Engineering bacterium for inhibiting synthesis of dodecanedioic acid as well as construction method and application of engineering bacterium

Abstract

The invention provides an engineering bacterium for inhibiting synthesis of dodecanedioic acid. The expression of related redox genes is knocked out or down-regulated in a genome of the engineering bacterium. The engineering bacterium can effectively reduce the production of a by-product dodecanedioic acid and prevent the peroxidation of 12-hydroxydodecanoic acid in the synthesis process of the nylon-12 monomer. The invention also provides a construction method of the engineering bacterium for inhibiting synthesis of dodecanedioic acid, a nylon-12 monomer is synthesized by utilizing a quorum sensing system in vibrio fischeri in escherichia coli, and the established escherichia coli containing a self-induction system can be used for producing the nylon-12 monomer from glucose; and the generation of a by-product dodecanedioic acid can be effectively reduced. When fermentation is carried out in a shake flask for 72 hours, the yield of the nylon-12 monomer is greater than 100 mg / L, and the yield of the by-product dodecanedioic acid is reduced to be less than 200 mg / L (the proportion in the product is less than 20%).

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial engineering, and particularly relates to an engineering bacterium for inhibiting the synthesis of dodecanedioic acid, a construction method thereof, and an application thereof. Background Art

[0002] Escherichia coli is widely used in fields such as enzyme engineering, metabolic engineering and pharmaceutical engineering, and is the most commonly used strain in genetic engineering. At present, the genetic modification strategy commonly used in Escherichia coli is mainly to carry out static regulation through gene overexpression and gene knockout. Although these strategies have improved the production performance of the strain to a certain extent, they are usually unable to balance the growth of the strain and the synthesis of the target product. In addition, although the inducible promoter commonly used in Escherichia coli can achieve the balance of growth and production to a certain extent, due to the expensive and toxic inducer, it is restricted in the large-scale production process. Therefore, it is necessary to develop a dynamic control element with wide adaptability to adapt to different industrial production processes.

[0003] Quorum sensing is a system used by microorganisms to regulate gene expression. The inducer is a signaling molecule secreted by the microorganism itself. As the microorganism grows and its population density increases, the concentration of the inducer accumulates. When the inducer concentration reaches a threshold, the expression of the relevant genes is induced. Quorum sensing systems vary among microorganisms. For example, the LuxI / LuxR system in Vibrio fischeri: as cell density increases, the amount of the signaling molecule acylhomoserine lactone (AHL) synthesized by the signaling molecule synthase LuxI increases. AHL freely enters and exits the cell along a concentration gradient, accumulating inside and outside the cell. When the AHL concentration reaches a certain threshold, AHL binds to the signaling molecule binding protein LuxR, forming a LuxR-AHL dimer. This dimer acts as an activator of the promoter Plux, thereby activating transcription of genes controlled by Plux.

[0004] Due to its excellent heat resistance, wear resistance, chemical resistance, UV resistance and scratch resistance, nylon 12 is widely used in the processing and manufacturing of pipes and automotive parts. At present, the industrial production process of nylon 12 monomer is synthesized using butadiene as raw material through a series of steps such as trimerization, catalytic hydrogenation, oxidation, ketoneization, and Beckmann rearrangement (opening of the N double bond and addition of groups). However, butadiene is produced by crude oil refining. Due to the non-renewable nature of crude oil and the environmental damage caused by chemical production, the chemical synthesis process of nylon 12 is limited. Therefore, it is necessary to develop an environmentally friendly, green and renewable 12 nylon monomer synthesis process. Currently, mild reaction conditions and environmentally friendly biosynthesis processes are highly favored.

[0005] Heterologous synthesis of nylon dodecanone in Escherichia coli requires the expression of four additional enzymes: 1. The lauroyl-ACP thioesterase (UcfatB) converts acetyl-CoA to dodecanoic acid; 2. Dodecanoic acid is converted to 12-hydroxy-12-decanoic acid under the catalysis of the cytochrome P450 enzyme (CYP153A-BM3); 3. 12-hydroxy-12-decanoic acid is finally converted to nylon dodecanone through the action of dehydrogenase (BsADH) and transaminase (CV2025). However, the current synthesis process of nylon dodecanone is prone to peroxidation in the cell, resulting in the production of a large amount of dodecanedioic acid as a byproduct. Summary of the Invention

[0006] The purpose of the present invention is to overcome the problem in the prior art that a large amount of by-product dodecanedioic acid is generated during the microbial synthesis of nylon dodecanone.

[0007] To this end, the present invention provides an engineered bacterium for inhibiting the synthesis of dodecanedioic acid, wherein the expression of the paoB encoding gene, the aldB encoding gene, the puuC encoding gene, the betB encoding gene, the patD encoding gene, the feaB encoding gene or the gabD encoding gene, the aldA encoding gene, the astD encoding gene, the putA encoding gene, the sad encoding gene, the dkgB encoding gene, the yeaE encoding gene, the yahK encoding gene, the yqhD encoding gene, the dkgA encoding gene, and the yqhC encoding gene is knocked out or downregulated in the genome of the engineered bacterium.

[0008] Specifically, the genetically engineered bacteria are prepared from Escherichia coli.

[0009] The present invention also provides a method for constructing an engineered bacterium for inhibiting the synthesis of dodecanedioic acid, comprising the following steps:

[0010] (1) Starting from Escherichia coli MG1655, the genes encoding luxI, luxR, and UcfatB and the promoters controlling the expression of these genes were integrated into the tesA gene site of the Escherichia coli MG1655 genome to construct strain M22;

[0011] (2) Based on the M22 strain, the same sequence as in step (1) was integrated into the ad hE gene locus to obtain strain M73;

[0012] (3) Based on the M73 strain, the CYP153A-BM3 encoding gene and the promoter controlling CYP153A-BM3 expression were integrated into the fadE site of Escherichia coli to obtain strain M209;

[0013] (4) Based on the M209 strain, the CYP153A-BM3 encoding gene and the promoter controlling CYP153A-BM3 expression were integrated into the pgi site of Escherichia coli to obtain the M220 strain;

[0014] (5) Based on the M220 strain, the expression of the paoB encoding gene, aldB encoding gene, puuC encoding gene, betB encoding gene, patD encoding gene, feaB encoding gene or gabD encoding gene, aldA encoding gene, astD encoding gene, putA encoding gene, sad encoding gene, dkgB encoding gene, yeaE encoding gene, yahK encoding gene, yq hD encoding gene, dkgA encoding gene, and yqhC encoding gene was inhibited to obtain the strain M230;

[0015] (6) The genes encoding CV2025 and BsADH and the promoters controlling the expression of CV2025 and BsAD H were cloned into the pCDFDuet plasmid to obtain a recombinant plasmid, which was then transformed into the strain M230 to obtain an engineered bacterium that inhibits the synthesis of dodecanedioic acid.

[0016] Specifically, the promoter for controlling the expression of luxI and UcfatB in the above step (1) is selected from P lux ; The promoter controlling luxR expression is selected from P tet .

[0017] Specifically, the promoter for controlling the expression of CYP153A-BM3 in the above steps (3) and (4) is selected from P lux .

[0018] Specifically, the promoter for controlling the expression of CV2025 and BsADH in the above step (6) is selected from P M12 .

[0019] The present invention also provides a method for producing nylon dodecanedioic acid monomer, comprising the following steps: inoculating the above-mentioned engineered bacteria that inhibit the synthesis of dodecanedioic acid into a culture medium for fermentation, and collecting the nylon dodecanedioic acid monomer in the fermentation product.

[0020] Specifically, the culture medium is TB culture medium supplemented with 4-40 g / L glucose, 0.1-1 g / L ferrous sulfate heptahydrate, 5-50 mM 5-aminolevulinic acid hydrochloride, 50-500 mM L-alanine, and 0.1-1 mM pyridoxal-5-phosphate.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] The engineered bacteria for inhibiting the synthesis of dodecanedioic acid provided by the present invention can effectively reduce the production of the byproduct dodecanedioic acid and prevent the overoxidation of 12-hydroxydodecanoic acid during the synthesis process of nylon dodecamonomer.

[0023] The method for constructing an engineered bacterium that inhibits dodecanedioic acid synthesis, provided by the present invention, utilizes the quorum sensing system of Vibrio fischeri to synthesize nylon dodecanedioic acid in Escherichia coli. The resulting E. coli, containing an autoinduction system, can produce nylon dodecanedioic acid from glucose while effectively reducing the production of the byproduct dodecanedioic acid. After 72 hours of shake flask fermentation, the yield of nylon dodecanedioic acid was greater than 100 mg / L, while the byproduct dodecanedioic acid yield was reduced to less than 200 mg / L (accounting for less than 20% of the product). DETAILED DESCRIPTION

[0024] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0025] The effects of the engineered bacteria for inhibiting the synthesis of dodecanedioic acid and the construction method thereof of the present invention are studied below through specific examples.

[0026] Example 1:

[0027] This embodiment provides an engineered bacterium that inhibits the synthesis of dodecanedioic acid, which is prepared by the following steps.

[0028] (1) Starting from Escherichia coli MG1655, the genes encoding luxI, luxR, and UcfatB and the promoters controlling their expression were integrated into the tesA gene locus of the E. coli genome to construct strain M22;

[0029] The promoters controlling the expression of luxI and UcfatB were selected from P lux ; The promoter controlling luxR expression is selected from P tet The genotype of strain M22 is tesA::P tet _luxR-P lux _luxI_UcfatB.

[0030] (2) Based on the M22 strain, the same sequence as in step (1) was integrated into the ad hE gene locus to obtain strain M73, whose genotype was tesA::P tet_luxR-P lux _luxI_Uc fatB, adhE::P tet _luxR-P lux _luxI_UcfatB;

[0031] (3) Based on the M73 strain, the gene encoding the cytochrome P450 enzyme (CYP153A-BM3) and the promoter controlling the expression of CYP153A-BM3 were integrated into the fadE site of Escherichia coli to obtain the strain M209;

[0032] The promoter for controlling the expression of cytochrome P450 enzyme (CYP153A-BM3) was selected from P lux The genotype of strain M209 is tesA::P tet _luxR-P lux _luxI_UcfatB, adhE::P tet _luxR-P lux _luxI_UcfatB, fadE::P lux _CYP153A-BM3.

[0033] (4) Based on the M209 strain, the CYP153A-BM3 encoding gene and the promoter controlling CYP153A-BM3 expression were integrated into the pgi site of Escherichia coli to obtain the M220 strain;

[0034] The promoter for controlling the expression of cytochrome P450 enzyme (CYP153A-BM3) was selected from P lux The strain M220 genotype is tesA::P tet _luxR-P lux _luxI_UcfatB, adhE::P tet _luxR-P lux _luxI_UcfatB, fadE::P lux _CYP153A-BM3, pgi::P lux _CYP153A-BM 3.

[0035] (5) Based on the M220 strain, the expression of the paoB encoding gene, aldB encoding gene, puuC encoding gene, betB encoding gene, patD encoding gene, feaB encoding gene, gabD encoding gene, aldA encoding gene, astD encoding gene, putA encoding gene, sad encoding gene, dkgB encoding gene, yeaE encoding gene, yahK encoding gene, yqhD encoding gene, dkgA encoding gene, and yqhC encoding gene was inhibited to obtain the strain M230;

[0036] The genotype of strain M230 is tesA::P tet _luxR-P lux _luxI_UcfatB, adhE::P tet _luxR-P lux _luxI_UcfatB, fadE::P lux _CYP153A-BM3, pgi::P lux _CYP153A-BM3, △paoB, △aldB, △puuC, △betB, △patD, △feaB, △gab D, △aldA, △astD, △putA, △sad, △dkgB, △yeaE, △yahK, △yqhD, △dkgA, △yqhC.

[0037] (6) The genes encoding CV2025 and BsADH and the promoters controlling the expression of CV2025 and BsADH were cloned into the pCDFDuet plasmid to obtain a recombinant plasmid. The promoters controlling the expression of CV2025 and BsADH were selected from P M12 , the gene and promoter were cloned into the pCDFDuet plasmid to construct the plasmid pCDFDuet-PM12_CV2025_BsADH.

[0038] The recombinant plasmid was transformed into M230 to obtain the engineered bacteria M240 that inhibits the synthesis of dodecanedioic acid.

[0039] The P lux The nucleotide sequence of luxI is shown in SEQ ID NO 1; the amino acid sequence of luxI is shown in SEQ ID NO 2; the amino acid sequence of luxR is shown in SEQ ID NO 3; the P tet The nucleotide sequence of P is shown in SEQ ID NO 4; M12The nucleotide sequence of UcfatB is shown in SEQ ID NO 5; the nucleotide sequence of UcfatB is shown in SEQ ID NO 6; the nucleotide sequence of CYP153A-BM3 is shown in SEQ ID NO 7; the nucleotide sequence of aldehyde dehydrogenase (PaoB) is shown in SEQ ID NO 8; the nucleotide sequence of acetaldehyde dehydrogenase B (AldB) is shown in SEQ ID NO 9; the nucleotide sequence of aldehyde dehydrogenase (PuuC) is shown in SEQ ID NO 10; the nucleotide sequence of betaine aldehyde dehydrogenase (BetB) is shown in SEQ ID NO 11; the nucleotide sequence of aldehyde dehydrogenase (PatD) is shown in SEQ ID NO 12; the nucleotide sequence of phenylacetaldehyde dehydrogenase (FeaB) is shown in SEQ ID NO 13; the nucleotide sequence of succinate semialdehyde dehydrogenase (GabD) is shown in SEQ ID NO 14; the nucleotide sequence of CV2025 is shown in SEQ ID NO 15; the nucleotide sequence of BsADH is shown in SEQ ID NO 16; the nucleotide sequence of the acetaldehyde dehydrogenase A (AldA) is shown in SEQ ID NO 17; the nucleotide sequence of the aldehyde dehydrogenase (AstD) is shown in SEQ ID NO 18; the nucleotide sequence of the 1-pyrrole-5-carboxylic acid dehydrogenase (PutA) is shown in SEQ ID NO 19; the nucleotide sequence of the succinate semialdehyde dehydrogenase (Sad) is shown in SEQ ID NO 20; the nucleotide sequence of the methylglyoxal reductase (DkgB) is shown in SEQ ID NO 21; the nucleotide sequence of the methylglyoxal reductase (YeaE) is shown in SEQ ID NO 22; the nucleotide sequence of the aldehyde reductase (YahK) is shown in SEQ ID NO 23; the nucleotide sequence of the aldehyde reductase (YqhD) is shown in SEQ ID NO 24; the nucleotide sequence of the methylglyoxal reductase (DkgA) is shown in SEQ ID NO 25; and the nucleotide sequence of the transcription activator (YqhC) is shown in SEQ ID NO 26.

[0040] SEQ ID NO 1:

[0041] ACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGT TATAGTCGAATAAA

[0042] SEQ ID NO 2:

[0043] <h2 style=";text-align:left;direction:ltr">MTIMIKKSDFLAIPSEEYKGILSLRYQVFKQRLEWDLVVENNLESDEYDNSNAEYIYACDDTENVSGCWRLLPTTGDYMLKSVFPELLGQQSAPKDP NIVELSRFAVGKNSSKINNSASEITMKLFEAIYKHAVSQGITEYVTVTSTAIERFLKRIKVPCHRIGDKEIHVLGDTKSVVLSMPINEQFKKAVLN*<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0044] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO3:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0045] <h2 style=";text-align:left;direction:ltr"> MKNINADDTYRIINKIKACRSNNDINQCLSDMTKMVHCEYYLLAIIYPHSMVKSDISILDNYPKKWRQYYDDANLIKYDPIVDYSNHSPINWNIFENNAVNKKSPNVIKEAKTSGLITGFSFP IHTANNGFGMLSFAHSEKDNYIDSLFLHACMNIPLIVPSLVDNYRKINIANNKSNNDLTKREKECLAWACEGKSSWDISKILGCSERTVTFHLTNAQMKLNTTNRCQSISKAILTGAIDCPYFKN*<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0046] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO4:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0047] <h2 style=";text-align:left;direction:ltr"> TCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGA GA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0048] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO5:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0049] <h2 style=";text-align:left;direction:ltr"> TTATCTCTGGCGGTGTTGACAAGAGATAACAACGTTGATATAATTGAGCCCTTTTGGTGCGTCAGTCAGTTTAAACCAGGAAACAGCT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0050] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO6:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0051] ATGCTGGAATGGAAACCAAAACCGAAACTGCCACAACTGCTGGACGATCACTTCGGCCTGCATGGTCTGGTGTTCCGTCGTACGTTCGCTATTCGCAGCTACGAAGTTGGTCCGGATCGCAGCACCTCTATCCTGGCTGTAATGAACCATATGCAGGAAGCGACCCTGAACCACGCTAAAAGCGTTGGTATTCTGGGCGACGGTTTCGGTACTACCCTGGAAATGTCCAAACGTGACCTGATGTGGGTGGTGCGTCGTACTCACGTGGCGGTTGAACGTTACCCGACTTGGGGCGATACTGTCGAAGTTGAATGTTGGATCGGCGCTTCCGGTAACAACGGTATGCGTCGCGATTTTCTGGTGCGCGATTGCAAAACCGGCGAAATTCTGACGCGCTGCACTTCCCTGTCTGTACTGATGAACACCCGCACCCGTCGCCTGTCTACCATCCCGGACGAAGTACGTGGTGAGATCGGTCCGGCGTTTATTGACAACGTGGCGGTTAAAGACGACGAAATCAAGAAGCTGCAGAAGCTGAACGACTCCACCGCAGACTATATCCAGGGTGGCCTGACCCCGCGCTGGAACGATCTGGACGTAAACCAGCACGTGAACAACCTGAAATACGTTGCCTGGGTTTTCGAAACCGTTCCGGACAGCATCTTCGAATCTCACCACATCTCTTCCTTCACCCTGGAGTACCGCCGTGAATGTACCCGCGATTCTGTTCTGCGTTCCCTGACTACCGTTTCCGGTGGTTCTTCTGAAGCTGGCCTGGTCTGTGATCATCTGCTGCAACTGGAAGGCGGTAGCGAGGTACTGCGTGCTCGCACTGAATGGCGCCCGAAACTGACGGATTCTTTCCGTGGCATTTCCGTGATCCCGGCAGAACCACGTGTT

[0052] SEQ ID NO7:

[0053] ATGCCTACCCTGCCGCGCACCTTCGACGACATCCAAAGCCGCCTGATCAATGCAACCTCTCGTGTTGTTCCGATGCAGCGCCA

[0054] GATCCAGGGTCTGAAATTTCTGATGTCCGCGAAACGTAAAACC

[0055] TTCGGTCCGCGCCGCCCAATGCCGGAATTCGTTGAGACCCCAA

[0056] TCCCGGATGTAAACACCCTGGCCCTGGAAGACATCGATGTTAG

[0057] CAATCCGTTCCTGTACCGTCAGGGCCAGTGGCGCGCATACTTT

[0058] AAGCGTCTGCGTGACGAGGCCCCGGTACATTACCAGAAAAACT

[0059] CCCCTTTCGGTCCGTTCTGGTCCGTGACTCGTTTCGAAGACATC

[0060] CTGTTCGTGGATAAAAGCCACGATCTGTTCAGCGCGGAACCTC

[0061] AGATCATCCTGGGCGATCCTCCTGAAGGTCTGTCCGTCGAGAT

[0062] GTTCATCGCGATGGATCCGCCGAAACACGACGTCCAGCGCTCT

[0063] TCTGTCCAGGGTGTTGTAGCGCCGAAAAACCTGAAAGAAATGG

[0064] AGGGTCTGATCCGCAGCCGTACCGGTGATGTTCTGGACTCTCT

[0065] GCCGACTGACAAACCGTTCAACTGGGTGCCGGCGGTTTCTAAA

[0066] GAACTGACCGGCCGTATGCTGGCTACCCTGCTGGACTTCCCGT

[0067] ATGAAGAACGTCATAAGCTGGTAGAATGGTCTGATCGTATGGC

[0068] TGGCGCTGCTTCCGCCACCGGCGGCGAATTCGCTGATGAAAAC

[0069] GCCATGTTCGACGATGCCGCTGATATGGCTCGTAGCTTCTCTCG

[0070] TCTGTGGCGTGACAAAGAAGCGCGTCGCGCCGCGGGCGAAGA

[0071] GCCGGGTTTCGACCTGATTAGCCTGCTGCAGTCCAACAAAGAA

[0072] ACCAAAGACCTGATTAACCGCCCTATGGAATTCATTGGCAACC

[0073] TGACCCTGCTGATCGTTGCCGGCAACGATACCACCCGCAATTC

[0074] TATGTCCGGTGGCCTGGTTGCTATGAACGAATTCCCGCGCGAA

[0075] TTCGAAAAGCTGAAGGCTAAACCGGAACTGATCCCAAACATG

[0076] GTTAGCGAGATCATCCGTTGGCAAACCCCGCTGGCATACATGC

[0077] GTCGTATCGCGAAACAGGATGTGGAACTGGGCGGTCAGACCAT

[0078] CAAGAAAGGCGACCGCGTTGTGATGTGGTATGCATCCGGCAAC

[0079] CGTGACGAGCGCAAATTCGACAACCCGGACCAGTTTATCATCG

[0080] ACCGTAAGGACGCACGCAACCACATGTCCTTCGGTTATGGTGT

[0081] TCATCGCTGTATGGGCAATCGCCTGGCTGAACTGCAGCTGCGC

[0082] ATTCTGTGGGAAGAAATCCTGAAACGTTTCGACAACATCGAAG

[0083] TAGTGGAAGAACCAGAACGTGTACAGTCCAACTTCGTTCGTGG

[0084] CTATTCTCGTCTGATGGTAAAGCTGACCCCGAACTCTGGTGGTT

[0085] CCGGTGGCTCTGGCGGCTCTATTCCATCTCCGTCCACCGAGCA

[0086] GTCTGCTAAGAAAGTGCGTAAAAAAGCTGAGAACGCACACAA

[0087] CACTCCGCTGCTGGTGCTGTACGGTTCTAACATGGGTACGGCT

[0088] GAAGGTACCGCTCGCGATCTGGCCGATATCGCGATGTCCAAAG

[0089] GTTTTGCGCCGCAGGTCGCCACGCTGGACTCTCATGCTGGTAA

[0090] CCTGCCGCGTGAGGGTGCTGTGCTGATCGTTACCGCCTCCTAC

[0091] AACGGTCACCCACCGGACAATGCAAAGGAATTCGTGGATTGGC

[0092] TGGATCAGGCGTCTGCGGATGAAGTCAAGGGTGTCCGCTATTC

[0093] CGTATTCGGCTGCGGTGACAAGAACTGGGCAACTACCTATCAG

[0094] AAGGTCCCGGCCTTCATCGACGAAACGCTGGCTGCTAAAGGCG

[0095] CAGAAAACATTGCGGAACGTGGTGAGGCAGACGCTAGCGATG

[0096] ACTTCGAGGGTACCTACGAGGAATGGCGCGAACATATGTGGA

[0097] GCGACCTGGCGGCGTACTTCAACCTGGACATCGAAAATTCCGA

[0098] AGAAAATGCCAGCACTCTGTCCCTGCAGTTTGTAGATTCCGCC

[0099] GCAGATATGCCGCTGGCTAAAATGCACCGTGCATTCAGCGCTA

[0100] ACGTTGTGGCCTCTAAAGAACTGCAGAAGCCAGGTTCTGCACG

[0101] CTCCACCCGTCACCTGGAAATCGAGCTGCCGAAAGAAGCTAGC

[0102] TACCAAGAAGGTGATCACCTGGGCGTTATCCCGCGCAACTACG

[0103] AGGGTATTGTGAACCGCGTTGCAACCCGTTTCGGTCTGGATGC

[0104] AAGCCAGCAGATCCGTCTGGAAGCCGAAGAAGAAAAACTGGC

[0105] TCACCTGCCGCTGGGCAAAACGGTCAGCGTGGAAGAACTGCTG

[0106] CAGTATGTTGAACTGCAGGATCCGGTTACCCGTACTCAGCTGC

[0107] GTGCGATGGCGGCAAAAACCGTGTGCCCACCTCACAAAGTAG

[0108] AGCTGGAGGTACTGCTGGAAAAACAGGCGTATAAGGAACAGG

[0109] TCCTGGCCAAACGTCTGACCATGCTGGAACTGCTGGAAAAATA

[0110] TCCGGCTTGCGAAATGGAATTCTCTGAATTCATCGCGCTGCTG

[0111] CCGTCCATGCGTCCGCGTTACTACTCTATCTCTTCCTCCCCACG

[0112] TGTAGATGAGAAACAGGCAAGCATTACGGTCTCTGTTGTAAGC

[0113] GGTGAAGCTTGGTCTGGTTACGGTGAATACAAAGGTATCGCGA

[0114] GCAACTATCTGGCTAACCTGCAGGAAGGTGACACTATTACCTG

[0115] TTTCGTGTCCACCCCTCAGTCCGGCTTTACTCTGCCGAAAGGCC

[0116] CGGAAACGCCGCTGATCATGGTTGGTCCTGGTACCGGCGTTGC

[0117] CCCGTTTCGCGGCTTCGTGCAGGCGCGCAAGCAGCTGAAGGAG

[0118] CAGGGTCAGAGCCTGGGTGAAGCTCACCTGTACTTCGGCTGCC

[0119] GTTCTCCGCACGAAGACTACCTGTACCAGAAAGAACTGGAAAA

[0120] CGCTCAGAACGAAGGTATCATCACGCTGCATACTGCCTTTTCT

[0121] CGTGTGCCGAACCAGCCGAAAACCTATGTTCAGCACGTCATGG

[0122] AGCAAGACGGTAAAAAACTGATCGAACTGCTGGACCAGGGTG

[0123] CCCACTTCTATATCTGCGGCGACGGTTCTCAAATGGCACCGGA

[0124] CGTGGAAGCGACCCTGATGAAGTCTTACGCGGAGGTTCATCAG

[0125] GTAAGCGAAGCGGACGCACGTCTGTGGCTGCAGCAGCTGGAA

[0126] GAAAAAGGTCGTTATGCGAAAGATGTTTGGGCGGGT

[0127] SEQ ID NO8:

[0128] ATGACCAATAATCCCCCTTCAGCACAGATTAAGCCCGGCGAGTATGGTTTCCCCCTCAAGTTAAAAGCCCGCTATGACAACTTTATTGGCGGCGAATGGGTAGCCCCTGCCGACGGCGAGTATTACCAGAATCTGACGCCGGTGACCGGGCAGCTGCTGTGCGAAGTGGCGTCTTCGGGCAAACGAGACATCGATCTGGCGCTGGATGCTGCGCACAAAGTGAAAGATAAATGGGCGCACACCTCGGTGCAGGATCGTGCGGCGATTCTGTTTAAGATTGCCGATCGAATGGAACAAAACCT

[0129] CGAGCTGTTAGCGACAGCTGAAACCTGGGATAACGGCAAACCC

[0130] ATTCGCGAAACCAGTGCTGCGGATGTACCGCTGGCGATTGACCA

[0131] TTTCCGCTATTTCGCCTCGTGTATTCGGGCGCAGGAAGGTGGGA

[0132] TCAGTGAAGTTGATAGCGAAACCGTGGCCTATCATTTCCATGAA

[0133] CCGTTAGGCGTGGTGGGGCAGATTATCCCGTGGAACTTCCCGCT

[0134] GCTGATGGCGAGCTGGAAAATGGCTCCCGCGCTGGCGGCGGGC

[0135] AACTGTGTGGTGCTGAAACCCGCACGTCTTACCCCGCTTTCTGT

[0136] ACTGCTGCTAATGGAAATTGTCGGTGATTTACTGCCGCCGGGCG

[0137] TGGTGAACGTGGTCAATGGCGCAGGTGGGGTAATTGGCGAATA

[0138] TCTGGCGACCTCGAAACGCATCGCCAAAGTGGCGTTTACCGGC

[0139] TCAACGGAAGTGGGCCAACAAATTATGCAATACGCAACGCAAA

[0140] ACATTATTCCGGTGACGCTGGAGTTGGGCGGTAAGTCGCCAAAT

[0141] ATCTTCTTTGCTGATGTGATGGATGAAGAAGATGCCTTTTTCGAT

[0142] AAAGCGCTGGAAGGCTTTGCACTGTTTGCCTTTAACCAGGGCG

[0143] AAGTTTGCACCTGTCCGAGTCGTGCTTTAGTGCAGGAATCTATC

[0144] TACGAACGCTTTATGGAACGCGCCATCCGCCGTGTCGAAAGCAT

[0145] TCGTAGCGGTAACCCGCTCGACAGCGTGACGCAAATGGGCGCG

[0146] CAGGTTTCTCACGGGCAACTGGAAACCATCCTCAACTACATTGA

[0147] TATCGGTAAAAAAGAGGGCGCTGACGTGCTCACAGGCGGGCGG

[0148] CGCAAGCTGCTGGAAGGTGAACTGAAAGACGGCTACTACCTCG

[0149] AACCGACGATTCTGTTTGGTCAGAACAATATGCGGGTGTTCCAG

[0150] GAGGAGATTTTTGGCCCGGTGCTGGCGGTGACCACCTTCAAAA

[0151] CGATGGAAGAAGCGCTGGAGCTGGCGAACGATACGCAATATGG

[0152] CCTGGGCGCGGGCGTCTGGAGCCGCAACGGTAATCTGGCCTAT

[0153] AAGATGGGGCGCGGCATACAGGCTGGGCGCGTGTGGACCAACT

[0154] GTTATCACGCTTACCCGGCACATGCGGCGTTTGGTGGCTACAAA

[0155] CAATCAGGTATCGGTCGCGAAACCCACAAGATGATGCTGGAGC

[0156] ATTACCAGCAAACCAAGTGCCTGCTGGTGAGCTACTCGGATAAACCGTTGGGGCTGTTCTGA

[0157] SEQ ID NO9:

[0158]

[0159] SEQ ID NO10:

[0160]

[0161] SEQ ID NO11:

[0162] ATGTCCCGAATGGCAGAACAGCAGCTTTATATACATGGTGGTTATACCTCCGCCACCAGCGGTCGCACCTTCGAGACCATTAACCCGGCCAACGGTAACGTGCTGGCGACCGTGCAGGCCGCCGGGCGCGAGGATGTCGATCGCGCCGTGAAAAGCGCCCAGCAGGGGCAAAAAATCTGGGCGTCGATGACCGCCATGGAGCGCTCGCGTATTCTGCGTCGGGCCGTTGATATTCTGCGTGAACGCAATGACGAACTCGCAAAACTGGAAACCCTCGACACCGGAAAAGCATATT

[0163] CGGAAACCTCAACCGTCGATATCGTTACCGGTGCGGACGTGCT

[0164] GGAGTACTACGCCGGGCTGATCCCGGCGCTGGAAGGCAGCCA

[0165] GATCCCGTTGCGTGAAACGTCCTTTGTGTATACCCGCCGCGAA

[0166] CCGCTGGGCGTAGTGGCAGGGATTGGCGCATGGAACTACCCGA

[0167] TCCAGATTGCCCTGTGGAAATCCGCCCCGGCGCTGGCGGCAGG

[0168] CAACGCAATGATTTTCAAACCGAGCGAAGTTACCCCGCTTACC

[0169] GCGTTAAAGCTGGCTGAAATTTACAGCGAAGCGGGCCTGCCGG

[0170] ACGGCGTATTTAACGTGTTGCCGGGCGTGGGCGCGGAGACCGG

[0171] GCAATATCTGACCGAGCATCCGGGCATTGCCAAAGTGTCATTT

[0172] ACCGGCGGTGTCGCCAGCGGCAAAAAAGTGATGGCTAACTCG

[0173] GCGGCCTCTTCCCTGAAAGAAGTGACCATGGAACTGGGCGGTA

[0174] AATCACCGCTGATCGTTTTCGATGATGCGGATCTCGATCTCGCC

[0175] GCCGATATCGCCATGATGGCAAACTTCTTCAGCTCCGGTCAGG

[0176] TGTGTACCAATGGCACCCGCGTCTTCGTTCCGGCGAAATGCAA

[0177] AGCCGCATTTGAGCAGAAAATTCTGGCGCGCGTTGAGCGCATT

[0178] CGCGCGGGCGACGTTTTCGATCCGCAAACTAACTTCGGCCCGC

[0179] TGGTCAGCTTCCCGCATCGCGATAACGTGCTGCGCTATATCGC

[0180] CAAAGGCAAAGAGGAAGGCGCGCGCGTACTGTGCGGCGGCGA

[0181] TGTACTGAAAGGCGATGGCTTCGATAACGGCGCATGGGTTGCA

[0182] CCGACAGTGTTCACCGATTGCAGCGACGATATGACCATCGTGC

[0183] GTGAAGAGATCTTCGGGCCAGTGATGTCCATTCTGACCTACGA

[0184] GTCGGAAGACGAAGTCATTCGCCGCGCTAACGATACCGACTAC

[0185] GGCCTGGCGGCGGGCATCGTGACAGCGGACCTGAACCGCGCG

[0186] CATCGCGTCATTCATCAGCTGGAAGCGGGTATTTGCTGGATCA

[0187] ACACCTGGGGCGAATCCCCGGCAGAGATGCCCGTTGGCGGCTA

[0188] CAAACACTCCGGCATTGGTCGCGAGAACGGCGTGATGACGCTC

[0189] CAGAGTTACACCCAGGTGAAGTCCATCCAGGTTGAGATGGCTA

[0190] AATTCCAGTCCATATTCTAA

[0191] SEQ ID NO12:

[0192]

[0193] SEQ ID NO13:

[0194]

[0195] SEQ ID NO14:

[0196]

[0197]

[0198] SEQ ID NO16:

[0199]

[0200] SEQ ID NO17:

[0201]

[0202] SEQ ID NO18:

[0203]

[0204] SEQ ID NO19:

[0205] ATGGGAACCACCACCATGGGGGTTAAGCTGGACGACGCGACGCGTGAGCGTATTAAGTCTGCCGCGACACGTATCGATCGCACACCACACTGGTTAATTAAGCAGGCGATTTTTTCTTATCTCGAACAACTGGAAAACAGCGATACTCTGCCGGAGCTACCTGCGCTGCTTTCTGGCGCGGCCAATGAGAGCGATGAAGCACCGACTCCGGCAGAGGAACCACACCAGCCATTCCTCGACTTTGCCGAGCAAATATTG

[0206] CCCCAGTCGGTTTCCCGCGCCGCGATCACCGCGGCCTATCGCCG

[0207] CCCGGAAACCGAAGCGGTTTCTATGCTGCTGGAACAAGCCCGC

[0208] CTGCCGCAGCCAGTTGCTGAACAGGCGCACAAACTGGCGTATC

[0209] AGCTGGCCGATAAACTGCGTAATCAAAAAAATGCCAGTGGTCG

[0210] CGCAGGTATGGTCCAGGGGTTATTGCAGGAGTTTTCGCTGTCAT

[0211] CGCAGGAAGGCGTGGCGCTGATGTGTCTGGCGGAAGCGTTGTT

[0212] GCGTATTCCCGACAAAGCCACCCGCGACGCGTTAATTCGCGAC

[0213] AAAATCAGCAACGGTAACTGGCAGTCACACATTGGTCGTAGCC

[0214] CGTCACTGTTTGTTAATGCCGCCACCTGGGGGCTGCTGTTTACT

[0215] GGCAAACTGGTTTCCACCCATAACGAAGCCAGCCTCTCCCGCT

[0216] CGCTGAACCGCATTATCGGTAAAAGCGGTGAACCGCTGATCCG

[0217] CAAAGGTGTGGATATGGCGATGCGCCTGATGGGTGAGCAGTTC

[0218] GTCACTGGCGAAACCATCGCGGAAGCGTTAGCCAATGCCCGCA

[0219] AGCTGGAAGAGAAAGGTTTCCGTTACTCTTACGATATGCTGGGC

[0220] GAAGCCGCGCTGACCGCCGCAGATGCACAGGCGTATATGGTTT

[0221] CCTATCAGCAGGCGATTCACGCCATCGGTAAAGCGTCTAACGGT

[0222] CGTGGCATCTATGAAGGGCCGGGCATTTCAATCAAACTGTCGGC

[0223] GCTGCATCCGCGTTATAGCCGCGCCCAGTATGACCGGGTAATGG

[0224] AAGAGCTTTACCCGCGTCTGAAATCACTCACCCTGCTGGCGCGT

[0225] CAGTACGATATTGGTATCAACATTGACGCCGAAGAGTCCGATCG

[0226] CCTGGAGATCTCCCTCGATCTGCTGGAAAAACTCTGTTTCGAGC

[0227] CGGAACTGGCAGGCTGGAACGGCATCGGTTTTGTTATTCAGGCT

[0228] TATCAAAAACGCTGCCCGTTGGTGATCGATTACCTGATTGATCTC

[0229] GCCACCCGCAGCCGTCGCCGTCTGATGATTCGCCTGGTGAAAG

[0230] GCGCGTACTGGGATAGTGAAATTAAGCGTGCGCAGATGGACGG

[0231] CCTTGAAGGTTATCCGGTTTATACCCGCAAGGTGTATACCGACG

[0232] TTTCTTATCTCGCCTGTGCGAAAAAGCTGCTGGCGGTGCCGAAT

[0233] CTAATCTACCCGCAGTTCGCGACGCACAACGCCCATACGCTGGC

[0234] GGCGATTTATCAACTGGCGGGGCAGAACTACTACCCGGGTCAGT

[0235] ACGAGTTCCAGTGCCTGCATGGTATGGGCGAGCCACTGTATGAG

[0236] CAGGTCACCGGGAAAGTTGCCGACGGCAAACTTAACCGTCCGT

[0237] GTCGTATTTATGCTCCGGTTGGCACACATGAAACGCTGTTGGCG

[0238] TATCTGGTGCGTCGCCTGCTGGAAAACGGTGCTAACACCTCGTT

[0239] TGTTAACCGTATTGCCGACACCTCTTTGCCACTGGATGAACTGG

[0240] TCGCCGATCCGGTCACTGCTGTAGAAAAACTGGCGCAACAGGA

[0241] AGGGCAAACTGGATTACCGCATCCGAAAATTCCCCTGCCGCGC

[0242] GATCTTTACGGTCACGGGCGCGACAACTCGGCAGGGCTGGATC

[0243] TCGCTAACGAACACCGCCTGGCCTCGCTCTCCTCTGCCCTGCTC

[0244] AATAGTGCACTGCAAAAATGGCAGGCCTTGCCAATGCTGGAAC

[0245] AACCGGTAGCGGCAGGTGAGATGTCGCCCGTTATTAACCCTGCG

[0246] GAACCGAAAGATATTGTGGGCTATGTGCGTGAAGCCACGCCGC

[0247] GTGAAGTAGAACAGGCGCTGGAAAGTGCGGTTAATAACGCGCC

[0248] AATCTGGTTTGCCACGCCTCCGGCTGAACGCGCAGCGATTTTGC

[0249] ACCGCGCTGCCGTGCTGATGGAAAGCCAGATGCAGCAACTGAT

[0250] TGGTATTCTGGTGCGTGAGGCCGGAAAAACCTTCAGTAACGCC

[0251] ATTGCCGAAGTGCGCGAAGCGGTCGATTTTCTCCACTACTACGC

[0252] CGGACAGGTGCGGGATGATTTCGCTAACGAAACCCACCGTCCA

[0253] TTAGGGCCTGTGGTGTGTATCAGTCCGTGGAACTTCCCGCTGGC

[0254] TATTTTCACCGGGCAGATCGCCGCCGCACTGGCGGCAGGTAAC

[0255] AGCGTGCTGGCAAAACCGGCAGAACAAACGCCGCTGATTGCC

[0256] GCGCAAGGGATCGCCATTTTGCTGGAAGCGGGTGTACCGCCAG

[0257] GCGTGGTGCAATTGCTGCCAGGTCGGGGTGAAACCGTGGGCGC

[0258] GCAACTGACGGGTGATGATCGCGTGCGCGGGGTGATGTTTACC

[0259] GGTTCAACCGAAGTCGCTACGTTACTGCAGCGCAATATCGCCAG

[0260] CCGCCTGGACGCTCAGGGTCGCCCTATTCCGCTCATCGCTGAAA

[0261] CCGGCGGCATGAACGCGATGATTGTCGATTCTTCAGCACTGACC

[0262] GAACAGGTCGTCGTGGATGTACTGGCCTCGGCGTTCGACAGTG

[0263] CGGGTCAGCGTTGTTCGGCGCTGCGCGTGCTGTGCCTGCAAGA

[0264] TGAGATTGCCGACCACACGTTGAAAATGCTGCGCGGCGCAATG

[0265] GCCGAATGCCGGATGGGTAATCCGGGTCGCCTGACCACCGATAT

[0266] CGGTCCAGTGATTGATAGCGAAGCGAAAGCCAATATTGAGCGC

[0267] CATATTCAGACCATGCGTAGCAAAGGCCGTCCGGTGTTCCAGGC

[0268] GGTGCGGGAAAACAGCGAAGATGCCCGTGAATGGCAAAGCGG

[0269] CACCTTTGTCGCCCCGACGCTGATCGAACTGGATGACTTTGCCG

[0270] AATTGCAAAAAGAGGTCTTTGGTCCGGTGCTGCATGTGGTGCG

[0271] TTACAACCGTAACCAGCTACCAGAGCTGATCGAGCAGATTAAC

[0272] GCTTCCGGTTATGGTCTGACGCTTGGCGTCCATACGCGCATTGA

[0273] TGAAACCATCGCCCAGGTCACTGGCTCGGCCCATGTTGGTAACC

[0274] TGTATGTTAACCGTAATATGGTGGGCGCAGTGGTTGGTGTGCAG

[0275] CCGTTCGGCGGCGAAGGGTTGTCCGGTACCGGGCCGAAAGCAG

[0276] GCGGTCCGCTCTATCTCTACCGTCTGCTGGCGAATCGCCCGGAA

[0277] AGTGCGCTGGCAGTGACGCTCGCGCGTCAGGATGCAAAGTATC

[0278] CGGTCGATGCGCAGTTGAAAGCCGCATTGACTCAGCCGCTAAA

[0279] TGCACTGCGGGAATGGGCAGCAAATCGTCCAGAATTGCAGGCG

[0280] TTATGTACGCAATATGGCGAGCTGGCGCAGGCAGGAACACAAC

[0281] GATTGCTGCCGGGGCCGACGGGTGAACGCAACACCTGGACGCT

[0282] GCTGCCGCGTGAGCGCGTGTTGTGTATTGCCGATGATGAGCAGG

[0283] ATGCGCTGACTCAGCTCGCCGCCGTGCTGGCGGTGGGCAGCCA

[0284] GGTACTGTGGCCGGATGACGCGCTGCATCGTCAGTTAGTGAAG

[0285] GCATTGCCATCGGCAGTCAGCGAACGTATTCAACTGGCGAAAG

[0286] CGGAAAATATAACCGCTCAACCGTTTGATGCGGTGATCTTCCAC

[0287] GGTGATTCGGATCAGCTTCGCGCATTGTGTGAAGCAGTTGCCGC

[0288] GCGGGATGGCACAATTGTTTCGGTGCAGGGTTTTGCCCGTGGC

[0289] GAAAGCAATATCCTTCTGGAACGGCTGTATATCGAGCGTTCGCT

[0290] GAGTGTGAATACCGCTGCCGCTGGCGGTAACGCCAGCTTAATG

[0291] ACTATAGGTTAA

[0292] SEQ ID NO20:

[0293]

[0294] ATGGCTATCCCTGCATTTGGTTTAGGTACTTTCCGTCTGAAAGACGACGTTGTTATTTCATCTGTGATAACGGCGCTTGAACTTGGTTATCGCGCAATTGATACCGCACAAATCTATGATAACGAAGCCGCAGTAGGTCAGGCGATTGCAGAAAGTGGCGTGCCACGTCATGAACTCTACATCACCACTAAAATCTGGATTGAAAATCTCAGCAAAGACAAATTGATCCCAAGTCTGAAAGAGAGCCTGCAAAAATTGCGTACCGATTATGTTGATCTGACGCTAATCCACTGGCCGTCACCAAACGATGAAGTCTCTGTTGAAGAGTTTATGCAGGCGCTGCTGGAAGCCAAAAAACAAGGGCTGACGCGTGAGATCGGTATTTCCAACTTCACGATCCCGTTGATGGAAAAAGCGATTGCTGCTGTTGGTGCTGAAAACATCGCTACTAACCAGATTGAACTCTCTCCTTATCTGCAAAACCGTAAAGTGGTTGCCTGGGCTAAACAGCACGGCATCCATATTACTTCCTATATGACGCTGGCGTATGGTAAGGCCCTGAAAGATGAGGTTATTGCTCGTATCGCAGCTAAACACAATGCGACTCCGGCACAAGTGATTCTGGCGTGGGCTATGGGGGAAGGTTACTCAGTAATTCCTTCTTCTACTAAACGTAAAAACCTGGAAAGTAATCTTAAGGCACAAAATTTACAGCTTGATGCCGAAGATAAAAAAGCGATCGCCGCACTGGATTGCAACGACCGCCTGGTTAGCCCGGAAGGTCTGGCTCCTGAATGGGATTAA*

[0295] SEQ ID NO22:

[0296] ATGCAACAAAAAATGATTCAATTTAGTGGCGATGTCTCACTGCCAGCCGTAGGGCAGGGAACATGGTATATGGGCGAAGATGCCAGTCAGCGCAAAACAGAAGTTGCTGCACTACGCGCGGGCATTGAACTCGGTTTAACCCTCATTGATACCGCCGAAATGTATGCCGATGGCGGTGCCGAAAAGGTGGTTGGGGAAGCATTAACCGGTCTGCGAGAGAAGGTCTTTCTCGTCTCTAAAGTCTATCCGTGGAATGCTGGCGGGCAAAAAGCGATAAATGCATGCGAAGCCAGTTTACGCCGTCTCAATACTGATTATCTCGATCTTTACTTATTACACTGGTCTGGCAGTTTCGCTTTTGAAGAGACTGTCGCAGCGATGGAAAAATTGATCGCCCAGGGAAAAATCCGCCGCTGGGGCGTTTCTAACCTTGATTATGCTGATATGCAGGAACTCTGGCAGCTGCCGGGGGGAAATCAGTGTGCCACTAATCAGGTGCTTTACCATCTCGGTTCACGAGGAATTGAGTACGATCTACTCCCCTGGTGCCAGCAACAGCAGATGCCGGTGATGGCTTACAGTCCGTTAGCCCAGGCCGGGCGGTTGCGCAATGGACTGTTAAAAAACGCGGTAGTCAACGAAATTGCACATGCTCACAATATCAGCGCGGCACAAGTATTGTTGGCGTGGGTGATCAGTCATCAGGGTGTGATGGCGATTCCAAAAGCGGCCACGATTGCCCATGTCCAACAAAATGCGGCTGTGCTTGAGGTCGAACTTTCTTCAGCGGAATTAGCTATGCTGGATAAGGCATATCCGGCACCAAAAGGAAAAACTGCGCTGGATATGGTGTGA

[0297] SEQ ID NO23:

[0298]

[0299] SEQ ID NO24:

[0300]

[0301] SEQ ID NO25:

[0302] ATGGCTAATCCAACCGTTATTAAGCTACAGGATGGCAATGTCATGCCCCAGCTGGGACTGGGCGTCTGGCAAGCAAGTAATGAGGAAGTAATCACCGCCATTCAAAAAGCGTTAGAAGTGGGTTATCGCTCGATTGATACCGCCGCGGCCTACAAGAACGAAGAAGGTGTCGGCAAAGCCCTGAAAAATGCCTCAGTCAACAGAGAAGAACTGTTCATCACCACTAAGCTGTGGAACGACGACCACAAGCGCCCCCGCGAAGCCCTGCTCGACAGCCTGAAAAAACTCCAGCTTGATTATATCGACCTCTACTTAATGCACTGGCCCGTTCCCGCTATCGACCATTATGTCGAAGCATGGAAAGGCATGATCGAATTGCAAAAAGAGGGATTAATCAAAAGCATCGGCGTGTGCAACTTCCAGATCCATCACCTGCAACGCCTGATTGATGAAACTGGCGTGACGCCTGTGATAAACCAGATCGAACTTCATCCGCTGATGCAACAACGCCAGCTACACGCCTGGAACGCGACACACAAAATCCAGACCGAATCCTGGAGCCCATTAGCGCAAGGAGGGAAAGGCGTTTTCGATCAGAAAGTCATTCGCGATCTGGCAGATAAATACGGCAAAACCCCGGCGCAGATTGTTATCCGCTGGCATCTGGATAGCGGCCTGGTGGTGATCCCGAAATCGGTCACACCTTCACGTATTGCCGAAAACTTTGATGTCTGGGATTTCCGTCTCGACAAAGACGAACTCGGCGAAATTGCAAAACTCGATCAGGGCAAGCGTCTCGGTCCCGATCCTGACCAGTTCGGCGGCTAA

[0303] SEQ ID NO26:

[0304] ATGCTACAAAATTGCGCACAATCAAATTGCCGCATTATTCCTAAGAAATTACGCGATATGAAACGTGAAGAGATTTGCCGCTTGCTGGCGGATAAAGTTAATAAACTGAAAAATAAAGAAAATAGTTTGTCAGGACTGTTGCCCGATGTGCGTTTGTTGTATGGCGAGACGCCTTTCGCACGTACACCGGTGATGTACGAGCCTGGCATCATAATTCTCTTTTCCGGGCATAAAATCGGTTATATCAATGAACGCGTGTTTCGTTATGATGCCAATGAATACCTGCTGCTGACGGTGCCGTTGCCGTTTGAGTGCGAAACCTATGCCACGTCAGAGGTGCCGCTGGCAGGGTTGCGTCTCAATGTCGATATTTTGCAGTTACAGGAACTGTTGATGGACATTGGCGAAGATGAGCATTTCCAGCCGTCGATGGCAGCCAGCGGGATTAACTCCGCCACGTTATCAGAAGAGATTTTATGCGCGGCGGAGCGGTTACTCGACGTGATGGAGCGACCACTGGATGCGCGTATTCTCGGCAAACAGATCATCCGCGAAATTCTGTACTACGTGCTGACCGGACCTTGCGGCGGCGCGTTACTGGCGCTGGTCAGTCGCCAGACTCACTTCAGTCTGATTAGCCGCGTGCTGAAACGGATTGAGAATAAATACACCGAAAACCTGAGCGTCGAGCAACTGGCGGCAGAAGCCAACATGAGCGTATCGGCGTTCCACCATAATTTTAAGTCTGTCACCAGTACCTCGCCGTTGCAGTATTTGAAGAATTACCGTCTGCATAAGGCGCGGATGATGATCATCCATGACGGCATGAAGGCCAGCGCAGCAGCGATGCGCGTCGGCTATGAAAGCGCATCGCAATTTAGCCGTGAGTTTAAACGTTACTTCGGTGTGACGCCGGGGGAAGATGCGGCAAGAATGCGGGCGATGCAGGGGAATTAA

[0305] Example 2:

[0306] The engineered strain M240 constructed in Example 1 was inoculated into TB medium additionally containing 5 g / L glucose, 0.1 g / L ferrous sulfate heptahydrate, 5 mM 5-aminolevulinic acid hydrochloride, 50 mM L-alanine, and 0.1 mM pyridoxal-5-phosphate. Fermentation was performed in a shake flask. At 72 h of fermentation, production of greater than 100 mg / L of nylon dodecamonomer was detected during the fermentation process. The yield of the byproduct dodecanedioic acid was reduced to less than 200 mg / L (less than 20% of the product), and overoxidation of 12-hydroxydodecanoic acid was prevented.

[0307] The above examples are merely illustrative of the present invention and do not limit the scope of protection of the present invention. Any design that is identical or similar to the present invention falls within the scope of protection of the present invention.

Claims

1. An engineered bacterium for inhibiting the synthesis of dodecanedioic acid, characterized in that: The genome of the engineered bacteria is knocked out or down-regulated with the expression of the paoB encoding gene, aldB encoding gene, puuC encoding gene, betB encoding gene, patD encoding gene, feaB encoding gene or gabD encoding gene, aldA encoding gene, astD encoding gene, putA encoding gene, sad encoding gene, dkgB encoding gene, yeaE encoding gene, yahK encoding gene, yqhD encoding gene, dkgA encoding gene, and yqhC encoding gene.

2. The engineered bacteria for inhibiting the synthesis of dodecanedioic acid according to claim 1, characterized in that: The genetically engineered bacteria uses Escherichia coli as the starting bacteria.

3. A method for constructing an engineered bacterium that inhibits the synthesis of dodecanedioic acid, characterized in that: The following steps are involved: (1) Starting from Escherichia coli MG1655, the genes encoding luxI, luxR, and UcfatB and the promoters controlling their expression were integrated into the tesA gene locus of the E. coli genome to construct strain M22; (2) Based on the M22 strain, the same sequence as in step (1) was integrated into the ad hE gene locus to obtain strain M73; (3) Based on the M73 strain, the CYP153A-BM3 encoding gene and the promoter controlling CYP153A-BM3 expression were integrated into the fadE site of Escherichia coli to obtain strain M209; (4) Based on the M209 strain, the CYP153A-BM3 encoding gene and the promoter controlling CYP153A-BM3 expression were integrated into the pgi site of Escherichia coli to obtain the M220 strain; (5) Based on the M220 strain, the expression of the paoB encoding gene, aldB encoding gene, puuC encoding gene, betB encoding gene, patD encoding gene, feaB encoding gene or gabD encoding gene, aldA encoding gene, astD encoding gene, putA encoding gene, sad encoding gene, dkgB encoding gene, yeaE encoding gene, yahK encoding gene, yq hD encoding gene, dkgA encoding gene, and yqhC encoding gene was inhibited to obtain the strain M230; (6) The genes encoding CV2025 and BsADH and the promoters controlling the expression of CV2025 and BsAD H were cloned into the pCDFDuet plasmid to obtain a recombinant plasmid, which was then transformed into the strain M230 to obtain an engineered bacterium that inhibits the synthesis of dodecanedioic acid.

4. The method for constructing an engineered bacterium for inhibiting the synthesis of dodecanedioic acid according to claim 3, wherein: The promoter for controlling the expression of luxI and UcfatB in step (1) is selected from P lux ; The promoter controlling luxR expression is selected from P tet .

5. The method for constructing an engineered bacterium for inhibiting the synthesis of dodecanedioic acid according to claim 3, wherein: The promoter for controlling the expression of CYP153A-BM3 in steps (3) and (4) is selected from P lux .

6. The method for constructing an engineered bacterium for inhibiting the synthesis of dodecanedioic acid according to claim 3, wherein: The promoter for controlling the expression of CV2025 and BsADH in step (6) is selected from P M12 .

7. Use of the engineered bacteria for inhibiting the synthesis of dodecanedioic acid according to any one of claims 1 to 2 or the construction method according to any one of claims 3 to 6 in the production of nylon dodecanedioic acid monomer.

8. A method for producing nylon dodecamonomer, characterized in that: The following steps are involved: The engineered bacteria for inhibiting the synthesis of dodecanedioic acid according to any one of claims 1 to 2 are inoculated into a culture medium for fermentation, and the nylon dodecamonomer in the fermentation product is collected.

9. The method for producing nylon dodecamonomer according to claim 8, wherein: The culture medium is a TB culture medium supplemented with 4-40 g / L glucose, 0.1-1 g / L ferrous sulfate heptahydrate, 5-50 mM 5-aminolevulinic acid hydrochloride, 50-500 mM L-alanine, and 0.1-1 mM pyridoxal-5-phosphate.