pro Gln Tyr Phe Gly Val Met Arg Arg Asp Gly Asp Gly Gly Asp Leu 260 265 270 His Trp Phe Thr Ala Pro Asn Gly Phe Gln Gly His Thr Leu Asn Ala 275 280 285 Phe Asp Asp Gly Glu Lys Val Tyr Ala Asp Met Thr Ser Thr Asn Gly 290 295 300 Asn Val Phe Tyr Phe Phe Pro Pro Ala Asp Gly Phe Val Pro Ser Pro 305 310 315 320 Glu Thr Leu Val Ser Gln Leu Val Arg Trp Thr Phe Asp Leu Ser Val 325 330 335 Lys Gly Gly Arg Leu Asp Met Ser Pro Leu Thr Pro Phe Pro Ala Glu 340 345 350 Phe Pro Arg Ile Asp Asp Arg Val Ala Leu Arg Pro His Arg His Gly 355 360 365 Trp Met Met Ala Met Asp Pro Thr Lys Pro Tyr Ala Glu Asp Arg Val 370 375 380 Gly Pro Arg Pro Phe Ala Phe Phe Asn Gln Leu Ala His Leu Asn Ile 385 390 395 400 Ala Thr Gly Lys Ile Gln Thr Trp Phe Ala Asp Glu Ala Ser Cys Phe 405 410 415 Gln Glu Pro Val Phe Val Pro Arg Thr Gly Ser Ser Arg Glu Gly Asp 420 425 430 Gly Tyr Leu Leu Ser Leu Val Asn Arg Leu Asp Glu Arg Thr Thr Asp 435 440 445 Met Val Val Leu Asp Ala Leu Arg Leu Gly Glu Gly Pro Val Ala Thr 450 455 460 Val Lys Leu Pro Leu Arg Met Arg Met Ala Leu His Gly Asn Trp Ser 465 470 475 480 Arg Ala Val Ser Pro Ser Ser Ile Lys Ala Val 485 490 <210> 13 <211> 1476 <212> DNA <213> Artificial Sequence <400> 13 atgaccgctc gttttccaaa cacccgtgaa tttactggcg cactgtatcg cccatctcgc 60 tttgaaggtg aagttttcga cctggaagtg gatggtcagc tgccgactga cattgatggt 120 accttcttct ccgtagctcc agatccggct ttcccgccga tgcgtgaaga tgacattttt 180 ttcaacggcg acggtgccgt gtctgcattc cgttttggtg gtggtcatgt ggatttccag 240 cgccgttacg tacgcactca gcgtctggaa gctcagcgtg cagctcgtcg ctccctgcat 300 ggtgtttacc gcaacccgag caccaatgac ccgtctgttc tgggcctgaa caactctacc 360 gcgaatacca acgttctgga acatgcgggt gtcctgctgg ccatgaaaga ggattctctg 420 ccatacgccc tggaccctct gactctggaa accaaaggcc tgtggaattt cggcggccaa 480 ctgaccgacg ctccgtttac tgctcatcca aagattgacc cgctgactgg tgacatgatc 540 gcctttggtt acgaggcgcg tggtgacggc tctcgtgata ttgtttatta cgagttcgat 600 gaacatggtg ctaaaacccg tgagatttgg gttcaggctc cggtgtctgc tatggttcac 660 gattttgccg tcactgagcg ttttgtggtc ttcccgatta tccctctgtc cgttgatgtt 720 gaacgcctgc gtcaaggtgg tcgtcatttc cagtggcaac cggatctgcc gcagtacttc 780 ggtgtgatgc gtcgtgacgg tgatggcggt gatctgcact ggttcaccgc tccgaacggt 840 ttccagggcc acacgctgaa cgctttcgac gacggtgaaa aagtttatgc ggacatgacc 900 agcactaacg gtaacgtatt ctacttcttt ccgccggctg atggttttgt tccgagcccg 960 gaaaccctgg ttagccagct ggtacgttgg actttcgatc tgagcgtgaa gggtggtcgt 1020 ctggacatgt ctccgctgac ccctttcccg gcggaatttc cgcgtattga cgaccgcgta 1080 gctctgcgtc cgcatcgtca cggttggatg atggcaatgg acccgactaa accgtacgcc 1140 gaagaccgtg ttggtccacg tccgttcgcg ttcttcaacc agctggctca tctgaacatc 1200 gctaccggca aaatccagac ttggttcgcc gacgaagcgt cctgttttca ggaacctgtg 1260 ttcgtcccgc gtaccggctc ttcccgtgaa ggtgacggtt atctgctgtc tctggttaac 1320 cgcctggacg aacgcaccac ggacatggtt gttctggatg cactgcgtct gggtgaaggt 1380 ccggttgcta ccgttaaact gccgctgcgt atgcgtatgg ctctgcacgg caactggtcc 1440 cgtgcggttt ctccgagctc catcaaagcg gtgtaa 1476 <210> 14 <211> 491 <212> PRT <213> Artificial Sequence <400> 14 Met Thr Ala Arg Phe Pro Asn Thr Arg Glu Phe Thr Gly Ala Leu Tyr 1 5 10 15 Arg Pro Ser Arg Phe Glu Gly Glu Val Phe Asp Leu Glu Val Asp Gly 20 25 30 Gln Leu Pro Thr Asp Ile Asp Gly Thr Phe Phe Ser Val Ala Pro Asp 35 40 45 Pro Ala Phe Pro Pro Met Arg Glu Asp Asp Ile Phe Phe Asn Gly Asp 50 55 60 Gly Ala Val Ser Ala Phe Arg Phe Gly Gly Gly His Val Asp Phe Gln 65 70 75 80 Arg Arg Tyr Val Arg Thr Gln Arg Leu Glu Ala Gln Arg Ala Ala Arg 85 90 95 Arg Ser Leu His Gly Val Tyr Arg Asn Pro Ser Thr Asn Asp Pro Ser 100 105 110 Val Leu Gly Leu Asn Asn Ser Thr Ala Asn Thr Asn Val Leu Glu His 115 120 125 Ala Gly Val Leu Leu Ala Met Lys Glu Asp Ser Leu Pro Tyr Ala Leu 130 135 140 Asp Pro Leu Thr Leu Glu Thr Lys Gly Leu Trp Asn Phe Gly Gly Gln 145 150 155 160 Leu Thr Asp Ala Pro Phe Thr Ala His Pro Lys Ile Asp Pro Leu Thr 165 170 175 Gly Asp Met Ile Ala Phe Gly Tyr Glu Ala Arg Gly Asp Gly Ser Arg 180 185 190 Asp Ile Val Tyr Tyr Glu Phe Asp Glu His Gly Ala Lys Thr Arg Glu 195 200 205 Ile Trp Val Gln Ala Pro Val Ser Ala Met Val His Asp Phe Ala Val 210 215 220 Thr Glu Arg Phe Val Val Phe Pro Ile Ile Pro Leu Ser Val Asp Val 225 230 235 240 Glu Arg Leu Arg Gln Gly Gly Arg His Phe Gln Trp Gln Pro Asp Leu 245 250 255 Pro Gln Tyr Phe Gly Val Met Arg Arg Asp Gly Asp Gly Gly Asp Leu 260 265 270 His Trp Phe Thr Ala Pro Asn Gly Phe Gln Gly His Thr Leu Asn Ala 275 280 285 Phe Asp Asp Gly Glu Lys Val Tyr Ala Asp Met Thr Ser Thr Asn Gly 290 295 300 Asn Val Phe Tyr Phe Phe Pro Pro Ala Asp Gly Phe Val Pro Ser Pro 305 310 315 320 Glu Thr Leu Val Ser Gln Leu Val Arg Trp Thr Phe Asp Leu Ser Val 325 330 335 Lys Gly Gly Arg Leu Asp Met Ser Pro Leu Thr Pro Phe Pro Ala Glu 340 345 350 Phe Pro Arg Ile Asp Asp Arg Val Ala Leu Arg Pro His Arg His Gly 355 360 365 Trp Met Met Ala Met Asp Pro Thr Lys Pro Tyr Ala Glu Asp Arg Val 370 375 380 Gly Pro Arg Pro Phe Ala Phe Phe Asn Gln Leu Ala His Leu Asn Ile 385 390 395 400 Ala Thr Gly Lys Ile Gln Thr Trp Phe Ala Asp Glu Ala Ser Cys Phe 405 410 415 Gln Glu Pro Val Phe Val Pro Arg Thr Gly Ser Ser Arg Glu Gly Asp 420 425 430 Gly Tyr Leu Leu Ser Leu Val Asn Arg Leu Asp Glu Arg Thr Thr Asp 435 440 445 Met Val Val Leu Asp Ala Leu Arg Leu Gly Glu Gly Pro Val Ala Thr 450 455 460 Val Lys Leu Pro Leu Arg Met Arg Met Ala Leu His Gly Asn Trp Ser 465 470 475 480 Arg Wing Val Be Pro Ser Ser Ile Lys Wing Val 485,490 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <400> 15 atgggttca cggcagagac aattaac 29 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <400> 16 ttagagcttc ttgagtact caataggtt aacacc 36 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <400> 17 ttagaagga gatataccat gggttcacg gcagagacac 40 <210> 18 <211> 59 <212> DNA <213> Artificial Sequence <400> 18 tcgagtgcgg ccgcaagctt ggatccttag agctcttga gtaaccaat aaggttaac 59 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <400> 19 ggagtagtcc atgtcgattg cattatg 27 <210> 20 <211> 46 <212> DNA <213> Artificial Sequence <400> 20 tgcaatcgac atggactact cctttggttg agaggcttga gtctgg 46 <210> 21 <211> 82 <212> DNA <213> Artificial Sequence <400> 21 gatcaacccg ggtacctaaa ctagaaataa ttttgtttaa ctttaagaag gagatatacc 60 atgggttcaa cggcagagac ac 82 <210> 22 <211> 56 <212> DNA <213> Artificial Sequence <400> 22 agtgcggccg caagcttact agtttagagc ttcttgagta actcaataag gttaac 56 <210> 23 <211> 42 <212> DNA <213> Artificial Sequence <400> 23 ccttcttctc cgtagctcca gatccggctt tcccgccgat gc 42 <210> 24 <211> 49 <212> DNA <213> Artificial Sequence <400> 24 cggatctgga gctacggaga agaaggtacc atcaatgtca gtcggcagc 49 <210> 25 <211> 46 <212> DNA <213> Artificial Sequence <400> 25 tgttggtcca cgtccgttcg cgttcttcaa ccagctggct catctg 46 <210> 26 <211> 44 <212> DNA <213> Artificial Sequence <400> 26 cgcgaacgga cgtggaccaa cacggtcttc ggcgtacggt ttag 44

Claims

1. A caffeic acid O-methyltransferase mutant, characterized in that, Based on wild-type caffeic acid O-methyltransferase, the mutant has a mutation at position 310 to Gly; the amino acid sequence of the mutant is shown in SEQ ID NO:

4.

2. An isolated polynucleotide encoding the caffeic acid O-methyltransferase mutant of claim 1.

3. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, The host cell contains the vector as described in claim 3.

5. The host cell as described in claim 4, characterized in that, The cell includes a ferulic acid synthesis pathway gene, thereby enabling the cell to synthesize ferulic acid; the ferulic acid synthesis pathway gene includes a gene encoding the following enzymes: tyrosine deaminase, p-coumarate 3-hydroxylase, and the caffeic acid O-methyltransferase mutant of claim 1.

6. The host cell as described in claim 5, characterized in that, The tyrosine deaminase is derived from Rhodotorula glutinis (Yeast). Rhodotorula glutinis The p-coumaric acid 3-hydroxylase mentioned is derived from Spanish yeast ( Saccharothrix espanaensis The amino acid sequence of the tyrosine deaminase is shown in SEQ ID NO: 6, and the amino acid sequence of the p-coumaric acid 3-hydroxylase is shown in SEQ ID NO:

8.

7. The host cell as described in claim 6, characterized in that, The coding sequence of the tyrosine deaminase is shown in SEQ ID NO: 5, and the coding sequence of the p-coumaric acid 3-hydroxylase is shown in SEQ ID NO:

7.

8. The host cell as described in claim 5, characterized in that, The cell also includes a vanillin synthesis pathway gene, enabling the cell to synthesize vanillin; the vanillin synthesis pathway gene includes: a gene encoding ferulic acid decarboxylase and a dioxygenase mutant; the gene encoding ferulic acid decarboxylase is the fdc gene; the gene encoding the dioxygenase mutant is the cso2 mutant gene.

9. The host cell as described in claim 8, characterized in that, The fdc gene is derived from Bacillus pumilus (… Bacillus pumilus The cso2 gene is derived from *Sigmoidospermum* (…). Caulobacter segnis The cso2 gene is a mutant gene that encodes enzymes with mutations A49P and Q390A; wherein the amino acid sequence of ferulic acid decarboxylase is shown in SEQ ID NO: 10, and the amino acid sequence of the dioxygenase mutant is shown in SEQ ID NO:

14.

10. The host cell as described in claim 9, characterized in that, The coding sequence of the ferulic acid decarboxylase is shown in SEQ ID NO: 9, and the coding sequence of the dioxygenase mutant is shown in SEQ ID NO:

13.

11. A method for producing the caffeic acid O-methyltransferase mutant of claim 1, characterized in that, Including the following steps: (1) Culturing the host cells according to any one of claims 4-10 to obtain a culture; (2) Isolate the caffeic acid O-methyltransferase mutant from the culture.

12. A method for improving the catalytic activity of caffeic acid O-methyltransferase, comprising: The amino acid corresponding to position 310 of SEQ ID NO: 2 in its amino acid sequence was mutated; The mutation at position 310 is Gly; the amino acid sequence of the mutant is shown in SEQ ID NO:

4.

13. The method as described in claim 12, characterized in that, When the caffeic acid O-methyltransferase mutant catalyzes the O-methylation reaction of a substrate, the substrate is a compound containing a phenolic hydroxyl group, and the compound containing the phenolic hydroxyl group is caffeic acid; during catalysis, a methyl group is obtained from a methyl donor.

14. The method as described in claim 13, characterized in that, The methyl donor includes S-adenosylmethionine.

15. The application of the caffeic acid O-methyltransferase mutant of claim 1, for catalyzing the O-methylation reaction of a substrate; or, for preparing a reagent or culture medium for catalyzing the O-methylation reaction of a substrate.

16. The application as described in claim 15, characterized in that, When the caffeic acid O-methyltransferase mutant catalyzes the O-methylation reaction of a substrate, the substrate is a compound containing a phenolic hydroxyl group, and the compound containing the phenolic hydroxyl group is caffeic acid; during catalysis, a methyl group is obtained from a methyl donor.

17. The application as described in claim 16, characterized in that, The methyl donor includes S-adenosylmethionine.

18. A method for synthesizing ferulic acid, characterized in that, The method includes: fermenting the host cell according to any one of claims 5-7 to generate ferulic acid.

19. A method for synthesizing vanillin, characterized in that, The method includes: fermenting the host cell according to any one of claims 8-10 to generate vanillin.

20. A composition for catalyzing O-methylation reactions, characterized in that, The composition contains: The caffeic acid O-methyltransferase mutant of claim 1; or The host cell according to any one of claims 4-10.

21. A kit for O-methylation of a substrate, characterized in that, It contains: The caffeic acid O-methyltransferase mutant of claim 1; or The host cell according to any one of claims 4-10; or The composition of claim 20.

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