Application of ROMT-9 mutant in the preparation of homoeriodictyol

Abstract

The present invention relates to the field of bioengineering, in particular to the application of ROMT-9 mutants in the preparation of homoeriodictyol. The present invention provides the application of His274 residue and / or Asp275 residue in the preparation of homoeriodictyol. The present invention confirms the catalytic ability of ROMT-9 in Saccharomyces cerevisiae and measures its conversion rate. More importantly, the present invention provides a general rational design and transformation strategy for enzymes, which effectively improves the conversion rate of ROMT-9 after using these strategies, and finally produces 0.57mmol / L of homoeriodictyol. It lays the foundation for building a cell factory for the efficient production of homoeriodictyol.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to application of a ROMT-9 mutant in the preparation of homoeriodictyol. Background Art

[0002] ROMT-9, a 3'-O-methyltransferase, has been shown to be a highly specific enzyme that can transfer the methyl group of S-adenosylmethionine (SAM) to eriodictyol in vitro to generate homoeriodictyol ( Figure 1 ). The in vitro conversion rate of ROMT-9 is low, only 52.4%1, and the in vivo catalytic ability of ROMT-9 has not been confirmed, so it is difficult to achieve efficient conversion of eriodictyol to homoeriodictyol in Saccharomyces cerevisiae.

[0003] At present, there is technology to express the enzyme in Saccharomyces cerevisiae, and after separation and purification, the enzyme is used in vitro to catalyze the conversion of eriodictyol into homoeriodictyol. Although the efficiency is low, there is no technology to improve the efficiency of the enzyme by enzyme modification. However, there have been reports on the prediction of the active site of ROMT-9. By comparing the sequence of ROMT-9 with the caffeic acid oxygen methyltransferase COMT from alfalfa, the oxygen methyltransferase AtOMT1 from Arabidopsis thaliana, the chalcone oxygen methyltransferase ChOMT from alfalfa, and the isoflavone oxygen methyltransferase IOMT from alfalfa, the possible substrate binding sites are predicted to be Met134, ASN135, LEU140, ALA160, HIS170, PHE176, PHE180, MET184, HIS187, VAL321, ILE324, MET325, and ASN329. In addition, Zhu et al. proposed a hypothetical catalytic mechanism for 3'-hydroxy-N-methyldodecyl-4'-O-methyltransferase HOMT from Coptis chinensis, which has a high sequence similarity with ROMT-9. 3 The above results laid the foundation for the rational design and modification of ROMT-9. Summary of the invention

[0004] In view of this, the present invention provides the use of ROMT-9 mutants in the preparation of homoeriodictyol. The present invention confirms the catalytic ability of ROMT-9 in Saccharomyces cerevisiae and measures its conversion rate. More importantly, the present invention provides a general rational design and transformation strategy for enzymes, which effectively improves the conversion rate of ROMT-9 after using these strategies, and finally produces 0.57mmol / L homoeriodictyol. It lays a foundation for the construction of a cell factory for the efficient production of homoeriodictyol.

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

[0006] The present invention provides the use of His274 residue and / or Asp275 residue in the preparation of homoeriodictyol.

[0007] The present invention also provides the use of His274 residue and / or Asp275 residue in preparing ROMT-9 mutants.

[0008] The present invention also provides a ROMT-9 mutant, which is obtained by mutation of one or more of Asn 135, Ile 324 and / or Asn 329 of wild-type ROMT-9.

[0009] In some embodiments of the present invention, in the above-mentioned ROMT-9 mutant, Asn 135 of wild-type ROMT-9 is substituted by Thr, Ala, Met, Trp, Leu, Tyr, Arg, Gln, Ile, Phe, Lys or His; and / or

[0010] The wild-type ROMT-9 Ile at position 324 is substituted by Met, Leu, Val, Cys, His, Ala, Gln, Phe, Ser, Gly or Thr.

[0011] In some embodiments of the present invention, in the above-mentioned ROMT-9 mutant, Asn at position 135 of the wild-type ROMT-9 is mutated to Thr; and / or Ile at position 324 of the wild-type ROMT-9 is replaced by Met.

[0012] In some embodiments of the present invention, the above-mentioned ROMT-9 mutant has:

[0013] (1), the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; or

[0014] (2) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or

[0015] (3) A sequence having a homology of more than 90% with the amino acid sequence shown in (1).

[0016] In some embodiments of the invention, SEQ ID NO:1 is: MGSTAADMAAAADEEACMYALQLASSSILPMTLKNAIELGLLETLQSAAVAGGGGGKAALLTPAEVADKLPSKANPAAADMVDRMLRLLASYNVVRCEMEEGADGKLSRRYAAAPVCKWLTPNEDGVSMAALALMTQDKVLMESWYYLKDAVLDGGIPFNKAYGMTAFEYHGTDARFNRVFNE GMKNHSVIITKKLLDLYTGFDAASTVVDVGGGVGATVAAVVSRHPHIRGINYDLPHVISEAPPFPGVEHVGGDMFASVPRGGDAILMKWILHDWSDEHCARLLKNCYDALPEHGKVVVVECVLPESSDATAREQGVFHVDMIMLAHNPGGKERYEREFRELARAAGFTGFKATYIYANAWAIEFTK*.

[0017] In some embodiments of the invention, SEQ ID NO:2 is: MGSTAADMAAAADEEACMYALQLASSSILPMTLKNAIELGLLETLQSAAVAGGGGGKAALLTPAEVADKLPSKANPAAADMVDRMLRLLASYNVVRCEMEEGADGKLSRRYAAAPVCKWLTPNEDGVSMAALALMTQDKVLMESWYYLKDAVLDGGIPFNKAYGMTAFEYHGTDARFNRVFNE GMKNHSVIITKKLLDLYTGFDAASTVVDVGGGVGATVAAVVSRHPHIRGINYDLPHVISEAPPFPGVEHVGGDMFASVPRGGDAILMKWILHDWSDEHCARLLKNCYDALPEHGKVVVVECVLPESSDATAREQGVFHVDMMMLAHNPGGKERYEREFRELARAAGFTGFKATYIYANAWAIEFTK*.

[0018] The present invention provides a nucleic acid molecule encoding the above-mentioned ROMT-9 mutant, which has:

[0019] (4), the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or

[0020] (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or

[0021] (6) A nucleotide sequence that is at least 40% identical to the nucleotide sequence shown in (4) or (5).

[0022] In some embodiments of the invention, SEQ ID NO:3 is: ATGGGTTCTACAGCAGCAGATATGGCAGCAGCAGCAGATGAAGAAGCTTGCATGTACGCATTGCAATTGGCTTCTTCTTCCATCTTGCCAATGACTTTGAAGAACGCTATCGAATTGGGCTTGTTGGAAACTTTGCAATCAGCAGCAGTCGCAGGAGGAGGAGGTAAAGCAGCTTTATTAACTCCAGCAGAAGTTGCAGATAAGTT ACCATCTAAGGCTAATCCAGCAGCAGCAGATATGGTGGATAGAATGTTGAGGTTGTTGGCTTCTTATAACGTCGTTAGGTGCGAAATGGAAGAAGGTGCAGACGGTAAACTATCTAGAAGATACGCAGCAGCTCCAGTTTGCAAATGGTTAACACCAAACGAAGATGGTGTTTCAATGGCAGCATTGGCTTTGATGACACAAGACAAGGTCT

[0023] TGATGGAATCTTGGTACTACTTGAAAGACGCAGTTTTGGACGGCGGTATCCTTTTA

[0024] ATAAGGCTTACGGCATGACAGCTTTTGAATATCACGGTACAGACGCTAGATTCAACA

[0025] GAGTTTTCAACGAAGGCATGAAGAACCATTCCGTTATCATCACCAGAAGTTGTTGG

[0026] ACTTGTACACCGGTTTTGATGCAGCTTCTACAGTTGTTGACGTTGGTGGAGGAGGTTG

[0027] GAGCTACAGTTGCTGCAGTTGTTTCCAGACACCCACATATTAGGGCATTAACTACG

[0028] ACTTGCCACACGTTATTTCAGAGCTCCACCATTTCCAGGAGTTGAACACGTTGGAG

[0029] GAGATATGTTTGCTTCAGTTCCAAGAGGAGGAGACGCTATTTTGATGAAGTGGATTT

[0030] TGCACGATTGGTCAGACGAACATTGCGCTAGATTGTTGAAGAATTGCTACGACGCAT

[0031] TACCAGAACACGGTAAAGTTGTCGTTGTTGAATGCGTTTTGCCAGAATCTTCAGACG

[0032] CTACAGCTAGAGAACAAGGAGTTTTTCACGTGGACATGATCATGTTGGCTCATAATC

[0033] CAGGCGGTAAAGAAAGATACGAGAGAGAATTCAGAGAATTGGCTAGAGCAGCAGG

[0034] TTTTACAGGTTTCAAGGCTACCTACATATATGCTAAGCCTTGGGCTATTGAATTCACTAAGTAA。

[0035]

[0036] The present invention also provides an expression cassette, comprising the above ROMT-9 mutant and / or the above nucleic acid molecule.

[0037] The present invention also provides a host for transforming and / or transfecting the above nucleic acid molecule and / or the above expression box.

[0038] In some embodiments of the present invention, the above-mentioned host comprises Saccharomyces cerevisiae.

[0039] In some embodiments of the present invention, in the above-mentioned host, the Saccharomyces cerevisiae includes: Yeri001.

[0040] In some embodiments of the present invention, in the above-mentioned host, the construction method of Yeri001 comprises the following steps: the original chassis is CEN.PK2-1D (available for purchase at BiobW: https: / / www.biobw.org / , number: Bio-110854), and the strain carries 4-p-coumaroyl-CoA ligase 4CL from parsley, chalcone synthase CHS from petunia, chalcone isomerase CHI from alfalfa, flavonoid 3'-monooxygenase F3'H from Arabidopsis thaliana, and cytochrome P450 reductase ATR1. The strain overexpressed the yeast endogenous acetyl-CoA carboxylase ACC1, knocked out the yeast endogenous citrate synthase CIT2, knocked out the yeast endogenous ferulic acid decarboxylase FDC1, replaced the homologous gene of the yeast endogenous essential gene enoyl reductase TSC13 with the ECR gene from apple, and fused the chalcone synthase CHS with the chalcone isomerase CHI using the universal enzyme fusion connector (GGGGS)1.

[0041] The present invention also provides the use of the ROMT-9 mutant, the nucleic acid molecule, the expression box or the host in the preparation of homoeriodictyol.

[0042] The present invention also provides a method for screening mutation sites, comprising the following steps:

[0043] S1: ROMT-9 is combined with the ligand SAM to obtain a complex;

[0044] S2: docking the complex with the substrate binding site of eriodictyol to obtain a docking result;

[0045] S3: Using HOMT as a template, sequence alignment was performed with the ROMT-9 to obtain the catalytic mechanism;

[0046] S4: Obtaining mutation sites according to the docking results and the catalytic mechanism.

[0047] In some embodiments of the present invention, in the above screening method, the ROMT-9 and the ligand SAM are obtained after dehydration and hydrogenation.

[0048] In some embodiments of the present invention, in the above screening method, the eriodictyol is obtained after hydrogenation and charge distribution.

[0049] In some embodiments of the present invention, in the above screening method, the complex is a rigid structure; and the eriodictyol is a flexible structure.

[0050] In some embodiments of the present invention, in the above screening method, the docking includes: semi-flexible docking.

[0051] In some embodiments of the present invention, in the above screening method, the substrate binding site includes one or more of: MET134, ASN135, LEU140, ALA160, HIS170, PHE176, PHE180, MET184, HIS187, VAL321, ILE324, MET325 or ASN329.

[0052] In some embodiments of the present invention, in the above screening method, the amino acids of the catalytic mechanism include: His274 and Asp275.

[0053] In some embodiments of the present invention, in the above screening method, the catalytic mechanism includes the following steps: electrons are transferred from the water molecule in the reaction center to the imidazole ring of the His274, and then to the Asp275, causing the 3'-hydroxyl group of eriodictyol to become an electron-rich group, and finally, the methyl group of SAM is nucleophilically attacked by the 3'-hydroxyl group of eriodictyol to form homoeriodictyol and s-adenosylhomocysteine ​​(SAH).

[0054] The present invention provides the use of His274 residue and / or Asp275 residue in the preparation of homoeriodictyol. The present invention confirms the catalytic ability of ROMT-9 in Saccharomyces cerevisiae and measures its conversion rate. More importantly, the present invention provides a general rational design and transformation strategy for enzymes, which effectively improves the conversion rate of ROMT-9 after using these strategies, and finally produces 0.57mmol / L of homoeriodictyol. This lays a foundation for building a cell factory for the efficient production of homoeriodictyol. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0056] Figure 1 It was shown that ROMT-9 catalyzes the conversion of eriodictyol into homoeriodictyol;

[0057] Figure 2 Show homology modeling results and reliable docking conformations; where: a shows homology modeling results; b, c, d show reliable docking results;

[0058] Figure 3 The putative catalytic mechanism of ROMT-9 is shown;

[0059] Figure 4 Show the results of one round of mutation and the results of two rounds of mutation; among them: a shows the results of one round of mutation; b shows the results of two rounds of mutation. DETAILED DESCRIPTION

[0060] The invention discloses application of a ROMT-9 mutant in preparing homoeriodictyol.

[0061] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0062] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0063] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0064] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0065] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly 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.

[0066] The present invention first determines the conversion rate of wild-type ROMT-9 in Saccharomyces cerevisiae, and then verifies in sequence according to Asn135, Ile324 and Asn329. After obtaining the N135T mutant, the mutant is used as a template for Ile324 mutation; after obtaining the N135T / I324M mutant, the mutant is used as a template for Asn329 mutation verification. The verification method is to insert the expression cassette P of the mutant into the TPI1 -ROMT9 mut -T CPS1 The mutant was constructed on a universal plasmid and expressed in the yeast strain Yeri001, which produces eriodictyol. The yield of high eriodictyol and the residual amount of eriodictyol after fermentation were determined, and the conversion rate was calculated. The effective mutant finally obtained was ROMT-9 N135T / I324M Compared with the previous technology of expressing methyltransferase in recombinant Escherichia coli to produce homoeriodictyol from eriodictyol, the present invention combines the rational design of the enzyme to improve the conversion rate of the enzyme, and even when the copy number of the enzyme expression cassette is low (1-2 copies), a high conversion rate (77.6%) can be achieved. In addition, the present invention provides a general strategy for enzyme modification, which can achieve efficient production of homoeriodictyol without a large amount of enzyme expression and regulation of the fermentation process.

[0067] In Examples 1 to 4 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0068] The present invention will be further described below in conjunction with embodiments:

[0069] Example 1

[0070] In this example, homology modeling was first performed using the SWISS-MODEL (www.swissmodel.expasy.org / ) website. The caffeic acid O-methyltransferase from sorghum (with SAM ligand) was used as a template, and the complex structure of ROMT-9 and the ligand SAM was selected (e.g. Figure 2 a). Then, the complex was molecularly docked with the substrate eriodictyol using Autodock software. Before molecular docking, the ROMT-9 and SAM complex was dehydrated and hydrogenated, and eriodictyol was pre-treated with hydrogenation and automatic charge distribution. The docking grid box coordinates were adjusted to surround all substrate binding sites: MET134, ASN135, LEU140, ALA160, HIS170, PHE176, PHE180, MET184, HIS187, VAL321, ILE324, MET325, ASN329. During the docking process, the ROMT-9 and SAM complex was regarded as a rigid structure, and the substrate eriodictyol was regarded as a flexible structure. The docking method was semi-flexible docking.

[0071] In order to reveal the true binding conformation of the enzyme and the substrate as much as possible, we screened the docking results according to the catalytic mechanism of HOMT. Since HOMT and ROMT-9 have a high sequence similarity, the catalytic mechanism of ROMT-9 can be inferred in combination with the sequence alignment results. It can be inferred that the oxygen-methylation reaction of eriodictyol involves the His274 and Asp275 residues of ROMT-9. Electrons are transferred from the water molecule in the reaction center to the imidazole ring of His274, and then to Asp275, causing the 3'-hydroxyl group of eriodictyol to become an electron-rich group. Finally, the methyl group of SAM is nucleophilically attacked by the 3'-hydroxyl group of eriodictyol to form homoeriodictyol and s-adenosylhomocysteine ​​(SAH) (such as Figure 3 Based on the above catalytic mechanism and docking result scores, we screened out three reliable conformations (such as Figure 2 b, c, d).

[0072] According to Figure 2 b, c, d show reliable conformations, and selected active site Asn135, Ile324, and Asn329 as candidate mutation sites. In order to find mutants that enhance substrate and enzyme binding ability, we used DiscoveryStudio TM Virtual amino acid saturation mutations were performed on these three sites. In the Effect column of the output results, "stabilizing" means that the mutation may enhance the ability of the enzyme to bind to the substrate. The point mutations with the result of "stabilizing" were experimentally verified.

[0073] Example 2

[0074] (1) Method for mutation of Asn135 to Ala135: The coding gene of ROMT-9 is divided into fragment A and fragment B for PCR, using KOD-FX high-fidelity enzyme. The upper primer of fragment A removes the part of amplified fragment A, and also carries 20bp homologous to the promoter; the lower primer introduces a point mutation closer to the 5' end, replacing the codon of Asn with the codon of Ala. The upper primer of fragment B introduces a point mutation closer to the 5' end, replacing the codon of Asn with the codon of Ala; the lower primer removes the part of amplified fragment B, and also carries 20bp homologous to the terminator. At the same time, it is necessary to ensure that fragments A and B have a homologous region of about 20bp after the introduction of the mutation. Therefore, when constructing P TPI1 -ROMT9 N135A -T CPS1When the primer sequence of fragment A is: actacaaaaaacacatacataaactaaaaGCATGGGTTCTACAGCAGC (as shown in SEQ ID NO:5), and the primer sequence is: GACCTTGTCTTGTGCCATCAAAGCCAATGC (as shown in SEQ ID NO:6); the primer sequence of fragment B is: ATTGGCTTTGATGGCACAAGACAAGGTCTTG (as shown in SEQ ID NO:7); and the primer sequence is: tgactattcaatcattgcgcGCTTACTTAGTGAATTCAATAGCCCAAG (as shown in SEQ ID NO:8).

[0075] (2) Then, the two fragments were overlap-PCRed using fragment A as the upper primer and fragment B as the lower primer. The PCR product was then PCR-polymerase chain reaction (PCR) with the linearized pRS413-P TPI1 -T CPS1 Perform seamless cloning and obtain episomal expression cassettes with mutants.

[0076] (3) The expression cassette was transformed into the yeast strain Yeri001 that produces eriodictyol (eriodictyol production is 0.75 mmol / L, as shown in Table 1, the original chassis is CEN.PK2-1D (available for purchase at BiobW: https: / / www.biobw.org / , number: Bio-110854), and the strain carries 4-p-coumaryl-CoA ligase 4CL from parsley, chalcone synthase CHS from petunia, chalcone isomerase CHI from alfalfa, and flavonoid 3'-monooxygenase from Arabidopsis thaliana. F3'H and cytochrome P450 reductase ATR1. The strain overexpressed the endogenous acetyl-CoA carboxylase ACC1, knocked out the endogenous citrate synthase CIT2, knocked out the endogenous ferulic acid decarboxylase FDC1, replaced the homologous gene of the endogenous essential gene enoyl reductase TSC13 of yeast with the ECR gene from apple, and fused the chalcone synthase CHS with the chalcone isomerase CHI using the universal enzyme fusion connector (GGGGS)1. The yield of homoeriocin was determined by fermentation for 72 hours, and compared with the strain with pRS413-P TPI1 -ROMT9-T CPS1 The yeast strains were compared to determine whether the mutant was effective.

[0077] Table 1 Yeri001 eriodictyol production

[0078] strains Eriochoride (mmol / 1) - Parallel 1 Eriochoride (mmol / 1) - Parallel 2 Yeri001 0.76682 0.7262

[0079] 4CL amino acid sequence: MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKVGDKSCLINGATGETFTYSQVELLSRKVASGLNKLGIQQGDTIMLLLPNSPEYFFAFLGASYRGAISTMANPFFTSAEVIKQLKASQAKLIITQACYVDKVKDYAAEKNIQIICIDDAPQDCLHFSKLMEADESEMPEVVINSDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYMHSEDVMICILPLFHIYSLNAVLCCGLRAGVTILIMQKFDIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDKYDLSSVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIVDPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRTTIDEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRTNGFTTTEEEIKQFVSKQVVFYKRIFRVFFVDAIPKSPSGKILRKDLRARIASGDLPK*。(as shown in SEQ ID NO:9)

[0080] CHS amino acid sequence: MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTYPDYYFRITNSEHKTDLKEKFKRMCEKSMIKKRYMHLTEEILKENPSMCEYMAPSLDARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAGAIIIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLEEAFRPLSISDWNSLFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNYGNMSSACVLFILDEMRKASAKEGLGTTGEGLEWGVLFGFGPGLTVETVVLHSVAT*. (As shown in SEQ ID NO:10)

[0081] CHI amino acid sequence: MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERGLTIEGNFIKFTAIGVYLEDIAVASLAAKWKGKSSEELLETLDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVMENCVAHLKSVGTYGDAEAEAMQKFAEAFKPVNFPPGASVFYRQSPDGILGLSFS PDTSIPEKEAALIENKAVSSAVLETMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN*. (As shown in SEQ ID NO:11)

[0082] F3’H Amino Acid Sequence: MATLFLTILLATVLFLILRIFSHRRNRSHNNRLPPGPNPWPIIGNLPHMGTKPHRTLSAMVTTYGPILHLRLGFVDVVVAASKSVAEQFLKIHDANFASRPPNSGAKHMAYNYQDLVFAPYGHRWRLLRKISSVHLFSAKALEDFKHVRQEEVGTLTRELVRVGTKPVNLGQLVNMCVVNALGREMIGRRLFGADADHKADEFRSMVTEMMALAGVFNIGDFVPSLDWLDLQGVAGKMKRLHKRFDAFLSSILKEHEMNGQDQKHTDMLSTLISLKGTDLDGDGGSLTDTEIKALLLNMFTAGTDTSASTVDWAIAELIRHPDIMVKAQEELDIVVGRDRPVNESDIAQLPYLQAVIKENFRLHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDPLAFKPERFLPGGEKSGVDVKGSDFELIPFGAGRRICAGLSLGLRTIQFLTATLVQGFDWELAGGVTPEKLNMEESYGLTLQRAVPLVVHPKPRLAPNVYGLGSG* (as shown in SEQ ID NO:12).

[0083] ATR1 amino acid sequence: MTSALYASDLFKQLKSIMGTDSLSDDVVLVIATTSLALVAGFV VLLWKKTTADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSIFFGTQTGTAEGFAKALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYGDGEPTDNAARFYKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKSVATPYTAVIPEYRVVTHDPRFTTQKSMESNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAPSRVHVTSALVYGPTPTGRIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVMVGPGTGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMDFIYEDELNNFVDQGVISELIMAFSREGAQKEYVQHKMMEKAAQVWDLIKEEGYLYVCGDAKGMARDVHRTLHTIVQEQEGVSSSEAEAIVKKLQTEGRYLRDVW*. (as shown in SEQ ID NO:13)

[0084] ECR amino acid sequence: MKVTVVSRSGREVVKGGLELSDSATVADLQDAIHKRTKKFYPA RQRLTLPVQPGSKERPVVLSYKKSLQDYISGNSDNLTVVFKDLGPQVSYRTLFFFEYLGPLILYPIFYYFPVYDYLGFKGDRVIHPVQTYALYYWCFHYFKRIMETFFVHRFSHATSPLSNVFRNCAYYWSFGA FIAYYLNHPLYTPVSDLQMKIGFGIGIICQISNFYCHILLRNLRSPDGGNGGYQIPRGFLFNIVTCANYTTEIYQWLGFNIATQTVAGYIFLIVAASIMTNWALAKHRRLKKIFDGKDGRPKYPRRWVILPPFL*. (As shown in SEQ ID NO:14)

[0085] Example 3

[0086] Except for the different primers, the construction methods of the other mutants are the same as in Example 2. The primers are shown in the following table:

[0087]

[0088]

[0089]

[0090] Second round of mutagenesis ROMT-9 N135T As template, the primers are shown in the following table:

[0091]

[0092]

[0093] Example 4

[0094] By transforming Yeri001 into pRS413-P TPI1 -ROMT9-T CPS1 After fermentation for 72h, 0.36mmol / L of eriodictyol and 0.25mmol / L of homoeriodictyol were obtained (as shown in Table 2). It can be seen that the in vivo conversion rate of ROMT-9 is about 41.2%. In this example, Asn135 was firstly subjected to a first round of point mutation to obtain ROMT-9. N135T The conversion rate is 62.6%, which is 51.9% higher than that of ROMT-9 ( Figure 4 a and Table 3), p<0.01. N135TAs a template for Ile324 mutation, a second round of point mutagenesis was performed, and the double mutant ROMT-9 N135T / I324M The conversion rate reached 77.6%, which is 89.3% higher than that of ROMT-9 (such as Figure 4 b and Table 4), p<0.05. N135T / I324M The Asn329 point mutation was verified as a template, but no effective mutant was found. (WT is wild type; N135T is Asn135 mutated to Thr135)

[0095] Table 2 Wild-type eriodictyol and high eriodictyol yields

[0096] Eriochoride (mmol / 1) - Parallel 1 Eriochoride (mmol / 1) - Parallel 2 0.3638 0.3594 High eriodictyol (mmol / 1) - Parallel 1 High eriodictyol (mmol / 1) - Parallel 2 0.2521 0.2541

[0097] Table 3 Results of one round of mutation

[0098] Mutants High eriodictyol (mmol / 1) - Parallel 1 High eriodictyol (mmol / 1) - Parallel 2 WT 0.25208 0.25413 N135T 0.38359 0.39274 N135A 0.07757 0.07616 N135M 0.02452 0.02446 N135W 0.02258 0.02324 N135L 0.02171 0.02194 N135Y 0.01928 0.01895 N135R 0.01879 0.01901 N135Q 0.01397 0.01413 N135I 0.00607 0.00638 N135F 0 0 N135K 0 0 N135H 0 0

[0099] Table 4 Results of the second round of mutation

[0100]

[0101]

[0102] This example confirms the catalytic ability of ROMT-9 in Saccharomyces cerevisiae and measures its conversion rate. More importantly, the present invention provides a general rational design and transformation strategy for enzymes, which effectively improves the conversion rate of ROMT-9 after using these strategies, and ultimately produces 0.57mmol / l of homoeriodictyol. This lays the foundation for building a cell factory for the efficient production of homoeriodictyol.

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ROMT-9 mutant, characterized in that It is composed of any one or more of mutants 1 to 3; The mutant 1 is a wild-type ROMT-9 in which Asn at position 135 is replaced by Thr; The mutant 2 is a wild-type ROMT-9 in which Asn at position 135 is replaced by Thr and Ile at position 324 is replaced by Met; The mutant 3 is a mutant in which the 135th Asn of the wild-type ROMT-9 is replaced by Thr and the 324th Ile of the wild-type ROMT-9 is replaced by Leu.

2. The ROMT-9 mutant according to claim 1, characterized in that Its sequence is: the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. A nucleic acid molecule encoding the ROMT-9 mutant according to claim 1 or 2, characterized in that: Its sequence is: the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:

4.

4. An expression cassette, characterized in that Comprising the nucleic acid molecule as described in claim 3.

5. A host, characterized in that Transformation and / or transfection of a nucleic acid molecule according to claim 3 and / or an expression cassette according to claim 4.

6. The host according to claim 5, characterized in that Including Saccharomyces cerevisiae.

7. Use of the ROMT-9 mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression cassette according to claim 4 or the host according to claim 5 or 6 in the preparation of homoeriodictyol.

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