Phenylpyruvate decarboxylase ARO10 mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a phenylpyruvate decarboxylase ARO10 protein mutant and application of the phenylpyruvate decarboxylase ARO10 protein mutant in preparation of tyrosol. The invention discloses an ARO10 mutant. The phenylpyruvate decarboxylase mutant is obtained by mutating one or more of the 335th site of phenylpyruvate decarboxylase ARO10 (an amino acid sequence shown as SEQ ID NO1) from yeast. Experiments prove that the mutated phenylpyruvate decarboxylase protein can improve the specific reaction on 4-hydroxyphenylpyruvic acid, so that the tyrosol generation capability of saccharomyces cerevisiae is improved. Glycosyl transferase ARO10 mutant protein is expressed in microorganisms, the capacity of the microorganisms for converting tyrosol is obviously enhanced, and the industrial application prospect is good.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the ARO10 mutant of phenylpyruvate decarboxylase and its applications. Background Technology

[0002] Tyrosol, a phenylethyl alcohol derivative, is one of the main active components of olive oil and can also be found in alcoholic beverages, metabolites of some microorganisms (such as yeast), and metabolites of other plants (such as green tea). Tyrosol exhibits a variety of biological activities, including antioxidant and anti-inflammatory activities, as well as some protective effects on the cardiovascular and nervous systems. In yeast, 4-hydroxyphenylpyruvate is decarboxylated by the phenylpyruvate decarboxylase ARO10 to form 4-hydroxyphenylacetaldehyde, which is then reduced to tyrosol by alcohol dehydrogenase. However, ARO10 is a thiamine diphosphate-dependent phenylpyruvate decarboxylase with broad substrate specificity, capable of catalyzing multiple reactions of the same type in Saccharomyces cerevisiae. Therefore, modifying the endogenous phenylpyruvate decarboxylase ARO10 in Saccharomyces cerevisiae using enzyme engineering methods to obtain mutants with high catalytic activity and substrate specificity for 4-hydroxyphenylpyruvate is of significant practical importance. Summary of the Invention

[0003] This invention addresses the shortcomings of phenylpyruvate decarboxylase ARO10 by providing an ARO10 mutant, engineered strain, and its application in tyrosol production. The phenylpyruvate decarboxylase is modified through site-directed mutagenesis to improve its reactivity with 4-hydroxyphenylpyruvate. The resulting mutant strain can effectively increase the yield of tyrosol in Saccharomyces cerevisiae.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] In a first aspect, the present invention provides a phenylpyruvate decarboxylase ARO10 mutant, which is obtained by mutating the isoleucine (I335) amino acid residue at position 335 of the amino acid sequence of phenylpyruvate decarboxylase ARO10 shown in SEQ ID No. 1 to other amino acid residues.

[0006] In some specific embodiments of the present invention, the mutation includes, but is not limited to, a combination of one or more mutations from G, A, V, L, M, W, F, P, S, T, C, Y, N, D, E, K, or H.

[0007] Secondly, the present invention also provides a nucleic acid molecule encoding the phenylpyruvate decarboxylase ARO10 mutant described above.

[0008] In some specific embodiments of the present invention, the nucleic acid molecule has:

[0009] (I) A nucleotide sequence as shown in any one of SEQ ID NO. 3 to 19; or

[0010] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0011] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0012] (IV) and nucleotide sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequences described in (I), (II) or (III).

[0013] Thirdly, the present invention also provides a recombinant expression vector, expression cassette, or construct containing the aforementioned nucleic acid molecule.

[0014] Fourthly, the present invention also provides a host, which is transfected or transformed with the recombinant expression vector, expression cassette or construct described above.

[0015] In some specific embodiments of the present invention, the host includes transgenic cell lines and / or recombinant strains;

[0016] Preferably, the recombinant strain includes an engineered strain of Saccharomyces cerevisiae.

[0017] Fifthly, the present invention also provides any of the following applications in the preparation of tyrosol;

[0018] (i) The phenylpyruvate decarboxylase ARO10 mutant;

[0019] (ii) the aforementioned nucleic acid molecules;

[0020] (iii) the recombinant expression vector, expression cassette, or construct described above;

[0021] (iv) The aforementioned host.

[0022] In a sixth aspect, the present invention also provides a method for preparing the phenylpyruvate decarboxylase ARO10 mutant, wherein the host is fermented to obtain a fermentation broth containing the phenylpyruvate decarboxylase ARO10 mutant.

[0023] In a seventh aspect, the present invention also provides a method for preparing tyrosol, using 4-hydroxyphenylpyruvic acid as a substrate, expressing the phenylpyruvic acid decarboxylase ARO10 mutant in Saccharomyces cerevisiae, using YPD medium at pH 5-9 as the reaction medium, fermenting at 30-37°C and 0-600 rpm, collecting the fermentation broth for separation and purification to obtain tyrosol.

[0024] The experiments of this invention demonstrate that expressing the ARO10 mutant phenylpyruvate decarboxylase in Saccharomyces cerevisiae can effectively increase the yield of tyrosol in Saccharomyces cerevisiae compared with wild-type phenylpyruvate decarboxylase ARO10, and has good prospects for industrial application. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0026] Figure 1 The recombinant strain of Saccharomyces cerevisiae, CEN.PK2-1C-308a::ARO4, is shown. K229L -ARO7 G141S -LEU2::-ARO3 D154N A comparison of tyrosol production in mutants expressing phenylpyruvate decarboxylase ARO10. Detailed Implementation

[0027] This invention discloses the ARO10 mutant of phenylpyruvate decarboxylase and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0028] The amino acid sequence (SEQ ID No. 1) of phenylpyruvate decarboxylase ARO10 in this invention is: *

[0029] One of the technical solutions of the present invention provides a phenylpyruvate decarboxylase mutant, which is obtained by replacing the 335th amino acid residue of the ARO10 amino acid sequence shown in SEQ ID No. 1 with other amino acid residues to form a new amino acid sequence. The mutant significantly improves the production of tyrosol in Saccharomyces cerevisiae compared with the ARO10 formed by the amino acid sequence shown in SEQ ID No. 1.

[0030] Specifically, the preferred ARO10 mutant is one in which the amino acid sequence shown in SEQ ID No. 1 is mutated to one of the following: the mutation of I335 includes, but is not limited to, a combination of one or more mutations of G, A, V, L, M, W, F, P, S, T, C, Y, N, D, E, K or H.

[0031] The gene encoding the protein is also within the scope of protection of this invention, and the gene sequence is shown in the sequence listing.

[0032] Table 1

[0033]

[0034]

[0035] SEQ ID NO.3 (ARO10-I335G nucleotide sequence)

[0036] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0037] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0038] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0039] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0040] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0041] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0042] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0043] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0044] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0045] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0046] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTTGTTCCTGCAGATT

[0047] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0048] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0049] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0050] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0051] TTCCACTGTTATGGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0052] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0053] GGTCTTGCATTTTGGAGTCGACATCAATGAAggaAATAATGGGCATTATACTTTTACT

[0054] TATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGAC

[0055] ACTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAA

[0056] GAACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTA

[0057] ACTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAG

[0058] CATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGA

[0059] TGTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTT

[0060] CGCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGC

[0061] CGCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGG

[0062] CAACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAG

[0063] CACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAA

[0064] GTTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACC

[0065] AGGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGA

[0066] GACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCA

[0067] CTGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGA

[0068] AGTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAG

[0069] CGGCACTTAAAAGAAATAAAAAATAG

[0070] SEQ ID NO.4 (Nucleotide sequence of ARO10 - I335A)

[0071] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0072] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0073] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0074] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0075] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0076] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT<000017o>

[0077] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0078] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0079] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0080] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0081] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT It should be noted that there seems to be a typo in the original text where "GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT" has a wrong line break in the original. It's adjusted in the translation for better presentation. Also, the "GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT" line in the original has a wrong "o" in "<000017o>" which is corrected to " " in the translation.

[0082] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0083] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0084] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0085] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0086] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0087] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0088] GGTCTTGCATTTTGGAGTCGACATCAATGAAgctAATAATGGGCATTATACTTTTACTT

[0089] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0090] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0091] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0092] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0093] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0094] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0095] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0096] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC

[0097] AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC

[0098] ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG

[0099] TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA

[0100] GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG

[0101] ACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCAC

[0102] TGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGAA

[0103] GTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAGC

[0104] GGCACTTAAAAGAAATAAAAAATAG

[0105] SEQ ID NO.5 (Nucleotide sequence of ARO10-I335V)

[0106] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0107] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAGATTGTTGTCCATC

[0108] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0109] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0110] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0111] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0112] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0113] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0114] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0115] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0116] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTTCCTGCAGATT

[0117] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0118] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0119] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0120] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0121] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0122] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0123] GGTCTTGCATTTTGGAGTCGACATCAATGAAgttAATAATGGGCATTATACTTTTACTT

[0124] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0125] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0126] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0127] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0128] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0129] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0130] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0131] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC

[0132] AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC

[0133] ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG

[0134] TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA

[0135] GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG

[0136] ACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCAC

[0137] TGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGAA

[0138] GTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAGC

[0139] GGCACTTAAAAGAAATAAAAAATAG

[0140] SEQ ID NO.6 (Nucleotide sequence of ARO10-I335L)

[0141] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0142] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0143] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0144] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0145] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0146] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0147] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0148] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0149] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0150] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0151] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0152] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0153] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0154] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0155] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0156] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0157] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0158] GGTCTTGCATTTTGGAGTCGACATCAATGAAttaAATAATGGGCATTATACTTTTACTT

[0159] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0160] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0161] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0162] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0163] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0164] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0165] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0166] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC

[0167] AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC

[0168] ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG

[0169] TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA

[0170] GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG

[0171] ACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCAC

[0172] TGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGAA

[0173] GTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAGC

[0174] GGCACTTAAAAGAAATAAAAAATAG

[0175] SEQ ID NO.7 (Nucleotide sequence of ARO10-I335M)

[0176] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0177] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0178] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0179] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0180] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0181] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0182] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0183] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0184] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0185] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0186] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0187] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0188] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0189] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0190] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0191] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0192] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0193] GGTCTTGCATTTTGGAGTCGACATCAATGAAatgAATAATGGGCATTATACTTTTACT

[0194] TATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGAC

[0195] ACTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAA

[0196] GAACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTA

[0197] ACTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAG

[0198] CATTATTACACAAGTTCACTTACAAGAGACGATGCCTAAATTTTTGAACCCTGGTGA

[0199] TGTTGTCGTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTT

[0200] CGCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGC

[0201] CGCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGG

[0202] CAACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAG

[0203] CACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAA

[0204] GTTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACC

[0205] AGGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGA

[0206] GACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCA

[0207] CTGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGA​​​​​​​​​​​​​​​​​​​​​​​​​​TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0217] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0218] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0219] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0220] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0221] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0222] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0223] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0224] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0225] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0226] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0227] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0228] GGTCTTGCATTTTGGAGTCGACATCAATGAAttgAATAATGGGCATTATACTTTTACTT

[0229] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0230] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0231] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0232] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0233] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0234] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0235] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0236] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC

[0237] AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC

[0238] ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG

[0239] TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA

[0240] GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG

[0241] ACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCAC

[0242] TGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGAA

[0243] GTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAGC

[0244] GGCACTTAAAAGAAATAAAAAATAG

[0245] SEQ ID NO.9 (Nucleotide sequence of ARO10-I335F)

[0246] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0247] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0248] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC<(

[0249] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0250] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0251] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT[[ID=]]

[0252] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0253] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0254] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0255] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0256] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTTGTTCCTGCAGATT

[0257] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0258] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0259] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0260] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0261] TTCCACTGTTATGGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0262] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0263] GGTCTTGCATTTTGGAGTCGACATCAATGAAttcAATAATGGGCATTATACTTTTACTT

[0264] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0265] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0266] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0267] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0268] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0269] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0270] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0271] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC

[0272] AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC

[0273] ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG

[0274] TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA

[0275] GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG

[0276] ACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCAC

[0277] TGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGAA

[0278] GTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAGC

[0279] GGCACTTAAAAGAAATAAAAAATAG

[0280] SEQ ID NO.10 (Nucleotide sequence of ARO10-I335P)

[0281] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC [[ID=​​​​​​​​​​​​​​​​​​​​​CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0290] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0291] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTTGTTCCTGCAGATT

[0292] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0293] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0294] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0295] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0296] TTCCACTGTTATGGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0297] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0298] GGTCTTGCATTTTGGAGTCGACATCAATGAAccaAATAATGGGCATTATACTTTTACT

[0299] TATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGAC

[0300] ACTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAA

[0301] GAACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTA

[0302] ACTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAG

[0303] CATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGA

[0304] TGTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTT

[0305] CGCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGC

[0306] CGCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGG

[0307] CAACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAG

[0308] CACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAA

[0309] GTTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACC

[0310] AGGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGA

[0311] GACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCA

[0312] CTGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGA

[0313] AGTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAG

[0314] CGGCACTTAAAAGAAATAAAAAATAG

[0315] SEQ ID NO.11 (Nucleotide sequence of ARO10-I33S)

[0316] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0317] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0318] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0319] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0320] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0321] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0322] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0323] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0324] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0325] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0326] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0327] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0328] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0329] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0330] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0331] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0332] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0333] GGTCTTGCATTTTGGAGTCGACATCAATGAAtccAATAATGGGCATTATACTTTTACTT

[0334] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0335] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0336] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0337] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0338] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0339] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0340] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0341] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC

[0342] AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC

[0343] ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG

[0344] TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA

[0345] GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG

[0346] ACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCAC

[0347] TGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGAA

[0348] GTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAGC

[0349] GGCACTTAAAAGAAATAAAAAATAG

[0350] SEQ ID NO.12 (ARO10-I335T nucleotide sequence)

[0351] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0352] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0353] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0354] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0355] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0356] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0357] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0358] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0359] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0360] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0361] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0362] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0363] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0364] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0365] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0366] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0367] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0368] GGTCTTGCATTTTGGAGTCGACATCAATGAAactAATAATGGGCATTATACTTTTACTT

[0369] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0370] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0371] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0372] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0373] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0374] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0375] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0376] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC

[0377] AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC

[0378] ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG

[0379] TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA

[0380] GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG<​​​​​​​​​​​​​​​​​

[0387] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAGATTGTTGTCCATC

[0388] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0389] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0390] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0391] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0392] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0393] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0394] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0395] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0396] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTTGTTCCTGCAGATT

[0397] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0398] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0399] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0400] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0401] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0402] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0403] GGTCTTGCATTTTGGAGTCGACATCAATGAAtgcAATAATGGGCATTATACTTTTACTT

[0404] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0405] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0406] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0407] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0408] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0409] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0410] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0411] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC<00,00840><00,00841>AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC<00,00842><00,00843>ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG<00,00844><00,00845>TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA<00,00846><00,00847>GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG<00,00848><00,00849>ACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCAC<00,00850><00,00851>TGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGAA<00,00852><00,00853>GTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAGC<00,00854><00,00855>GGCACTTAAAAGAAATAAAAAATAG<00,00856>​​​​​​​​​​GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0424] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0425] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0426] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0427] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0428] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0429] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0430] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0431] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0432] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0433] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0434] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0435] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0436] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0437] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0438] GGTCTTGCATTTTGGAGTCGACATCAATGAAtatAATAATGGGCATTATACTTTTACTT

[0439] ATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGACA

[0440] CTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAAG

[0441] AACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTAA

[0442] CTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAGC

[0443] ATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGAT

[0444] GTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTTC

[0445] GCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGCC

[0446] GCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGGC

[0447] AACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAGC

[0448] ACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAAG

[0449] TTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACCA

[0450] GGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGAG

[0451] ACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCAC

[0452] TGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGAA

[0453] GTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAGC

[0454] GGCACTTAAAAGAAATAAAAAATAG

[0455] SEQ ID NO.15 (Nucleotide sequence of ARO10-I335N)

[0456] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0457] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0458] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0459] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0460] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0461] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0462] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0463] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0464] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0465] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0466] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0467] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0468] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0469] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0470] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0471] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0472] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0473] GGTCTTGCATTTTGGAGTCGACATCAATGAAaatAATAATGGGCATTATACTTTTACT

[0474] TATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGAC

[0475] ACTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAA

[0476] GAACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTA

[0477] ACTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAG

[0478] CATTATTACACAAGTTCACTTACAAGAGACGATGCCTAAATTTTTGAACCCTGGTGA

[0479] TGTTGTCGTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTT

[0480] CGCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGC

[0481] CGCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGG

[0482] CAACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAG

[0483] CACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAA

[0484] GTTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACC

[0485] AGGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGA

[0486] GACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCA

[0487] CTGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGA

[0488] AGTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAG

[0489] CGGCACTTAAAAGAAATAAAAAATAG

[0490] SEQ ID NO.16 (Nucleotide sequence of ARO10 - I335D)

[0491] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0492] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0493] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0494] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0495] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0496] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0497] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0498] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0499] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0500] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0501] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0502] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0503] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0504] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0505] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0506] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0507] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0508] GGTCTTGCATTTTGGAGTCGACATCAATGAAgacAATAATGGGCATTATACTTTTACT

[0509] TATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGAC

[0510] ACTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAA

[0511] GAACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTA

[0512] ACTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAG

[0513] CATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGA

[0514] TGTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTT

[0515] CGCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGC

[0516] CGCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGG

[0517] CAACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAG

[0518] CACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAA

[0519] GTTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACC

[0520] AGGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGA

[0521] GACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCA

[0522] CTGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGA

[0523] AGTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAG

[0524] CGGCACTTAAAAGAAATAAAAAATAG

[0525] SEQ ID NO.17 (Nucleotide sequence of ARO10-I335E)

[0526] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0527] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0528] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0529] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC​​​​​​​​​

[0533] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0534] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0535] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0536] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTTCCTGCAGATT

[0537] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0538] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0539] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0540] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0541] TTCCACTGTTATGGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0542] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0543] GGTCTTGCATTTTGGAGTCGACATCAATGAAgaaAATAATGGGCATTATACTTTTACT

[0544] TATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGAC

[0545] ACTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAA

[0546] GAACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTA

[0547] ACTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAG

[0548] CATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGA

[0549] TGTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTT

[0550] CGCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGC

[0551] CGCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGG

[0552] CAACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAG

[0553] CACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAA

[0554] GTTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACC

[0555] AGGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGA

[0556] GACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCA

[0557] CTGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGA

[0558] AGTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAG

[0559] CGGCACTTAAAAGAAATAAAAAATAG

[0560] SEQ ID NO.18 (Nucleotide sequence of ARO10-I335K)

[0561] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0562] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0563] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0564] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0565] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0566] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0567] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0568] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0569] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0570] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0571] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTTGTTCCTGCAGATT

[0572] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0573] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0574] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0575] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0576] TTCCACTGTTATGGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0577] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0578] GGTCTTGCATTTTGGAGTCGACATCAATGAAaagAATAATGGGCATTATACTTTTACT

[0579] TATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGAC

[0580] ACTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAA

[0581] GAACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTA

[0582] ACTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAG

[0583] CATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGA

[0584] TGTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTT

[0585] CGCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGC

[0586] CGCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGG

[0587] CAACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAG

[0588] CACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAA

[0589] GTTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACC

[0590] AGGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGA

[0591] GACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCA

[0592] CTGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGA

[0593] AGTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAG

[0594] CGGCACTTAAAAGAAATAAAAAATAG

[0595] SEQ ID NO.19 (Nucleotide sequence of ARO10-I335H)

[0596] ATGGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGTTTCC

[0597] AACCGATCAGCAACAATTCCGTTTGGTGAATACATATTTAAAAGATTGTTGTCCATC

[0598] GATACGAAATCAGTTTTCGGTGTTCCTGGTGACTTCAACTTATCTCTATTAGAATATC

[0599] TCTATTCACCTAGTGTTGAATCAGCTGGCCTAAGATGGGTCGGCACGTGTAATGAAC

[0600] TGAACGCCGCTTATGCGGCCGACGGATATTCCCGTTACTCTAATAAGATTGGCTGTT

[0601] TAATAACCACGTATGGCGTTGGTGAATTAAGCGCCTTGAACGGTATAGCCGGTTCGT

[0602] TCGCTGAAAATGTCAAAGTTTTGCACATTGTTGGTGTGGCCAAGTCCATAGATTCGC

[0603] GTTCAAGTAACTTTAGTGATCGGAACCTACATCATTTGGTCCCACAGCTACATGATT

[0604] CAAATTTTAAAGGGCCAAATCATAAAGTATATCATGATATGGTAAAAGATAGAGTC

[0605] GCTTGCTCGGTAGCCTACTTGGAGGATATTGAAACTGCATGTGACCAAGTCGATAAT

[0606] GTTATCCGCGATATTTACAAGTATTCTAAACCTGGTTATATTTTTGTTCCTGCAGATT

[0607] TTGCGGATATGTCTGTTACATGTGATAATTTGGTTAATGTTCCACGTATATCTCAACA

[0608] AGATTGTATAGTATACCCTTCTGAAAACCAATTGTCTGACATAATCAACAAGATTAC

[0609] TAGTTGGATATATTCCAGTAAAACACCTGCGATCCTTGGAGACGTACTGACTGATAG

[0610] GTATGGTGTGAGTAACTTTTTGAACAAGCTTATCTGCAAAACTGGGATTTGGAATTT

[0611] TTCCACTGTTATGGGAAAATCTGTAATTGATGAGTCAAACCCAACTTATATGGGTCA

[0612] ATATAATGGTAAAGAAGGTTTAAAACAAGTCTATGAACATTTTGAACTGTGCGACTT

[0613] GGTCTTGCATTTTGGAGTCGACATCAATGAAcacAATAATGGGCATTATACTTTTACT

[0614] TATAAACCAAATGCTAAAATCATTCAATTTCATCCGAATTATATTCGCCTTGTGGAC

[0615] ACTAGGCAGGGCAATGAGCAAATGTTCAAAGGAATCAATTTTGCCCCTATTTTAAAA

[0616] GAACTATACAAGCGCATTGACGTTTCTAAACTTTCTTTGCAATATGATTCAAATGTA

[0617] ACTCAATATACGAACGAAACAATGCGGTTAGAAGATCCTACCAATGGACAATCAAG

[0618] CATTATTACACAAGTTCACTTACAAAAGACGATGCCTAAATTTTTGAACCCTGGTGA

[0619] TGTTGTCGTTTGTGAAACAGGCTCTTTTCAATTCTCTGTTCGTGATTTCGCGTTTCCTT

[0620] CGCAATTAAAATATATATCGCAAGGATTTTTCCTTTCCATTGGCATGGCCCTTCCTGC

[0621] CGCCCTAGGTGTTGGAATTGCCATGCAAGACCACTCAAACGCTCACATCAATGGTGG

[0622] CAACGTAAAAGAGGACTATAAGCCAAGATTAATTTTGTTTGAAGGTGACGGTGCAG

[0623] CACAGATGACAATCCAAGAACTGAGCACCATTCTGAAGTGCAATATTCCACTAGAA

[0624] GTTATCATTTGGAACAATAACGGCTACACTATTGAAAGAGCCATCATGGGCCCTACC

[0625] AGGTCGTATAACGACGTTATGTCTTGGAAATGGACCAAACTATTTGAAGCATTCGGA

[0626] GACTTCGACGGAAAGTATACTAATAGCACTCTCATTCAATGTCCCTCTAAATTAGCA

[0627] CTGAAATTGGAGGAGCTTAAGAATTCAAACAAAAGAAGCGGGATAGAACTTTTAGA

[0628] AGTCAAATTAGGCGAATTGGATTTCCCCGAACAGCTAAAGTGCATGGTTGAAGCAG

[0629] CGGCACTTAAAAGAAATAAAAAATAG

[0630] The application of the mutant described in this invention includes in vivo application in *Saccharomyces cerevisiae*. This in vivo application involves mutating the amino acid sequence of the phenylpyruvate decarboxylase in the *Saccharomyces cerevisiae* genome, followed by fermentation to obtain the metabolite.

[0631] Specifically, it includes the following steps:

[0632] 1) A mutant strain was formed by replacing the isoleucine amino acid residue at position 335 of the ARO10 gene encoding phenylpyruvate decarboxylase in the genome of a Saccharomyces cerevisiae strain with other amino acid residues.

[0633] 2) The above-obtained Saccharomyces cerevisiae expressing the ARO10 mutant protein of phenylpyruvate decarboxylase was cultured to obtain Saccharomyces cerevisiae seed culture.

[0634] 3) Inoculate the brewer's yeast seed liquid into YPD fermentation medium and ferment to obtain fermentation broth.

[0635] 4) The yield of tyrosol was determined using high performance liquid chromatography.

[0636] This invention compares mutant and wild-type strains by mutating different ARO10 amino acid sites on the genome of *Saccharomyces cerevisiae* and comparing the tyrosol production differences between different mutant strains and wild-type strains. This experiment uses high-performance liquid chromatography (HPLC) to detect the amount of tyrosol produced to determine the conversion rate; specific experimental methods and data are described in the invention's methodology. The *Saccharomyces cerevisiae* strain expressing the phenylpyruvate decarboxylase ARO10 mutant of this invention exhibits significantly higher tyrosol production than the wild type, showing promising application potential.

[0637] The phenylpyruvate decarboxylase ARO10 mutant provided by this invention and the raw materials and reagents used in its application are all commercially available.

[0638] The present invention will be further illustrated below with reference to the embodiments:

[0639] Example 1

[0640] 1) The gene encoding phenylpyruvate decarboxylase ARO10 (SEQ.ID.NO.2) was inserted into the vector PUC19-ARO10-HR containing 1000bp homologous arms upstream and downstream of the ARO10 genome to form an expression cassette.

[0641] SEQ ID NO.2 (ARO10 nucleotide sequence)

[0642]

[0643] 2) Point mutation was used to obtain the ARO10 mutant, and PCR amplification was performed using the primers in the table below.

[0644] Table 2

[0645]

[0646]

[0647] The PCR amplification conditions are as follows:

[0648] The PCR product underwent a pre-denaturation process at 95℃ for 10 min, followed by 26 cycles of 98℃ for 30 s, 58℃ for 30 s, and 72℃ for 5 min. A final extension at 72℃ for 5 min was performed. The product was then treated with DpnI for 2 hours, and the PCR product was recovered. It was then transformed into *E. coli* DH5α, and plasmid extraction and sequencing verification confirmed its correctness before use.

[0649] 3) In CEN.PK2-1C-308a:∷ARO4 K229L -ARO7 G141S -LEU3∷ARO3 D154N In the genome of the -ΔARO10-HA strain, CRISPR / Cas9-mediated genome knock-in technology was used. The gene knock-in method uses a plasmid carrying the gene for the Cas9 protein and a sequence for transcribing sgRNA. The transcribed sgRNA binds to the Cas9 protein. Only a spacer gene paired with the target gene and a homologous recombination fragment need to be introduced into the plasmid. The reconstructed plasmid is then introduced into the cell. Because the designed homologous recombination fragment carries the ARO10 mutant expression cassette of phenylpyruvate decarboxylase, the target gene that was cut in the yeast eukaryotic cell is repaired by homologous recombination using the homologous arm on the plasmid as a template, thereby completing the gene knock-in.

[0650] Methods for constructing gene knock-in vectors:

[0651] This study employed a two-step method, GoldenGate assembly and GibsonAssembly assembly, to construct the vector.

[0652] 1. GoldenGate Assembly

[0653] GoldenGate assembly is generally used to assemble the pCas-lacZ plasmid to be used with Cas9 and the target spacer fragment. The pCas-lacZ gRNA has two BsaI restriction sites at its tip, so the spacer fragment also needs to have BsaI restriction sites added, leaving sticky ends complementary to the vector. Therefore, the method of assembling the spacer and pCas-lacZ using GoldenGate is described below.

[0654] 1) Obtaining double-stranded DNA from the spacer: Synthesize a pair of primers with sticky ends complementary to the vector and containing BsaI restriction sites (front primer: as shown in SEQ ID NO. 54: 5'-CTTTGGTCTCACTTT-GCGTAATCTGGAACATCGTA-GTTTAGAGACCTTTC-3', back primer: as shown in SEQ ID NO. 55: 5'-GAAAGGTCTCTAAAC-TACGATGTTCCAGATTACGC-AAAGTGAGACCAAAG-3'). Prepare the reaction mixture as follows: 1 μl of front primer (100 μM), 1 μl of back primer (100 μM), 1 μl of T4 DNA ligase buffer (NEB), and add ddH2O to bring the volume to 10 μl. Finally, place the prepared reaction mixture into a PCR instrument and react according to the following program to obtain the double-stranded DNA fragment. After the reaction is complete, incubate at 95℃ for 5 minutes, then slowly cool the PCR instrument (1℃ / min) to 25℃.

[0655] 2) GoldenGate assembly: First, dilute the double-stranded DNA obtained in the previous step 10 times with ddH2O, then prepare the reaction according to the system in Table 3, and react according to the procedure in Table 4 (about 4 hours).

[0656] Table 3 GoldenGate reaction system

[0657]

[0658] Table 4. GoldenGate's reaction procedure

[0659]

[0660] 2. Gibson Assembly

[0661] 1) Obtaining the linearized vector: The linearized vector is obtained using restriction endonucleases or PCR. The digestion system is prepared according to the instructions of the corresponding NEB enzyme, generally consisting of 5 μl of 10× reaction buffer, 1 μl of restriction endonuclease (double digestion, adding 1 μl of the corresponding enzyme to each enzyme), 1 μg of vector DNA, and ddH2O to bring the total volume to 50 μl. The prepared digestion system is then incubated at 37°C for 1-3 hours using a thermal cycler (PCR instrument). For single digestion, 1 μl of CIP can be added after digestion, and the mixture can be incubated at 37°C for 1 hour before recovery.

[0662] 2) Obtaining DNA to be assembled: The target gene is amplified by PCR, and at least 15 nt of the same sequence (homologous arm) as the ends of other DNA fragments to be assembled is added to the primers. The DNA with the homologous arm is then recovered.

[0663] 3) Assembly: Using the commercially available Gibson Assembly kit ( The HiFi DNAAssembly Cloning Kit assembles DNA fragments (including linearized vectors) with homologous arms. The kit is prepared according to the manufacturer's instructions (5 μl GA Mix, 50-100 ng of each DNA fragment, add ddH2O to bring the volume to 10 μl), and incubate at 50°C for 1 hour.

[0664] sgRNA: GCGTAATCTGGAACATCGTA (as shown in SEQ ID NO.56)

[0665] Homologous arm:

[0666] Left homologous arm: (as shown in SEQ ID NO.57)

[0667] 。

[0668] Right homologous arm: (as shown in SEQ ID NO.58)

[0669] 。

[0670] Thus, an engineered Saccharomyces cerevisiae strain expressing the above mutant of phenylpyruvate decarboxylase ARO10 was obtained.

[0671] Example 2: Strain Construction (Transformation-Validation)

[0672] 1. Inoculate with the target strain (CEN.PK2-1C-308a::ARO4) K229L -ARO7 G141S -LEU3∷ARO3 D154N -ΔARO10-HA) was added to 4ml of YPD and incubated overnight at 30℃;

[0673] 2. Take 2ml of bacterial culture into 50ml of YPD, incubate at 30℃ for 6h with shaking, OD600=0.8-1.0;

[0674] 3. Centrifuge at 3000 rpm for 2 minutes, then discard the supernatant;

[0675] 4. Boil the ssDNA sample for 10 minutes and then quickly place it in ice;

[0676] 5. Resuspend the bacterial cells in 50ml of sterile water and centrifuge.

[0677] 6. Resuspend in 1 ml of 100 mM LiAc, aspirate the supernatant at 12000 rpm for 15 seconds;

[0678] 7. Resuspend the cells to a final volume of 500 μl, add approximately 400 μl of 100 mM LiAc, divide into 50 μl portions per tube, centrifuge, and discard the supernatant;

[0679] 8. Add the "conversion mixture" in the following order:

[0680] 240μl PEG3350 (50%)

[0681] 36μl 1M LiAc

[0682] 10 μl ssDNA (10 mg / ml) (Manufacturer: Solarbio, Product Number: Cat.NO.H1060)

[0683] 50 μl of Cas9 plasmid containing a spacer and ARO10 mutations and upstream and downstream homologous arms.

[0684] 9. Vortex each reaction tube until the cells are completely mixed;

[0685] 10. Keep warm at 30℃ for 30 minutes;

[0686] 11. Place in a 42℃ heat shock for 25 minutes;

[0687] 12. Incubate in YPD medium for 4 hours at 30°C and 220 rpm;

[0688] 13. Coated plate YPD-G418 resistance plate.

[0689] The above describes the transformation process;

[0690] 14. Clones grow on the plates to be transformed. Select single colonies and PCR is performed to obtain the target fragment. Sanger sequencing is used to verify whether the ARO10 mutant with phenylpyruvate decarboxylase is correctly knocked into the Saccharomyces cerevisiae.

[0691] 15. Select the successfully verified clones and incubate them in YPD for 24 hours at 30°C and 220 rpm.

[0692] Example 3 Fermentation Method

[0693] 1. Culture the Saccharomyces cerevisiae strain on YPD plates to activate the Saccharomyces cerevisiae strain;

[0694] 2. Pick a single clone into 4 ml of YPD liquid and incubate for 18 hours until OD. 600 = Between 1 and 10;

[0695] 3. Transfer 4 ml of YPD culture medium to 50 ml of YPD medium at a ratio of 1.5:50, and incubate at 30°C and 220 rpm for 96 hours.

[0696] 4. Collect the supernatant of the fermentation broth to obtain a fermentation broth containing tyrosol.

[0697] Example 4 Detection Method

[0698] The detection methods of the present invention all utilize high performance liquid chromatography to detect the content of tyrosol.

[0699] General HPLC conditions: Fermentation broth / reaction solution loading volume: 10 μL. Mobile phases: Phase A is 0.1% formic acid aqueous solution, and Phase D is acetonitrile. High-performance liquid chromatography (HPLC) was performed using an Agilent 1260 system with an Agilent EC-C18 column (Agilent, 4 μm, 4.6 mm × 150 mm). The mobile phases were: A (0.1% v / v) formic acid aqueous solution and D (chromatographic grade acetonitrile). The flow rate was: 0-11 min: 94% A solution and 6% D solution; 11-14 min: 94% A solution decreased linearly to 4%, and 6% D solution increased linearly to 96%; 14-16 min: 4% A solution and 96% D solution; 16-21 min: 94% A solution and 6% D solution. Detection wavelength: 224 nm; column oven temperature: 35 °C; flow rate: 1 mL / min. The peak elution time of tyrosol was 10.67 min.

[0700] The following table shows the tyrosol yields of ARO10 mutants expressing different phenylpyruvate decarboxylases. A t-test was used to analyze the differences between the mutants and the wild type, and the results are shown in the figure below. Figure 1 As shown:

[0701] Table 5

[0702] ARO10 Tyrosol production (mg / L) p-value Analysis of significant differences wild type 428±1.44 1 No significant difference I335G 793±3.83 0.0000000237336130627 P<0.01, highly significant difference I335A 838±10.06 0.0000006436 P<0.01, highly significant difference I335V 610±4.72 0.00000080960782605576 P<0.01, highly significant difference I335L 468±9.14 0.00358331561169367 P<0.01, highly significant difference I335M 465±5.18 0.000632297889955527 P<0.01, highly significant difference I335W 466±4.14 0.000253684 P<0.01, highly significant difference I335F 591±17.77 0.0002854642 P<0.01, highly significant difference I335P 738±9.02 0.00000112425350814688 P<0.01, highly significant difference I335S 830±3.80 0.0000000181 P<0.01, highly significant difference I335T 759±10.16 0.000001615 P<0.01, highly significant difference I335C 719±22.47 0.0000523863450628734 P<0.01, highly significant difference I335Y 587±21.77 0.0006868038 P<0.01, highly significant difference I335N 636±11.96 0.0000213844 P<0.01, highly significant difference I335D 745±6.83 0.00000052630 P<0.01, highly significant difference I335E 1077±16.47 0.00000085038382660502 P<0.01, highly significant difference I335K 896±7.83 0.0000001401 P<0.01, highly significant difference I335H 756±4.26 0.0000000630 P<0.01, highly significant difference

[0703] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A phenylpyruvate decarboxylase ARO10 mutant, characterized in that, The phenylpyruvate decarboxylase ARO10 mutant was obtained by mutating the isoleucine (I335) amino acid residue at position 335 of the amino acid sequence of phenylpyruvate decarboxylase ARO10 shown in SEQ ID No. 1 to other amino acid residues.

2. The phenylpyruvate decarboxylase ARO10 mutant as described in claim 1, characterized in that, The mutations include, but are not limited to, mutations that are one or more of the following: G, A, V, L, M, W, F, P, S, T, C, Y, N, D, E, K, or H.

3. A nucleic acid molecule encoding the phenylpyruvate decarboxylase ARO10 mutant as described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, It has the following characteristics: (I) A nucleotide sequence as shown in any one of SEQ ID NO. 3 to 19; or (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or (IV) and nucleotide sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequences described in (I), (II) or (III).

5. A recombinant expression vector, expression cassette, or construct, characterized in that, It contains the nucleic acid molecule as described in claim 3 or 4.

6. The host, characterized in that, Transfection or transformation may be performed on the recombinant expression vector, expression cassette, or construct as described in claim 5.

7. The host as described in claim 6, characterized in that, The host includes transgenic cell lines and / or recombinant strains; Preferably, the recombinant strain includes an engineered strain of Saccharomyces cerevisiae.

8. Any of the following applications in the preparation of tyrosol; (i) The phenylpyruvate decarboxylase ARO10 mutant as described in claim 1 or 2; (ii) The nucleic acid molecule as described in claim 3 or 4; (iii) The recombinant expression vector, expression cassette, or construct as described in claim 5; (iv) The host as described in claim 6 or 7.

9. The method for preparing the phenylpyruvate decarboxylase ARO10 mutant as described in claim 1 or 2, characterized in that, Fermentation is carried out on the host as described in claim 6 or 7 to obtain a fermentation broth containing the phenylpyruvate decarboxylase ARO10 mutant as described in claim 1 or 2.

10. A method for preparing tyrosol, characterized in that, Using 4-hydroxyphenylpyruvic acid as a substrate, the phenylpyruvic acid decarboxylase ARO10 mutant as described in claim 1 or 2 was expressed in Saccharomyces cerevisiae. YPD medium with pH 5-9 was used as the reaction medium, and fermentation was carried out at 30-37°C and 0-600 rpm. The fermentation broth was collected, separated, and purified to obtain tyrosol.

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