KR102608905B1 - Transformed microorganism producing nonanedioic acid and a method for producing nonanedioic acid using the same - Google Patents

Abstract

The present invention relates to a transformed microorganism that produces nonanedioic acid and a method for producing nonanedioic acid using the same. Additionally, the present invention relates to a method of producing nonanedioic acid (NDA) from nonanoic acid by manipulating the genes of an E. coli strain. The method according to the present invention has a significant advantage over the existing nonanedioic acid production method in that it not only enables E. coli to grow rapidly in nonanoic acid, but also enables E. coli to overproduce nonanedioic acid, a high value-added substance, from nonanoic acid. provides. Therefore, the E. coli of the present invention can be usefully used to produce nonanedioic acid.

Description

Transformed microorganism producing nonanedioic acid and method for producing nonanedioic acid using the same {TRANSFORMED MICROORGANISM PRODUCING NONANEDIOIC ACID AND A METHOD FOR PRODUCING NONANEDIOIC ACID USING THE SAME}

The present invention relates to a transformed microorganism that produces nonanedioic acid and a method for producing nonanedioic acid using the same. Additionally, the present invention relates to a method of producing nonanedioic acid (NDA) from nonanoic acid by manipulating the genes of an E. coli strain.

Medium chain α,ω-dicarboxylic acids (MCDCA) are organic compounds with two terminal carboxyl functional groups (C8 to C14). It is widely applied in the polymer industry for the production of various products such as nylon and other polyamides, polyesters, and resins, as well as in cosmetic ingredients. To date, MCDCA is mainly produced by chemical synthesis or ozonolysis of unsaturated fatty acids. A solid metal catalyst is used to chemically synthesize MCDCA by direct hydroxylation of the ω-carbon of the alkyl chain. However, these solid metal catalysts suffer from low efficiency and poor selectivity. Ozonolysis of unsaturated fatty acids is an alternative method for MCDCA production due to its high selectivity and efficiency. However, this method has several limitations, including high energy input process, harsh reaction conditions, and by-product formation.

Bioconversion of fatty acids to MCDCA and its derived compounds is a promising alternative to the existing MCDCA production process. ω-oxidation using heme-containing cytochrome P450 enzymes has been used for the production of dicarboxylic acids because it directly hydroxylates the terminal alkane chains of fatty acids.

Similarly, introduction of the Pseudomonas putida GPo1 ω-oxidation pathway system into Escherichia coli was studied to ω-oxidize medium chain fatty acids (MCFA, C ₈ to C ₁₄ ) with high efficiency and regioselectivity. P. The ω-oxidation pathway system of putida GPo1 includes an alkane hydroxylase (AlkBGT) system as part of the alkane degradation pathway. This AlkBGT system has high specificity for oxidation of the terminal position of MCFA.

Meanwhile, nonanedioic acid (NDA) is a nine-carbon chain long dicarboxylic acid used in plastic and acne medication. NDA is a natural product found in crop and animal products. The value of NDA as a raw material for use in cosmetics has increased due to its antibacterial and anti-inflammatory effects, along with its whitening effect on blemishes and freckles. Therefore, biological production of NDA by ω-oxidation of nonanoic acid becomes a feasible and attractive method.

E. coli can grow using long-chain fatty acids through the β-oxidation pathway. FadR is an inhibitor of fatty acid degradation (β-oxidation), and its deletion enhances β-oxidation. However, several studies have reported that fadR -deleted E. coli strains do not grow well when using MCFA as the sole carbon source. This low growth may be due to the toxicity of MCFA to E. coli and the low substrate specificity of the acyl-CoA synthetase FadD for MCFA.

For large-scale production of MCDCA, direct supply of fatty acids as precursors could be an effective strategy, but due to the toxicity of MCFAs, an alternative to existing strategies is the use of methylated fatty acids or long-chain fatty acids as precursors, which show little toxicity. However, this method generally requires both esterification of fatty acids and conversion of monoesterified-carboxylic acids to α,ω-DCA and is therefore ultimately unsuitable for the production of MCDCA.

US 2018-0112241 A1 US 2020-0277634 A1

Accordingly, in order to develop a method of producing nonanedioic acid from nonanoic acid by manipulating the genes of an E. coli strain, the present inventors evolved a specific E. coli strain to adapt to the laboratory and then compared it to the wild type strain, showing the fastest growth on nonanoic acid, the sole carbon source. The present invention was completed by obtaining the E. coli mutant shown and confirming a total of six mutations from this E. coli mutant.

In order to achieve the above object, one aspect of the present invention is, i) the fadR gene is deleted, and ii) transformed E. coli in which at least one selected from the group consisting of AcrR, FadD, DppA, Crp, e14 prophage, and YeaR is mutated. provides.

Another aspect of the present invention includes i) culturing the transformed E. coli; and ii) obtaining nonanedioic acid produced from the transformed E. coli.

Another aspect of the present invention includes i) deleting the fadR gene of wild-type E. coli; ii) culturing the E. coli in a minimal medium in which nonanoic acid is the sole carbon source for more than 10 days; and iii) repeatedly subculturing for more than 90 days. It provides a method of producing E. coli that produces nonanedioic acid from nonanoic acid.

Another aspect of the present invention provides E. coli produced by the above method.

Another aspect of the present invention includes i) cultivating the E. coli; and ii) obtaining nonanedioic acid produced from E. coli.

The method according to the present invention has a significant advantage over the existing nonanedioic acid production method in that it not only enables E. coli to grow rapidly in nonanoic acid, but also enables E. coli to overproduce nonanedioic acid, a high value-added substance, from nonanoic acid. provides. Therefore, the E. coli of the present invention can be usefully used to produce nonanedioic acid.

Figure 1 is a graph showing the growth curves of E. coli wild type MG1655 (WT), fadR -deleted E. coli (MRD) grown in M9 minimal medium, and 10 mutants (C1 to C10) isolated after applying MRD to laboratory adaptive evolution. .
Figure 2 shows E. coli wild-type MG1655 (WT), fadR -deleted E. coli (MRD) grown in M9 minimal medium, and C6 (MRE), which showed the fastest growth among 10 mutants isolated after applying MRD to laboratory adaptive evolution. This is a graph showing the growth curve.
Figure 3A shows growth curves of MRE, MRE-derived single mutation restoration strain, and MRD-Pf; Figure 3b is a graph showing cell viability of MRE, MRD, and MRD-derived single mutation-introduced strains cultured in nonanoic acid.
Figure 4A shows a graph showing NDA concentrations in MRE-BGT, MRD-BGT, and MRD-EB; Figure 4b is a graph showing the NDA concentration of MRE-BGT, MRD-BGT and MRD-derived single mutation-introduced fadE -deletion alkBGT expression strain at 24 hours after biotransformation.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides transformed E. coli in which i) the fadR gene is deleted, and ii) one or more selected from the group consisting of AcrR, FadD, DppA, Crp, e14 prophage, and YeaR are mutated.

The term “ fadR ” gene used in the present invention is a gene encoding FadR, an inhibitor of fatty acid degradation (β-oxidation). The FadR and the fadR gene encoding it may have the sequence disclosed in Gene ID: 948652.

The method of deleting part or all of the gene encoding the enzyme or polypeptide, etc. can be performed, for example, by transforming a cassette for gene deletion into the parent strain using the Cre/loxP recombination system, and can be performed on the chromosome in E. coli. This can be done by replacing the gene encoding the target protein endogenous in the chromosome with a gene or marker gene with some nucleic acid sequences deleted through an insertion vector. The method of modifying the expression control sequence is carried out by inducing mutations in the expression control sequence by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence, or a combination thereof to further weaken the activity of the expression control sequence, or to further weaken the activity of the expression control sequence. This can be accomplished by replacing the nucleic acid sequence with an active nucleic acid sequence. The expression control sequence includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation. In addition, the method of modifying the base sequence on the chromosome encoding the enzyme or polypeptide induces a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further weaken the activity of the protein. This can be done by doing so, or by replacing it with an improved base sequence to have weaker activity.

The term "AcrR" used in the present invention refers to a potential regulatory protein of the acrAB gene, and the AcrR and the acrR gene encoding it may have the sequence disclosed in Gene ID: 945516. The AcrR mutation may be one in which adenine (A), the 567th base of the base sequence shown in SEQ ID NO: 42, is deleted.

The term “FadD” as used in the present invention is a protein that catalyzes the esterification as well as the transport of exogenous long-chain fatty acids to CoA thioester for degradation or incorporation into phospholipids. The FadD and the fadD gene encoding it may have the sequence disclosed in Gene ID: 946327. In the FadD mutation, guanine, the 90th base, and cytosine, the 94th base, in the nucleotide sequence of the fadD and yeaY intergenic region ( yeaY / fadD intergenic region) shown in SEQ ID NO: 43 are adenine. ) may have been replaced.

The term "DppA" used in the present invention refers to a dipeptide transport protein (plasmic dipeptide transport protein), and the DppA and the dppA gene encoding it may have the sequence disclosed in Gene ID: 948062. The DppA mutation may be one in which the mobile genetic element IS5 is inserted at base 1570 of the base sequence shown in SEQ ID NO: 44. The DppA mutation may be the base sequence represented by SEQ ID NO: 51.

The term "Crp" used in the present invention refers to CRP-cAMP activated transcriptional regulator CRP (cAMP-activated global transcriptional regulator CRP). The Crp and the crp gene encoding it may have the sequence disclosed in Gene ID: 947867. there is.

The Crp mutation may be the insertion of the mobility gene IS5 (SEQ ID NO: 52) at the 125th base of the nucleotide sequence of the crp and yhfA intergenic region (yhfA/crp intergenic region) shown in SEQ ID NO: 45. The intergenic region of crp and yhfA may be the base sequence represented by SEQ ID NO: 45.

The term "e14 prophage" used in the present invention has several genes and corresponds to 1195432 bp to 1210646 bp of the E. coli genome. The e14 prophage mutation may be one in which the icd-icdC gene of the nucleotide sequence shown in SEQ ID NO: 46 is deleted.

The term "YeaR" used in the present invention is a protein with an unknown function that has been reported to be induced in response to nitrate. YeaR and the yeaR gene encoding it may have the sequence disclosed in Gene ID: 946317. The YeaR mutation may be one in which the mobility gene IS186 (SEQ ID NO: 54) is inserted into the 115th base of the nucleotide sequence shown in SEQ ID NO: 47. The YeaR mutation may be the base sequence represented by SEQ ID NO: 53.

Another aspect of the present invention includes i) culturing the transformed E. coli; and ii) obtaining nonanedioic acid produced from the transformed E. coli. Specifically, the method includes deleting the fadE gene in E. coli; ii) inserting an expression vector containing an alkBGT gene and an IPTG-inducible promoter into the E. coli; iii) cultivating the E. coli; iv) culturing the E. coli after treating with IPTG; and v) obtaining nonanedioic acid produced from the E. coli.

In the method for producing nonanedioic acid, refer to the description of the above-mentioned transformed E. coli for the transformed E. coli.

The term “ fadE ” gene used in the present invention is a gene encoding acyl-CoA dehydrogenase. The acyl-CoA dehydrogenase and fadE genes may have sequences disclosed in Gene ID: 949007.

The term " alkBGT " gene used in the present invention is an operon consisting of three genes, alkB (SEQ ID NO: 48), alkG (SEQ ID NO: 49), and alkT (SEQ ID NO: 50), and is a gene encoding alkane monooxygenase. . The alkane monooxygenase is involved in the reaction of adding oxygen by oxidizing the carbon terminal portion of the intermediate carbon chain length. The alkBGT "gene may be derived from P. putida GPo1.

The term “cultivation” used in the present invention may refer to a series of actions of growing the transformed E. coli under appropriately artificially controlled environmental conditions in order to produce nonanedioic acid from the transformed E. coli. In the present invention, the method of culturing the transformed E. coli can be performed using methods widely known in the art. The medium used for culture may contain one or more substrates that can be metabolized into nonanedioic acid, for example, under aerobic conditions in a typical medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc., temperature, The requirements of the specific strain must be met in an appropriate manner while adjusting pH, etc. The culture may be culturing transformed E. coli in the presence of nonanoic acid.

Carbon sources that can be used include glucose as the main carbon source, and sugars and carbohydrates such as xylose, sucrose, lactose, fructose, maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut oil, etc. It may include oils and fats, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid.

The culture may be characterized as cultivating yeast in the presence of glucose.

These substances can be used individually or in mixtures. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used. You can. These nitrogen sources can be used alone or in combination. The medium may include, as ingredients, monopotassium phosphate, dipotassium phosphate and the corresponding sodium-containing salts. Agents that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. Additionally, inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate can be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins may be used.

Typically, E. coli can be cultured (grown) in an appropriate medium at temperatures ranging from about 20°C to about 37°C. The medium in the present invention may be, for example, a broth containing ammonium sulfate and dextrose as a carbon/energy source or a conventional commercially prepared medium. Other defined or synthesized media can also be used, and media suitable for the growth of specific microorganisms are known to those skilled in the art of microbiology or fermentation science.

Nonanedioic acid produced through the transformed E. coli can be isolated from the culture medium using methods known in the art. This separation method may be centrifugation, filtration, ion exchange chromatography, gas chromatography, or crystallization. For example, the culture can be centrifuged at low speed to remove biomass, and the resulting supernatant can be separated through gas chromatography.

Another aspect of the present invention includes i) deleting the fadR gene of wild-type E. coli; ii) culturing the E. coli in a minimal medium in which nonanoic acid is the sole carbon source for more than 10 days; and iii) repeatedly subculturing for more than 90 days. It provides a method of producing E. coli that produces nonanedioic acid from nonanoic acid.

In step i), a step of deleting the fadE gene of wild-type E. coli may be additionally included.

In step ii), nonanoic acid may be present at a concentration of at least 3 g/L.

In step ii), the minimal medium may be M9 minimal medium to which NaH ₂ PO ₄ , KH ₂ PO ₄ , NaCl, NH ₄ Cl, MgSO _4· 7H ₂ O and CaCl ₂ are added. In one embodiment of the present invention, 5 g/L, NaH ₂ PO ₄ 6.78 g/L, KH ₂ PO ₄ 3.0 g/L, NaCl 0.5 g/L, NH ₄ Cl 1.0 g/L, MgSO _4· 7H ₂ O M9 minimal medium supplemented with 0.49 g/L and 0.011 g/L CaCl ₂ was used.

Another aspect of the present invention provides E. coli produced by the above method. The E. coli may be characterized in that the fadR gene is deleted and one or more selected from the group consisting of AcrR, FadD, DppA, Crp, e14 prophage, and YeaR are mutated.

The E. coli may have an additional deletion of the fadE gene.

Another aspect of the present invention includes i) cultivating the E. coli; ii) It provides a method for producing nonanedioic acid comprising the step of obtaining nonanedioic acid produced from E. coli. Specifically, the method includes the steps of i) inserting an expression vector containing an alkBGT gene and an IPTG-inducible promoter into the E. coli; ii) cultivating the E. coli; iii) culturing the E. coli after treating with IPTG; and iv) obtaining nonanedioic acid produced from E. coli.

The culturing method and method for obtaining nonanedioic acid are the same as described above in the method for producing nonanedioic acid using transformed E. coli.

Hereinafter, the present invention will be explained in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to these only.

Experimental Example 1. Microbial strains and plasmids

E. coli DH10B was used for plasmid construction, and E. coli MG1655 K-12 (see Blattner et al., Science 277, 5331, 1453-1474 (1997)) was used for all genetic modifications, including heterologous gene expression for laboratory adaptive evolution and biotransformation. It was used as a parent strain for . The constructed strains and plasmids are shown in Table 1 below. Deletion of chromosomal genes was performed using the λ-recombinant system, and the primers used are shown in Table 2 below.

strain Genotype and description WT E. coli MG1655 K-12 F-λ ilvG - rfb -50 rph -1 MRD WT with ΔfadR ::FRT MREs MRD evolved to grow on media using 3 g/L nonanoic acid as the sole carbon source MRD-a MRD with acrR * MRD-Pf MRD with P fadD * MRD-e MRD with e14 prophage deletion MRD-y MRD with yeaR * MRD-Pc MRD with P crp * MRD-d MRD with dppA * MRE-a MRE with acrR restored MRE-Pf MRE with P fadD restored MRE-y MRE with yeaR restored MRE-Pc MRE with P crp restored MRE-d MRE with restored dppA MRD-BGT MRD with ΔfadE ::FRT and A6c- alkBGT MRE-BGT MRE with ΔfadE ::FRT and A6c- alkBGT MRD-EB MRD with six MRE mutations, ΔfadE ::FRT and A6c- alkBGT MRD-aFB MRD-a with ΔfadE ::FRT and A6c- alkBGT MRD-PfFB MRD-Pf with ΔfadE ::FRT and A6c- alkBGT MRD-eFB MRD-e with ΔfadE ::FRT and A6c- alkBGT MRD-yFB MRD-y with ΔfadE ::FRT and A6c- alkBGT MRD-PcFB MRD-Pc with ΔfadE ::FRT and A6c- alkBGT MRD-dFB MRD-d with ΔfadE ::FRT and A6c- alkBGT MRE-aFB MRE-a with ΔfadE ::FRT and A6c- alkBGT MRE-PfFB MRE-Pf with ΔfadE ::FRT and A6c- alkBGT MRE-yFB MRE-y with ΔfadE ::FRT and A6c- alkBGT MRE-PcFB MRE-Pc with ΔfadE ::FRT and A6c- alkBGT MRE-dFB MRE-d with ΔfadE ::FRT and A6c- alkBGT plasmid pBbA6c- rfp p15A replicon, carrying the P _LlacO-1 promoter and rfp , Cm ^R pBbA6c- alkBGT △rfp :: A6c- rfp with alkBGT , Cm ^R

The asterisk (*) indicates the corresponding mutation in the evolved strain, MRE.

primer Sequence (5'-3') purpose sequence number fadR del H1 F TCTGGTATGATGAGTCCAACTTTGTTTTGCTGTGTTATGGAAATCTCACTGTGTAGGCTGGAGCTGCTTC fadR knockout One fadR del H2 R AACAAACAAAAAACCCCTCGTTTGAGGGGTTTGCTCTTTAAACGGAAGGGAATTCCGGGGATCCGTCGACC fadR knockout 2 fadR seq F GGGCGAGTTTGTACCAGTCGG fadR sequencing 3 fadR seq R GCGCGGCGACCGTTCGCTG fadR sequencing 4 fadE del H1 F CCATATCATCACAAGTGGTCAGACCTCCTACAAGTAAGGGGCTTTTCGTTGTGTAGGCTGGAGGCTGCTTC fadE knockout 5 fadE del H2 R TTACCGGCTTCAACTTTCCGCACTTTCTCCGGCAACTTTACCGGCTTCGATTCCGGGGATCCGTCGACC fadE knockout 6 fadE seq F AAAAGTTAGCCAGCGTTTTCCGCCGC fadE sequencing 7 fadE seq R ACGTTGGGAGATGAGACGTATCAGG fadE sequencing 8 P fadD * MAGE Oligo ATATTAACTCATCATACCAGCTTGATAATTACCCAACGAAAAGTTTGTGAAGCGCGTCACTATTTATTTTTATCTTTACCGTAAGAATGC P fadD mutagenesis 9 P fad D back ATATTAACTCATCATACCAGCTTGATAATTACCCAACGAAAAGGTTGCGAAGCGCGTCACTATTTATTTTTATCTTTACCGTAAGAATGC P fadD wild type mutagenesis 10 P fadD * tetA H1P1 F CTGTTTCTGCATTCTTACGGTAAAGATAAAAATAAATAGTGACGCGCTTCGTGTAGGCTGGAGCTGCTTCG P fadD mutagenesis 11 P fadD * tetA H2P4 R TAACATAATATTAACTCATCATACCAGCTTGATAATTACCCAACGAAAAGATTCCGGGGATCCGTCGACC P fadD mutagenesis 12 acrR tetA H1P1 F CGGCCTGATGGAAAACTGGCTCTTTGCCCCGCAATCTTTTGATCTTAAAAGTGTAGGCTGGAGCTGCTTCG acrR mutagenesis 13 acrR tetA H2P4 R GGCACAGGAGATACATCTCCAGTAAGATGGCAACGTAATCGGCGGGCTTCTATTCCGGGGATCCGTCGACC acrR mutagenesis 14 acrR *MAGE Oligo GAGATACATCTCCAGTAAGATGGCAACGTAATCGCGGGCTTCTTTTTAAGATCAAAAGATTGCGGGGCAAAGAGCCAGTTTTCCATCAGG acrR mutagenesis 15 acrR back ATATTAACTCATCATACCAGCTTGATAATTACCCAACGAAAAGGTTGCGAAGCGCGTCACTATTTATTTTTATCTTTACCGTAAGAATGC acrR wild type mutagenesis 16 acrR seq F CCTGTTAGGGCTAAGCTGCC acrR sequencing 17 acrR seq R CGCCGTTTCTGGCATAACA acrR sequencing 18 e14 H1-P4 AAAGTAAATCCTGGCTCTATTATTCTTCTCCGCTGAGATGATGCTGCGCCAATTCCGGGGATCCGTCGACC e14 prophage deletion 19 e14 H2-P1 CGCCTTCCATACCTTTAACAATCAGGTCAGCCGCTTCAGTCCAACCCATAGTGTAGGCTGGAGCTGCTTCG e14 prophage deletion 20 e14 MAGE oligo TCCATACCTTTAACAATCAGGTCAGCCGCTTCAGTCCAACCCATATGGGCCAGCATCATCTCAGCGGAGAGAATAATAGAGCCAGGATTT e14 prophage deletion 21 e14 seq F GTAATTCCCCTGTCTTCAGG e14 prophage sequencing 22 e14 seq R AACAACGGGCGTGTTATACG e14 prophage sequencing 23 yeaR H1-P4 CACCTGCCGGAATATTCGAACGTCATCTTGATAAAGGAACGCGCCCGGGGATTCCGGGGATCCGTCGACC yeaR mutagenesis 24 yeaR H2-P1 TCAGCGTAGCCGAGATATTTGACCGCCCCATGCATAACGGAAAGGCGTGGGTGTAGGCTGGAGCTGCTTCG yeaR mutagenesis 25 yeaR IS186 FP CACCTGCCGGAATATTCGAA yeaR mutagenesis 26 yeaR IS186 RP TCAGCGTAGCCGAGATATTT yeaR mutagenesis 27 PyhfA/crpH1-P4 CCTGGGTCATGCTGAAGCGAGACACCAGGAGACACAAAGCGAAAGCTATGATTCCGGGGATCCGTCGACC PyhfA/crp mutagenesis 28 PyhfA/crpH2-P1 CTTTGCATACATGCAGTACATCAATGTATTACTGTAGCATCCTGACTGTTGTGTAGGCTGGAGCTGCTTCG PyhfA/crp mutagenesis 29 PyhfA/crp IS5 FP CCTGGGTCATGCTGAAGCGA PyhfA/crp mutagenesis 30 PyhfA/crp IS5 RP CTTTGCATACATGCAGTACA PyhfA/crp mutagenesis 31 dppAH1-P4 CCGTGTTTGAACCGGTACGTAAAGAAGTTAAAGGCTATGTGGTTGATCCAATTCCGGGGATCCGTCGACC dppA mutagenesis 32 dppAH2-P1 TTGTATGGCTTTTAATTATTCGATAGAGACGTTTTCGAAGTGATGTTTGCGTGTAGGCTGGAGCTGCTTCG dppA mutagenesis 33 dppAIS5FP CCGTGTTTGAACCGGTACGT dppA mutagenesis 34 dppA IS5 RP TTGTATGGCTTTTAATTATT dppA mutagenesis 35 dppA IS5 seq FP GCCTCTGAACAAGGCTCCAA dppA sequencing 36 dppA IS5 seq RP ACAGCGCCACCGGGTATACA dppA sequencing 37 PyhfA/crp IS5 seq FP TTATCGCCTGAGTTGCCGTC PyhfA/crp sequencing 38 PyhfA/crp IS5 seq RP AACCATTCGAGAGTCGGGTC PyhfA/crp sequencing 39 yeaR IS186 seq FP CTTCGCTGATATGGTGCTAA yeaR Sequencing 40 yeaR IS186 seq RP CTTTCCGTTGTTGCGCACCT yeaR Sequencing 41

All primers were purchased from Macrogen (Korea).

Experimental Example 2. Chemicals, enzymes and culture conditions

Unless otherwise specified, reagents used in the present invention were purchased from Sigma-Aldrich (St. Louis, MO, USA). Restriction enzymes and Phusion high-fidelity DNA polymerase (Thermo Fischer Scientific) and DNA ligase (New England Biolabs) were used for cloning and genetic modification.

The strain was cultured in LB (Luria-Bertani) medium (composition per liter: 5 g of yeast extract, 10 g of peptone, and 10 g of NaCl). For laboratory adaptation evolution, M9 minimal medium (composition per liter: 6.78 g NaH ₂ PO ₄ , 3 g KH ₂ PO ₄ , 0.5 g NaCl, 1 g NH ₄ Cl, 0.49 g MgSO ₄ 7H ₂ O , and 0.011 g of CaCl ₂ ) were used. The medium was supplemented with 0.5% Tween-80 and 3 g/L nonanoic acid. The strain was cultured under aerobic conditions at 37°C and with agitation on a shaker at 200 rpm. Ampicillin (100 mg/L), kanamycin (50 mg/L), and chloramphenicol (30 mg/L) were used as antibiotics.

Experimental Example 3. Laboratory adaptive evolution

A single colony of the MRD strain with the fadR gene deleted on the LB plate was inoculated into 3 ml of LB broth. First, it was diluted 1:100 in 5 ml of fresh M9 minimal medium supplemented with 3 g/L nonanoic acid as the sole carbon source. When the optical density (OD ₆₀₀ ) of the MRD strain cultured overnight reached 2.5, it was diluted 1:100 in 5 ml of the same M9 minimal medium, inoculated, and cultured. This process was repeated 30 times and subcultured.

After the subculture, a single colony of the strain cultured on the LB plate was inoculated into 3 ml of LB broth and cultured at 37°C for 16 hours. The strain was diluted 1:100 and inoculated in 5 ml of M9 minimal medium supplemented with 4 g/L of glucose. After culturing for 24 hours, the strain was diluted 1:25 and inoculated in 25 ml of fresh M9 minimal medium supplemented with 0.5% Tween-80 and 3 g/L nonanoic acid. OD ₆₀₀ was measured, and the strain with the highest OD ₆₀₀ was named “MRE” strain and selected for further study.

Experimental Example 4. Whole genome sequencing

Genomic DNA was isolated from MRE strains using the GeneAll DNA Isolation Kit (GeneAll Biotechnology Co., Ltd., Cat. No. 103-102) according to the manufacturer's manual, and next-generation sequencing (NGS) (Macrogen, Korea) was used on an Illumina HiSeq 2000 platform. Sequenced. At this time, the genome of wild-type E. coli strain MG1655 (NCBI number NC_000913.3) was used as a reference for genome assembly. All mutations were confirmed by Sanger DNA sequencing.

Experimental Example 5. Cell viability measurement

Strains cultured overnight in LB broth medium were washed twice with new M9 minimal medium. Afterwards, the cells were re-distributed to a 96-well plate using M9 minimal medium supplemented with 3 g/L nonanoic acid to reach 8Х10 ⁷ cells, and then cultured for 30 minutes at 37°C and 200 rpm. After 30 minutes, the culture was serially diluted and plated on LB agar plates. After incubation overnight at 37°C, individual colonies were counted. Colony forming units (CFU) were then determined. All treatments were performed in triplicate to ensure data reproducibility.

Experimental Example 6. RNA sequencing

Strains cultured overnight in M9 minimal medium supplemented with 4 g/L glucose were diluted 1:25 and inoculated in new M9 minimal medium supplemented with 1 g/L nonanoic acid, and incubated at 37°C and 200 rpm. Cultured. RNA samples were prepared from cultures when OD ₆₀₀ reached 0.5. To isolate RNA from the strain, the RNA was first stabilized by treatment with RNAprotect® Bacteria Reagent (Qiagen #76506). Afterwards, to remove rRNA, riboPooL Probes (Pan-prokaryote) (siTOOLS BIOTECH) was used, and pure RNA was obtained through RNA Clean & Concentrator (Zymoresearch R1013). An RNA library was constructed using the KAPA Stranded RNA-seq Library Preparation Kit (Roche KK8400), and Nextseq was used for sequencing. Sequencing result files were analyzed with Bowtie, samtools, and cufflinks, and visualized with metascope. Gene expression was calculated as log2 fold change, where a change in gene expression greater than 4-fold (2-fold in log2 scale) was considered significant. At this time, for GO (Gene Ontology) term analysis, GO Consortium ( http://geneontology.org/ ) and PANTHER were used.

Experimental Example 7. Biotransformation

A single colony of the recombinant strain or MRE strain was inoculated into 5 ml of LB broth medium and cultured overnight at 37°C and 200 rpm. The next day, the cultured strain was inoculated (1:100) into 400 ml of LB broth medium and treated with 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at an OD ₆₀₀ of 0.7 to 0.8 to induce expression of the target gene. was induced and cultured for 16 hours at 30°C and 200 rpm.

Afterwards, the strain was centrifuged at 4°C and 2,600Хg for 15 minutes and resuspended in 0.1 M potassium phosphate buffer (pH 7.4). The strain was concentrated and used until OD ₆₀₀ reached 35.

Biotransformation was performed by mixing a 96% stock solution of nonanoic acid in dimethyl sulfoxide (DMSO) with 10 mL of 0.1 M potassium phosphate buffer (pH 7.4) and 1 /L: 2.4 g FeCl ₃ ·6H ₂ O, 0.3 g CoCl ₂ ·H ₂ O, 0.15 g CuCl ₂ ·2H ₂ O, 0.3 g ZnCl ₂ , 0.3 g Na ₂ MO ₄ ·2H ₂ O , 0.075 g of H ₃ BO ₃ , and 0.495 g of MnCl ₂ ·4H ₂ O) in a 250 ml shake flask. 0.4% glycerol, a co-substrate, was supplemented, and the process was performed on a shaker at 30°C and 200 rpm. Samples were collected at different time points for product quantification and confirmation using gas chromatography (GC).

Experimental Example 8. Product and data analysis

For quantification and confirmation of nonanedioic acid (NDA), 500 ㎕ of the supernatant was dissolved in 50 ㎕ of HCl at a concentration of 12 N and 50 ㎕ of nonadecanoate (Sigma-Aldrich) at a concentration of 1 g/L. Added.

Afterwards, nonanedioic acid was extracted twice using 500 μl of ethyl acetate and vigorous vortexing. After centrifugation, 500 μl of the organic layer was collected and mixed with 100 μl of MeOH and 50 μl of trimethylsilyl-diazomethane (Sigma-Aldrich). Nonanedioic acid methyl ester (NDAME) was analyzed using a gas chromatograph (Agilent 7890A) equipped with a flame ionization detector (FID) and a DB-5 column (30 mХ0.25 mm, Agilent Technologies).

Gas chromatography analysis was performed under the following conditions:

Injection volume, 2 μl;

Inlet at 270°C with a split ratio of 1:10;

detector temperature, 300°C;

Carrier gas, N ₂ ;

Flow rate, 0.82 mL min ^-1 ;

The oven temperature was maintained at an initial temperature of 60°C for 2 minutes, then raised to 180°C at 20°C min ^-1 and held for 2 minutes, then raised to 300°C at 20°C min ^-1 and held for 6 minutes.

The concentration of the target compound was determined using a calibration curve (R ² > 0.99).

Example 1. fadR Preparation and laboratory adaptation evolution of genetically deleted Escherichia coli (MRD) strains

Deletion of the fadR gene of E. coli upregulates the expression of related enzymes such as FadA, FadD, and FadE to derepress its β-oxidation pathway. In this example, the MRD strain, which is E. coli with the fadR gene deleted, was laboratory adapted. By evolving it, we wanted to obtain a mutant that uses nonanoic acid as the sole carbon source and grows faster.

Specifically, to produce an MRD strain in which the fadR gene was deleted in the MG1655 strain, the lambda-red recombination system and the FLP-mediated site-specific recombination system were used. Specifically, the kan cassette was PCR amplified using primers (SEQ ID NOs: 1 and 2) having sequences homologous to the fadR gene and pKD13 as a template. After expression from pSIM5 using the lambda Red recombination system, the fadR gene was deleted by replacing it with the kan cassette through homologous recombination. Afterwards, the substituted kan cassette was removed by Flp-FRT recombination using pCP20.

As described in Experimental Examples 2 and 3 above, the MRD strain was repeatedly subcultured in M9 minimal medium supplemented with 3 g/L nonanoic acid to evolve laboratory adaptation. Specifically, after 30 repeated subcultures (90 days), 10 single adapted strains (C1 to C10) were randomly isolated. The degree of growth of the isolated C1 to C10 strains in 3 g/L nonanoic acid was confirmed by the method of Experiment 5. At this time, wild-type E. coli (WT) and MRD that were not subjected to laboratory adaptive evolution were used as controls.

As a result, as shown in Figure 1, strain C6 (hereinafter referred to as MRE strain) showed the fastest growth among the 10 strains. The growth performance of the MRE strain was greatly improved compared to the wild-type E. coli and MRD strains, and in particular, OD ₆₀₀ reached 2.7 at 120 hours of incubation. In addition, the degree of growth in 3 g/L nonanoic acid was further confirmed for the wild-type E. coli, MRD strain, and MRE strain. As a result, as shown in Figure 2, after repeated subculture 30 times, the OD ₆₀₀ of the MRE strain reached 1.5 in 72 hours of culture. On the other hand, it was confirmed that both wild-type E. coli and MRD strains did not grow at all until 120 hours of culture. This suggests that the growth of the E. coli strain was inhibited by nonanoic acid.

Through this, an MRD strain with a deletion of the fadR gene was adapted and evolved in the laboratory to obtain an MRE strain that grows rapidly even in the presence of nonanoic acid.

Example 2. Confirmation of mutations in MRE strains

Next Generation Sequencing (NGS) was performed to confirm any mutations that occurred in the MRE strain.

As a result, it was confirmed that six mutations occurred in the MRE strain compared to the MRD strain. Information on each mutation is shown in Table 3 below. All mutations were confirmed using Sanger DNA sequencing.

gene gene product sequence change ^a Protein and nucleotide changes ^b acrR HTH-type transcriptional regulator AcrR ΔA Coding (567/648 nt) crp/yhfA cAMP-activated broad transcriptional regulator
CRP/OsmC family protein YhfA IS5(-) Non-coding(-177 nt)/(-126 nt) dppA Periplasmic dipeptide transport protein IS5(-) Coding (1570-1573/1608 nt) fadD Long-chain fatty acid-CoA ligase C→A & G→A Non-coding (-111 nt) & (-115 nt) icd-icdC e14 prophage Big DEL (1-15,204/15,204 nt) yeaR protein of unknown function IS186(-) Coding (115-120/360 nt)

DEL, deletion

^a (-), negative for the sequence of MG1655 available at NCBI (NC_000913.3).

^b Mutations in non-coding regions are annotated by location upstream of the start codon(s) of adjacent genes

As shown in Table 3, it was confirmed that adenosine (A) was deleted at position 567 of the base sequence of the acrR gene. The deletion changed the 26 subsequent amino acids into 53 different amino acids.

In addition, two mutations occurred in the CRP-cAMP regulation of the MRE strain: (1) insertion of IS5 into the intergenic region of crp and yhfA (P _crp* ); (2) Two nucleotide changes in the CRP-cAMP binding site of the fadD promoter (P _fadD* ).

Furthermore, there were two additional mobile element insertion mutations in the MRE strain: (1) IS5 insertion at position 30 upstream from the stop codon of the dppA gene, a periplasmic binding protein gene; (2) IS186 insertion at the 115th nucleotide of the unannotated gene yeaR . The IS5 insertion occurred in the middle of the dppABCDF operon, altering its transcription initiation from the dppA promoter.

Finally, it was confirmed that the 15.2 kbp cryptic prophage element e14 prophage was deleted in the MRE strain.

Example 3. Comparison of growth ability and nonanedioic acid production ability in nonanoic acid according to mutation.

In order to analyze whether the mutations identified in Example 2 contribute to the growth of the MRE strain in nonanoic acid, the cell growth of the recombinant strain derived from the MRE strain in which each single mutation was restored to the wild-type gene was analyzed by the method of Experimental Example 5. (Figure 3a).

Specifically, the lambda-red recombination system and the tetA double selection system (see doi: 10.1371/journal.pone.0181501) were used to prepare a recombinant strain derived from an MRE strain in which each single mutation was restored to the wild-type gene. Specifically, the tetA cassette was PCR amplified using P3-BCD2-tetA as a primer and template containing homologous sequences outside the mutation of the acrR, fadD, dppA, crp, and yeaR genes. The primers for each gene used were acrR primer (SEQ ID NO: 13 and 14) , fadD primer (SEQ ID NO: 11 and 12) , dppA primer (SEQ ID NO: 32 and 33) , crp primer (SEQ ID NO: 28 and 29) , and yeaR primer (SEQ ID NO: Same as numbers 24 and 25). After expression from pSIM5 using the lambda Red recombination system, the mutations were replaced with the tetA cassette through homologous recombination. Afterwards, it was replaced with an oligo or cassette containing the wild-type sequence using the lambda Red recombination system. The oligos for each gene used are acrR oligo (SEQ ID NO: 16) and fadD oligo (SEQ ID NO: 10), and the primers for cassette amplification are dppA primer (SEQ ID NO: 34 and 35) , crp primer (SEQ ID NO: 30 and 31) , and yeaR primer. (SEQ ID NO: 26 and 27). E. coli substituted with wild type was selected using nickel.

As a result, it was confirmed that the P _fadD* mutation is important for MRE growth on nonanoic acid. The MRE-Pf strain, in which the P _fadD* mutation was restored from the MRE strain, failed to grow in nonanoic acid with a final OD ₆₀₀ exceeding 0.1. On the contrary, the MRD-Pf strain in which the P _fadD* mutation was introduced into the MRD strain reached a final OD ₆₀₀ of 1.0.

In addition, based on the growth curves of the MRE-a strain and the MRE-d strain, it was confirmed that acrR and dppA mutations also contribute to the growth of the MRE strain in nonanoic acid. The acrR* mutation was restored in the MRE strain. The MRE-a strain and the MRE-d strain in which the dppA* mutation was restored from the MRE strain reduced the growth of the MRE strain to 59% and 82%, respectively, at 120 hours in nonanoic acid. Other factors such as toxicity tolerance and favorable membrane transport were found to be beneficial for strain growth in nonanoic acid.

Through this, it was confirmed that the P _fadD* mutation is an important mutation for the growth of the strain in nonanoic acid, while other mutations may also partially contribute to the growth of the MRE strain.

Meanwhile, in order to identify mutations related to nonanoic acid toxicity resistance in the MRE strain, strains into which each single mutation was introduced into the MRD strain were prepared, cultured in 3 g/L nonanoic acid for 30 minutes, and then colony forming units (CFU) were measured. Cell viability was assessed by counting (Figure 3b).

Specifically, the lambda-red recombination system and the tetA double selection system (see doi: 10.1371/journal.pone.0181501) were used to prepare recombinant strains derived from MRD strains introducing each single mutation. Specifically, the tetA cassette was PCR amplified using P3-BCD2-tetA as a template and primers with homologous sequences outside the positions where mutations in acrR, fadD, dppA, crp, yeaR, and e-prophage occurred. The primers for each gene used were acrR primer (SEQ ID NO: 13 and 14) , fadD primer (SEQ ID NO: 11 and 12) , dppA primer (SEQ ID NO: 32 and 33) , crp primer (SEQ ID NO: 28 and 29) , and yeaR primer (SEQ ID NO: Nos. 24 and 25), icd-icdC primer (SEQ ID Nos: 19 and 20). After expression from pSIM5 using the lambda Red recombination system, the mutations were replaced with the tetA cassette through homologous recombination. Afterwards, it was replaced with an oligo or cassette containing the mutant sequence using the lambda Red recombination system. The oligos for each gene used are acrR oligo (SEQ ID NO: 15) and fadD oligo (SEQ ID NO: 9), and the primers for cassette amplification are dppA primer (SEQ ID NO: 34 and 35) , crp primer (SEQ ID NO: 30 and 31) , and yeaR primer. (SEQ ID NO: 26 and 27). E. coli substituted by mutations were selected using nickel.

As a result, as shown in Figure 3b, the survival rate of the MRD-a strain, which introduced the acrR mutation into the MRD strain, increased 2.8 times compared to the MRD strain. In addition, the MRD-Pf strain, which introduced the P _fadD* mutation into the MRD strain, showed a survival rate 3.3 times higher than that of the MRD strain.

Additionally, the MRD-e strain with a deletion of the gene encoding the e14 prophage of the MRD strain showed slightly increased nonanoic acid toxicity, due to the close relationship between secretive prophages and the strain's response to stresses such as acid stress or hydrogen peroxide stress. showed resistance.

Through this, it was confirmed that laboratory adaptation evolution not only improved fatty acid metabolism but also acquired toxic resistance to nonanoic acid.

Example 4. Nonanedioic acid production using MRE strains

To produce nonanedioic acid from nonanoic acid using the MRE strain, the acyl-CoA dehydrogenase gene ( fadE ) was deleted from the MRE strain to prevent nonanoic acid degradation by the β-oxidation pathway, and then biotransformed. The strain with the fadE gene deleted was prepared in the same manner as Example 1 using primers (SEQ ID NOs: 5 and 6).

In addition, in order to use the AlkBGT system derived from P. putida GPo1, the alkBGT gene was expressed under the control of the P _LlacO-1 promoter on the pBbA6c plasmid and introduced into the MRE strain and MRD strain with the fade gene deleted, respectively, producing the MRE-BGT strain. and MRD-BGT strains were prepared. Biotransformation of nonanedioic acid was performed using the following strains: MRE-BGT, MRD-BGT, and MRD-EB (MRD-BGT with all six MRE mutations introduced). The MRD-EB strain was prepared by introducing the acrR*, PfadD*, dppA*, crp*, e-prophage*, and yeaR* mutant genes of the MRE strain in the same manner as in Example 3.

As a result, as shown in Figure 4a, the MRE-BGT strain produced 221 mg/L of nonanedioic acid at 24 hours, which was 12.8 times higher than the production of the MRD-BGT strain (17.3 mg/L). Confirmed. In addition, the nonanedioic acid production of the MRD-EB strain was similar to that of the MRE-BGT strain, and it was confirmed that the mutations introduced therefrom (all six MRE mutations) increased the production of nonanedioic acid by the strain.

Additionally, in order to analyze mutations related to nonanedioic acid production, the fade gene of the mutant strain derived from the MRD strain of Example 3 was deleted and then transformed with plasmid pBbA6c- alkBGT . Nonanedioic acid production of the resulting strains was compared and analyzed (Figure 4b).

As a result, the strains (MRD-PcFB, MRD-eFB, MRD-dFB, MRD-PfFB, MRD-aFB, and MRD-yFB) into which each single mutation was introduced had significantly higher nonanedioic acid production compared to the MRD-BGT strain. increased significantly. In particular, among the strains into which each single mutation was introduced, the nonanedioic acid production of the MRD-PcFB strain into which the P _crp* ( crp promoter mutation) mutation was introduced significantly increased to about 68.6% of the nonanedioic acid production of the MRE-BGT strain. Through this, it was confirmed that the P _crp* mutation is important for nonanedioic acid production.

<110> UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> TRANSFORMED MICROORGANISM PRODUCING NONANEDIOIC ACID AND A METHOD FOR PRODUCING NONANEDIOIC ACID USING THE SAME <130> FPD/202012-0001 <160> 54 <170> KoPatentIn 3.0 <210 > 1 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_fadR del H1 F <400> 1 tctggtatga tgagtccaac tttgttttgc tgtgttatgg aaatctcact gtgtaggctg 60 gagctgcttc 70 <210> 2 <2 11> 70 < 212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_fadR del H2 R <400> 2 aacaaaaaa aacccctcgt ttgaggggtt tgctctttaa acggaaggga attccgggga 60 tccgtcgacc 70 <210> 3 <211> 21 <212> DNA <21 3> Artificial Sequence <220> <223> nucleotide sequence for primer_fadR seq F <400> 3 gggcgagttt gtaccagtcg g 21 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_fadR seq R <400> 4 gcgcggcgac cgttcgctg 19 <210> 5 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_fadE del H1 F <400> 5 ccatatcatc acaagtggtc agacctccta caagtaaggg gcttttcgtt gt gtaggctg 60 gagctgcttc 70 <210> 6 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_fadE del H2 R <400> 6 ttacgcggct tcaactttcc gcactttctc cggcaacttt accggcttcg attccgggga 60 tccgtcgacc 70 <210> 7 < 211> 25 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_fadE seq F <400> 7 aaaagttagc cagcgtttcc gccgc 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_fadE seq R <400> 8 acgttgggag atgagacgta tcagg 25 <210> 9 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PfadD* MAGE Oligo <400> 9 atattaactc atcataccag cttgataatt acccaacgaa aagtttgtga agcgcgtcac 60 tatttatttt tatctttacc gtaagaatgc 90 <210> 10 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PfadD back <400> 10 atattaactc atcataccag cttgataatt acccaacgaa aaggttgcga agcgcgtcac 60 tatttatttt tatctttacc gtaagaatgc 90 <210> 11 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PfadD* tetA H1P1 F <400> 11 ctgtttctgc attcttacgg taaagataaa aataaatagt gacgc gcttc gtgtaggctg 60 gagctgcttc g 71 <210> 12 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PfadD* tetA H2P4 R <400> 12 taacataata ttaactcatc ataccagctt gataattacc caacgaaaag attccgggga 60 tccgtcgacc 70 <210> 13 <2 11 > 71 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_acrR tetA H1P1 F <400> 13 cggcctgatg gaaaactggc tctttgcccc gcaatctttt gatcttaaaa gtgtaggctg 60 gagctgcttc g 71 <210> 14 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_acrR tetA H2P4 R <400> 14 ggcacaggag atacatctcc agtaagatgg caacgtaatc gcgggcttct attccgggga 60 tccgtcgacc 70 <210> 15 <211> 90 <212> DNA <213> Artificial Sequence <220 > <223> nucleotide sequence for primer_acrR* MAGE Oligo <400> 15 gagatacatc tccagtaaga tggcaacgta atcgcgggct tctttttaag atcaaaagat 60 tgcggggcaa agagccagtt ttccatcagg 90 <210> 16 <211> 90 <212> DNA <213> Artificial Sequence <2 20> <223> nucleotide sequence for primer_acrR back <400> 16 atattaactc atcataccag cttgataatt acccaacgaa aaggttgcga agcgcgtcac 60 tatttatttt tatctttacc gtaagaatgc 90 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_acrR seq F <400 > 17 cctgttaggg ctaagctgcc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_acrR seq R <400> 18 cgccgtgttc tggcataaca 20 <210> 19 <211> 70 < 212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_e14 H1-P4 <400> 19 aaagtaaatc ctggctctat tattctctcc gctgagatga tgctgcgcca attccgggga 60 tccgtcgacc 70 <210> 20 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_e14 H2-P1 <400> 20 cgccttccat acctttaaca atcaggtcag ccgcttcagt ccaacccata gtgtaggctg 60 gagctgcttc g 71 <210> 21 <211> 90 <212> DNA <213> Artificial Sequence <2 20> <223 > nucleotide sequence for primer_e14 MAGE oligo <400> 21 tccatacctt taacaatcag gtcagccgct tcagtccaac ccatatggcg cagcatcatc 60 tcagcggaga gaataataga gccaggattt 90 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> nucleotide sequence for primer_e14 seq F <400> 22 gtaattcccc tgtcttcagg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_e14 seq R <400> 23 aacaacgggc gtgttatacg 20 <210> 24 <211 > 70 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_yeaR H1-P4 <400> 24 cacctgccgg aatattcgaa cgtcatcttg ataaaggaac gcgcccgggg attccgggga 60 tccgtcgacc 70 <210> 25 <211> 71 <21 2> DNA < 213> Artificial Sequence <220> <223> nucleotide sequence for primer_yeaR H2-P1 <400> 25 tcagcgtagc cgagatattt gaccgcccca tgcataacgg aaaggcgtgg gtgtaggctg 60 gagctgcttc g 71 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220 > <223> nucleotide sequence for primer_yeaR IS186 FP <400> 26 cacctgccgg aatattcgaa 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_yeaR IS186 RP <400> 27 tcagcgtagc cgagatattt 20 <210> 28 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PyhfA/crp H1-P4 <400> 28 cctgggtcat gctgaagcga gacaccagga gacacaaagc gaaagctatg attccgggg a 60 tccgtcgacc 70 <210 > 29 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PyhfA/crp H2-P1 <400> 29 ctttgcatac atgcagtaca tcaatgtatt actgtagcat cctgactgtt gtgtaggctg 60 gagctgcttc g 71 <210> 30 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PyhfA/crp IS5 FP <400> 30 cctgggtcat gctgaagcga 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence < 220> <223> nucleotide sequence for primer_PyhfA/crp IS5 RP <400> 31 ctttgcatac atgcagtaca 20 <210> 32 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_dppA H1-P4 <400> 32 ccgtgtttga accggtacgt aaagaagtta aaggctatgt ggttgatcca attccgggga 60 tccgtcgacc 70 <210> 33 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_dppA H2-P1 <4 00> 33 ttgtatggct tttaattatt cgatagagac gttttcgaag tgatgtttgc gtgtaggctg 60 gagctgcttc g 71 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_dppA IS5 FP <400> 34 ccgtgtttga accggtacgt 20 <2 10> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_dppA IS5 RP <400> 35 ttgtatggct tttaattatt 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> nucleotide sequence for primer_dppA IS5 seq FP <400> 36 gcctctgaac aaggctccaa 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_dppA IS5 seq RP <400> 37 acagcgccac cgggtataca 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PyhfA/crp IS5 seq FP <400> 38 ttatcgcctg agttgccgtc 20 <210> 39 <211> 2 0 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_PyhfA/crp IS5 seq RP <400> 39 aacattcga gagtcgggtc 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220 > <223> nucleotide sequence for primer_yeaR IS186 seq FP <400> 40 cttcgctgat atggtgctaa 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for primer_yeaR IS186 seq RP <400 > 41 ctttccgttg ttgcgcacct 20 <210> 42 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for acrR <400> 42 atggcacgaa aaaccaaaca agaagcgcaa gaaacgcgcc aacacatcct cgatgtggct 60 ctacgtcttt tctcacagca gggggtatca tccacctcgc tgggcgagat tgcaaaagca 120 gctggcgtta cgcgcggtgc aatctactgg cattttaaag acaagtcgga tttgttcagt 180 gagatctggg aactgtcaga atccaatatt ggtgaactag agcttgagta tcaggcaaaa 240 ttccctggcg atccactctc agtattaaga gagatattaa ttcatgttct tgaatccacg 300 gtgacagaag aacggcgtcg attattgatg gagattatat tccacaaatg cgaatttgtc 360 ggagaaatgg ctgttgtgca acaggcacaa cgtaatctct gtctggaaag ttatgaccgt 420 atagaacaaa cgttaaaaca ttgtattgaa gcgaaaatgt tgcctgcgga tttaatgac g 480 cgtcgcgcag caattattat gcgcggctat atttccggcc tgatggaaaa ctggctcttt 540 gccccgcaat cttttgatct taaaaaagaa gcccgcgatt acgttgccat cttactggag 600 atgtatctcc tgtgccccac gcttcgtaat cctgccacta acgaataa 648 <210> 43 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for fa dD/yeaY intergenic region <400> 43 ttgcttgttt ttaaagaaaa agaaacagcg gctggtccgc tgtttctgca ttcttacggt 60 aaagataaaa ataaatagtg acgcgcttcg caaccttttc gttgggtaat tatcaagctg 120 gtatgatgag ttaatattat gttaacggca tgtatatcat ttggggttgc gatgacgacg 180 aacacgcatt ttagagg tga agaa 204 <210> 44 <211> 1608 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for dppA < 400> 44 atgcgtattt ccttgaaaaa gtcagggatg ctgaagcttg gtctcagcct ggtggctatg 60 accgtcgcag caagtgttca ggctaaaact ctggtttatt gctcagaagg atctccggaa 120 gggtttaacc cgcagctgtt tacctccggc accacctatg a cgcctcttc cgtcccgctt 180 tataaccgtc tggttgaatt taaaatcggc accaccgaag tgatcccggg cctcgctgaa 240 aagtgggaag tcagcgaaga cggtaaaacc tataccttcc atctgcgtaa aggtgtgaag 300 tggcacgaca ataaagaatt caaaccgacg cgtga actga acgccgatga tgtggtgttc 360 tcgttcgatc gtcagaaaaa cgcgcaaaac ccgtaccata aagtttctgg cggcagctac 420 gaatacttcg aaggcatggg cttgccagag ctgatcagtg aagtgaaaaa ggtggacgac 480 aacaccgttc agtttgtgct gactcgcccg gaagcgccgt tcctcgctga cctggcaatg 540 gacttcgcct ctattctgtc aaaagaata t gctgatgcga tgatgaaagc cggtacaccg 600 gaaaaaactgg acctcaaccc aatcggaacc ggtccgttcc agttacagca gtatcaaaaa 660 gattcccgta tccgctacaa agcgtttgat ggctactggg gcaccaaacc gcagatcgat 720 acgctggttt tctctattac ccctgacgct tccgtgcgtt acgcgaaatt gcagaagaat 780 gaatgccagg tgatgccgta cccgaacccg gcagatatcg ctcgcatgaa gcaggataaa 840 tccatcaatc tgatggaaat gccggggctg aacgtcggtt atctctcgta taacgtgcag 900 aaaaaaccac tcgatgacgt gaaagttcgc caggctctga cctacgcggt gaacaaagac 960 gcgatcatca aagcggttta tcagggcgcg ggcgtatcag cgaa aaacct gatcccgcca 1020 accatgtggg gctataacga cgacgttcag gactacacct acgatcctga aaaagcgaaa 1080 gccttgctga aagaagcggg tctggaaaaaa ggtttctcca tcgacctgtg ggcgatgccg 1140 gtacaacgtc cgtataaccc gaacg ctcgc cgcatggcgg agatgattca ggcagactgg 1200 gcgaaagtcg gcgtgcaggc caaaattgtc acctacgaat ggggtgagta cctcaagcgt 1260 gcgaaagatg gcgagcacca gacggtaatg atgggctgga ctggcgataa cggggatccg 1320 gataacttct tcgccaccct gttcagctgc gccgcctctg aacaaggctc caactactca 1380 aaatggtgct acaaaccgtt tgaagatctg attcaaccgg cgcgtgctac cgac gaccac 1440 aataaacgcg ttgaactgta caaacaagcg caggtggtga tgcacgatca ggctccggca 1500 ctgatcatcg ctcactccac cgtgtttgaa ccggtacgta aagaagttaa aggctatgtg 1560 gttgatccat taggcaaaca tcacttcgaa aacgtctcta tcgaataa 1608 <210> 45 <211> 301 < 212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for yhfA/crp intergenic region <400> 45 ttttgctact ccactgcgtc aattttcctg acagagtacg cgtactaacc aaatcgcgca 60 acggaaggcg acctgggtca tgctgaagcg agacaccagg a gacacaaag cgaaagctat 120 gctaaaacag tcaggatgct acagtaatac attgatgtac tgcatgtatg caaaggacgt 180 cacattaccg tgcagtacag ttgatagccc cttcccaggt agcgggaagc atatttcggc 240 aatccagaga cagcggcgtt atctggctct ggagaaagct tataacagag gataaccgcg 300 c 301 <210> 46 <211> 15204 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for icd -icdC <400> 46 catgggttgg accgaagcgg ctgacttaat tgttaaaggt atggaaggcg caatcaacgc 60 gaaaaccgta acctatgact tcgagcgtct gatggatggc gctaaactgc tgaaatgttc 120 agagtttggt gacgcgatca tcgaaaacat gtaatgccgt agtttgttaa atttattaac 180 gggagcgtaa cgctcccgtt gtt ttttgtt aggctgctaa cggttatcaa aattttatca 240 aaaaaagtta tcaaaacccc tcggtagttt tggggtaggc tggccggtca ggtggtagtt 300 ctactactag tctcccacat agatattcct tagcttttta ttattgctgg cggacgctcg 360 ttaatattta aggtcttcat tgattaagac atccccaaag ttagttatgt attcactgtt 420 attaggacaa ttatgaatta ccactcctta cacccgctca aatattgtta aattgccggt 480 tttgtatcaa ctactcaccc gggactcgcc aggggacagc caacaggcat tgggtgcaat 540 caccttagcg ttcaggtaca tgcggaatgt aaaaaaggcc gcgagcgcgg ccccttcaca 600 tacatcttta gtactgagac tgtttaacct agggaattat tatcgt atta tattgcatta 660 caactcccaa cagtgacata tgaacttcct gctttactcc acgactttaa tatttcaagg 720 ccatgtgaag aacatatttc ttgcactgcg agttcatcga gtaatccata gtaagaaaca 780 ctttttgaat ttttgtaaat gtatccgtaa agatgttttc ttcc tgtttc gtattttttg 840 aagtatgagc ttttatattg atttacaata agagtctcac ctcctgattt tagtaatctc 900 ttgatgctaa gaactatttt gtcgattgta tcccgacaag gaacggcaga gagaacatta 960 gagcaaagga tgaaatcgta acccccaatt attttgtcga catcctcgaa agcaactgta 1020 tttgcatttt tataatatcg tgggacatag tcaataattt tagttttaat tcctctaata 1080 atttgctctc tttcaagttg ccttttcgag tctagaaaag taacttcatc aaatttactg 1140 attaattcat cagaatatct aagttttccg cagccaaaat caagggcgtg gccatttttc 1200 tcaatgcttc taatatactc gcaaagatat ctagagggca tcgtatgagg ttt tgctgca 1260 ttctctgagc gaatattaac tccgtgcata ttatagttca aagcaagtac cattcaatta 1320 atgttattt tagtgaaaaa ttcttttatt ttatcgtccg ggatttttcc gacttgactt 1380 tcagttctgg gcttaactgt caaagcaaag ataataccac tcactgagaa tgataaaatt 1440 acaaaaaaga ataaagacag caaagaacct tcaacatgaa aaatatccat ttgtttgcaa 1500 aaaaagatta ttaggaagga aattaatgca attatcgaaa attcaaaaaaa tatccaaaaa 1560 tagtatactt tattccagaa gagttcaata taatgtttgt cttcaatttt tcttacttca 1620 gggtaatata gattgctcat tacattgtga gcttcatctt tatttaattt tctgttgact 1680 ccagctctcc gtgataacgg ttttataatt agatgcttat cccaaagata tcgcacccga 1740 agtagtttgg ctgcattgtt atgtaggtct aacgcaccgc taattaaata tgcaaaaatc 1800 gcaaaagcac aaggtaatat accaaaagat agtgcccaat taataaagct ctcatgatct 1860 ttaataggtg ggacatactt ggttggtaat gttattaaag ggatgtattc gtaaataact 1920 agaagtat gc aatatattag cgtatttaag aaagttgctt tatgaagttt aggcaacatt 1980 tcattataat ttttgggggg ttcgaacata ttattcacct gagtcacgct ttaggatagt 2040 gtaatggtaa tatttaatta agtgtcatta tatacttttc agtaggttag ttacaatttt 2100 ttgta tctgt tcaggctgac ctagcttcgc tgacagacaa tattgtgatc agtagcacgt 2160 atcgaggagg agtagcgcta caaatttgac gctgggtgag aatctgaaat tgatagaaat 2220 gaaataatga aataatgaaa tgatgaaatg atgaaatgat gaaatgatga aatgatgaaa 2280 tgatgacaga gtgtccagtg ggcacggatg gtgtcttacg acatgcttac cttaatcgtt 2340 cccagt gtgc ctatagcaga tattctaaac atgtcgataa ttcattacgc atatagtatc 2400 gaacatagaa aaaactgaag attcatctta ttttgtatat actacctagc ccaacaatgt 2460 agaggttaac gaaaaaatgcg ctcaccaatt tgtcatcttt tctcagcaat taattcatca 2520 ccattta aga ttgcaccaga gaaggagcaa gatcttaaaa cgatagttga cgacaaaaaaa 2580 attataattt cagttgtgag tgaacctggt tttaatatcc gagtcaggaa gaatgagagt 2640 aataattcac atgaaatagt tctaacagta gcttcacttg aatatatttg ggcattttcc 2700 aatttctttt gggtttttac gcaagagtac tccaaatctc agaaaaataa tgatgagcac 2760 tttgatttaa caggaaaaaa taggct taaa aagtctgatg aacttcttaa atgggcaagg 2820 aaaaacttgc aaacaacagg ttgcgaatca tggcctaaaa aatgtcccaa gccagaagca 2880 tatttacaag gaagcgaaga ctcacaagtt gctagcgaga tatttctttg tgctattgct 2940 tggattcttc atcatgaaat a agtcatgtt gttttacagc atccattggt cactacagca 3000 ttctccactc aagaggagcg tgaagcagat tcacatgcta caaaatggat attaggcaac 3060 ctgtatgaat ccgctcctga attaaagaaa cgtgcacttg gcattgctac ggcagtgctt 3120 tgtatacaaa gcttagaagt tgaaaattac ttctgtttac aaaatacaca cccagctgca 3180 tatgagcgta tatattcgaa tatttcatgc tacc ctgtcg gaaatgaaga gttgattgaa 3240 gctctatgta cagtgatgct tcaatatctt ttccatggca aaaatatcaa tgtgaatcta 3300 gatggggagt ccttttcatc gattttaggt gatcttctct gtgatatttc acgtcttacc 3360 agtaactgat atggctgtcc gcc gctcgct taaagtggac tttttagttt ttatcatgtg 3420 cggtgagaaa ttcaatgtgg cgttgagatg cttaaaggtt cacaacgcta ctttgctcca 3480 tcctttacct cgatcatcat gataacgatc ggtttgttgt tgtgttttat gaccaagtag 3540 tttttgtgtg tctaacccct gttctttata cagacgttca gataaagacc tttgctcatg 3600 gaatgtcgca ggtgaaccct ctcccc agtc aattcttgct aaatctctcg ctttactaaa 3660 attcatcgtc aatgtattgg ctttaacctg cgctccgcgc tctgcttgtg aagttgaacg 3720 aaaaaaatgc actaagtatg cactgactgc atagtcacgg cagcgggcta ctacatcgcg 3780 taaactccag ttaatcgcat tgaggcgaag agaaagagga attgcgattt tgctcccggt 3840 cttttcctga atgacatgaa gatgatcatc ccaaatatcg ctaaatttca tacgcgaaat 3900 atcacctaac cgctgaccag taaccagcgc taacagcatg gcatttccca tgtaacgatg 3960 agtagcgtct gcgatatcga agatttttt ccattcttca aggctcagcc gttgtcgggt 4020 aatttttctt cttggttgtt tagtggctaa tgctgggtta tagc caggag gtacttctcc 4080 gtagtgctgc gcctctttga aaacatcaat caggacggag cgaactactt gtgccattct 4140 tggccgccca gcggcgatat actcatcaag caattgtgct atatctctga catcaacggc 4200 tgagatcaac ttcattcctg ctcgttctct gagcaaggat actggtttag ctttttgttt 4260 ataggtgttg agtcttatat caccactttt aagcctgtca tcctggatcg cttgatagcg 4320 atctaaccag gttgacgttg tgatagcctt tcctttgctg g ttgcgatcc tgtcactgat 4380 agccagaatc tgccgggttc tttgttcagc caggcgagtg ttggcctcag tggcaatagc 4440 gatagcttca gcttcgtttg ttcccaaagc atggaatttt cctgtcactg gatgcttata 4500 ccgccaatag actttattta ccttcctact ataaagcgga tataagttag ggactgaaac 4560 attattctta cgcggtctgg ctgccattac tcaaaatccg ttgcaaaagt aatgagtcat 4620 ttttcttgat tacaggtgtt accaactccc caactaactc ggcgtcctca cgcactcgcc 4680 ataaccggcc ttgtttcatg gccggtggac aaaataaatt ctgcttagca taacgacgca 4740 atgtggacac acttggagga ttacttctgt atttttcagc agcccattct tcaagagtta 4800 acat ttgaag catatgcgat caccttatta ctacactaac tgcttagtct cagcatatcg 4860 accctgcacg gtcggttagt ttctccacaa aacagagaag agcacctgtg gccacagcta 4920 tcaggatggg tcgggttatt aacccgtcat ccggggatac tcttctctgt tttgtaaaaa 49 80 gggcggtacc agaaaggact aaggaaaaaaa ctggtaccgc caagactaca cacagcataa 5040 agttgtggtg tcgggtgccc ccggtgcctg gcgaaggttg cacaccaggc gggtgggtat 5100 ccacagaagg tcgattgtca gcctcaacct taacccgcgt gcgctgagcc gcattcacca 5160 caacgctaag gattctctct ggttgaaaat acttagctgt tatgtgcctg tcttttcacc 5220 acttca ggct cggtggtatg ctggagttct cacacagcca gcaagcaagg aaacttaatg 5280 aaccagtttt atgttcacgt tcgtctattt gaagacacag ccgaacagac caaaaaattt 5340 gaagaattaa tgcttaactt tctgtaccag aaaacagtta aagagtctga cgatagctgc 5400 t gcagactga ttccagaggg atatatcctc aaaagtacaa tgaactgcca acaaatcctt 5460 gatcaaacat tttcaattgc taacagtgcc ggtgttgacg caaatatatt tgtctgtaaa 5520 tttgaacaaa gcgcatgctt acttccgtct gcttccttag ttggtaacga tttcgttcat 5580 tacgatctta cgcctaagcc catcaagctc gattcttaaa gccttaacca ttgtgtcgtg 5640 ataaacacgg ctcacct tct ctccattgca tggcagaggg gtgagtgtgt tagccatgaa 5700 attcatgaac tcggttcgac caggggcttg cgccccgcaa gtctttaatg cctgttttgc 5760 taacaaaaatg cgggcctcag tgcctgcatt tggctctatc tgctgcaaac gtttagcgtc 58 20 ttccagcaac aatgcgatca catgcttcaa attctgctca ttcatctatt ctctccactg 5880 aaatcatccg ctaacgaatc atcccggtct tcgtacgtac cgggcgggct acttcgtggg 5940 cgtcctgcct gtttgttgtt tctcttgggt acattatgta tctcaaaggt acattgtcaa 6000 gtataaaaaa acctgccgaa gcaggttcat aaacattgat taggctttga ttttgtatct 6060 tcttggtttt cct gagaaaa tcacagtacc aattatagag caattaccgt tgatcttaat 6120 gtaaggctca ggccagtttg ggtttaacgc tttgagataa cgctgtgtcc catcttctat 6180 caaccttttg aaggtggttt cacctgtatc gtgcatcaat gcaataacgt cgtcaccgtg 6240 gcaggcaggt acttcaggat cgacaaaaat catgtctccc gggcggtact catcaatcat 6300 tgaatcacct attacccgca agatataagt catttcccca cagggtacag ggcagggata 6360 cgtttctgct gtgctcaaat caacctcaga atatccaact tctttccatg ctccggcctg 6420 tacccatgat atgacaggga ctaatgtgat ttgtttatta gtgattgaaa catcaggttt 6480 ttttgtgatg ttcgttgtct ggtgttcttg atcgagccat cctacaggca ggtcgaaaca 6540 tttttcgatg tgtcgtgcca tgctgtcacc gatattttta gtagcaccat ctcccataaa 6600 cctgctggtc tgggttggct cgcgatcaat catagtggca aaggaagaat tcccgccaac 6660 accatctctc agttt tctgg cgttagaccg ccggatgtca tggattgttt tcataacgaa 6720 attaaaaccc ttgtaccgtt aaggtacaag tatcttgaag gttcatttca atcatgtaat 6780 atgtacaccg gaggtacata ttgtatgaaa gcgtattggg actctttaac caaagaacag 6840 cagggcgagt tggccggaaa agttggctca acacctggct acttacggct ggttttcaat 6900 ggctataaaa aagccagttt tgtgctggct aaaaaacttg agcaatacac atcagg tgca 6960 attacgaaat ctgacttaag accggatatc tatccgaaag attagcagaa cactttcaat 7020 ttttaaccac agaacgatga ggctaatcgt gggtaagcat cactggaaaa tagaaaaaca 7080 gcctgagtgg tacgtgaaag ctgtcagaaa aactatcgcg gcgttgccga gtggttacgc 7140 tgaagcggct gactggctcg atgtaacaga aaacgcttta ttcaaccgcc ttcgtgcaga 7200 tggcgatcag attttcccgc tgggatgggc aatggtttta cagcgtgctg gtggcactca 7260 cttcattgct gatgctgtgg cgcagtctgc aaatggcgtc tttgtgtctc ttcctgacgt 7320 cgaggatgtg gacaacgccg atatta gcgtctg ctg gaagtcattg aacagatcgg 7380 cagttattca aaacagattc gttcagcaat cgaagacggt gtagtggaac cgcatgagaa 7440 gacagcaatt aacgacgagc tgtatctctc aatttcgaag ctgcaggagc atgcagcact 7500 tgtctacaaa attttttgca ttt cagaaag taatgacgcc cgcgagtgtg cagctccggg 7560 cgtcgtggcg tcgattgctt ctggttgtgg agaaactaac gcatgaacag tttaacaaca 7620 cactaccgtc gctcgcaact gattgcgctt cctgtaccgg gtggaaaaagc gaaggtggaa 7680 tattgctatg cagtgaatgt accaggtgac agggaaattg taacccacag ctttgcagag 7740 tgggctgtgg gtgatttcaa ccggcagaag gagacagtcc tttgcgacaa gttaaccgct 7 800 ggttcaaaga tcactacgga gtgcccgtca gagtcattcg ttgggagccg gaaacacaac 7860 gggttatcta cctccgcgaa ggttatgagc atgaatgctt cagcccgctc gaacagtttc 7920 gtcgtaaatt cagggaaata gaggtcggtc atgagcacta aattaacc gg ctatgtatgg 7980 gatggttgcg ctgcatcagg catgaagtta tccagcgtgg caattatggc ccgcctggct 8040 gatttcagta atgacgaagg tgtgtgctgg ccatcaattg aaaccatgc ccgtcagatt 8100 ggcgcgggga tgagtaccgt cagaacggct atcgcacggc tggaagcaga aggctggtta 8160 acgcgtaagg cgcgtcgcca gggtgatggt tcatcacccc actgtgccgt ggtggatgaa 822 0 tatcacgagc acgccacaga tgcgctttac accacgatgc ttaccgggat gggggcgcga 8280 cgccagccac tgatgtgggc cattaccacc gccgggtaca acattgaggg gccgtgctac 8340 gacaaacggc gggaagtcat cgagatgctc aacggctcgg tgccaaacga tgaact gttc 8400 gggatcatct ataccgttga tgaaggtgac gactggaccg acccgcaggt gctggaaaaaa 8460 gccaatccaa atattggcgt gtcggtttat cgcgaatttt tgttaagtca gcagcagcgt 8520 gcgaaaaata acgcccgtct ggcaaacgtc tttaaaacaa aacacctcaa tatctgggcg 8580 tcggcgcgtt cggcgtattt caacctggtg agctggcaga gctgcgagga taaatcactg 8640 acccttga gc agttcgaggg gcagccgtgc attctggcct ttgacctggc gcgtaagctg 8700 gatatgaaca gcatggcgcg actttatacc cgcgagattg acggtaaaac gcattactac 8760 agtgtggccc cgcgtttctg ggtaccgtat gacacggtgt acagcgtcga gaaaaatgaa 8820 gatcgccgga cagccgaacg ctttcagaaa tgggtggaaa tgggcgttct gaccgttacc 8880 gatggtgcgg aggtggatta tcgctacatc ctcgaagagg ccaaagcggc gaacaaaaatc 8940 agcccggtca gtgagtcacc catcgacccc ttcggggcga ccgggctgtc acatgacctt 9000 gctgatgaag acctgaaccc cgtcaccatc attcagaact acaccaacat gtccgatccg 9060 atgaaagagc tggaagcggc gattgaat cg gggcgctttc atcatgacgg caatcccatc 9120 atgacctggt gtatcggcaa cgtggtcggc aaaaccattc cgggtaacga tgatgtggtg 9180 aagcccgtca aggagcaggc ggaaaacaaa atcgatggtg cagttgcgct gattatggcg 9240 gttggcagag ccatgctgta cgagaaagaa gacacgctgt ctgatcacat tgagtcctac 9300 gggatccgct cgctttaact gaggtaatta tgatcatgct gattctcgcg cctctggtgg 9360 gcgtgctggg tgcgcttttg ctggcgtatg gtgcctggct gatttatccc ccggcgggtt 9420 ttgttgttgc cggggcgctg tgcctgttct ggtcgtggct ggtggcgcga tatctcgacc 9480 gtacacagtc g tctgtcggc ggaggtaaat agtgttcttt tcgggattat ttcaacgaaa 9540 aagtgacgca ccggtgacca cgccagcaga gctggcggat gccatcgggc tgtcgtatga 9600 cacctatacc ggaaagcaga tcagcagtca gcgggctatg cgactgacgg cggtttttt c 9660 ctgcgtcaga gtgctggcag agtcggtcgg gatgttgccc tgcaatctgt atcacctgaa 9720 cggcagcctg aagcagagag ccaccggcga acgtctgcat aaactgatct ccacgcatcc 9780 caatggctat atgacgccgc aggagttctg ggagctggtg gtcacctgtc tgtgcctgag 9840 gggaaacttt tacgcctaca aagtgaaagc atttggcgaa gtggctgaac tgctgcccgt 9900 cgatcccggc tgtgtggtat atgcgctggg aa ggtgtcag cgatggcctg aaggtgaccg 9960 ccgggagtgt tattcagcgc gatgacctgg tgcagtacac gacaactgac gatgcaacca 10020 gctccggtgg tgtcctgcgc gtgccgatcg cctgctcaag tgcaggtgcg gtcggtaacg 10080 ctg acgacgg tacggcatta atcctggtca cgccggtgaa tggtctgccg tcttccggtg 10140 tggctgacac cctgacaggc ggatttgata ctgaagagct ggaaacgtgg cgcgcccgcg 10200 tcattgagcg gtattactgg acgccgcagg gcggggctga cggggactat gtcgtctggg 10260 ctaaagaagt gcccggcatt acccgcgcat ggacataccg tcacttgatg ggaacgggaa 10320 ctgtcggtgt gatgattgcc agcagtgacc tgattaatcc cattccggaa gaat caacgg 10380 aaacggcggc aagacaacat atcgggccac tggccccggt ggcaggctct gatttgtatg 10440 tgttcaggcc ggtggcacat acggtggatt ttcatatccg cgtgacgccg gacacaccag 10500 aaatacgggc tgccattacc gcggagttgc gttcgtt cct gctgcgtgat ggttatccgc 10560 agggagaact caaggtatcg cgtatcagtg aggcgatttc cggtgcgaac ggggaataca 10620 gccatcagtt gcttgcaccg gtggacaata tctccattgc gaaaaacgaa ctggcggtac 10680 tggggacgat ttcatggacg tgacaaacga tgattacatc cgcctgttat cggcactgtt 10740 gccgcccggt ccggtgtggt cagccagcga tccggcgatt gccggtgcgg caccgtcatt 10800 aacccgtgtt catcagcgtg cggatgccct gatgcgggag ctggatccgc gcaccaccac 10860 tgaactgata aaccgctggg agcgtctgtg cggtctgccg gatgaatgta ttccggcggg 10920 aacgcagacc cttcgccagc gtcagcaacg gctgg atgcg aaggttaacc tggcgggcgg 10980 catcaacgag gatttttatc ttgcacagct tgctgccctg ggcagaccag atgccaccat 11040 cacgcgatac gacaaaagca ctttcacctg ctcatcggcc tgtactgacg cggtgaatgc 11100 gccggaatgg cggtattact ggcaggtcaa catgccagcc accaccaact ccacctggat 11160 gacatgtggc gatccctgtg attccgcact gcgtatctgg ggtgacaccg ttgtcgagtg 11220 tgtgcttaac aaactctgcc cgtcgcatac ctacgtaatt tttaaatatc cggagtaatc 11280 catgcatcgt atagacacga aaaccgcgca gaaggataag ttcggcgcgg gtaagaacgg 11340 ttttacccgt ggtaaccccc agaccggcac gcctgccacc gatctggatg atgactact t 11400 tgacatgttg caggaggaac tttgcagcgt ggtggaggca tccggtgcca gcctggagaa 11460 ggggcggcac gaccagttac ttaccgcact tcgcgcgctg ctgttaagcc gcaagaatcc 11520 gtttggcgat atcaaatcgg atggcactgt gcaaacggct ctcgaaaacc ttggtttggg 11580 agaaggagca aaactcaatg cagcaacggc tacattagga cgcaccggtt tcatagctat 11640 accggttatg attgg tggta ttgagcaatc agtaatcatt cagtgggggt ggaatgccgc 11700 aaaagcatct gcctctgggg gggatggaaa tacagttgta ttcccggttg cgtttaataa 11760 tgcctgtgtt gccgttgttg caaattatga caatgtcagc gcacctatca atgcagtggc 11820 aacgggggga tatacaacca cttcgttttt attacggtgc gcagctcaaa cgggtagtta 11880 ttactataac tggattgcta ttgggtatta agatgaaaat atactgttgc ttaaataccg 11940 ttggtttttt tatggatggc tgtggcgtca ttccgccaga ttctaaagaa ataacggcag 12000 aacactggca gtcattatta aaatctcaag ctgaaggagg cgtgatcgat ttttctgttt 12060 ttcctccttc tattaa agag gttatccgta ctcatgatga tgaagtcgca gatgcgaact 12120 ttcaaaagca gatgcttatc tctgatgcaa ctgattttat caatagcaga cagtggcagg 12180 gtaaggctgc attgggaaga cttaaagaag atgagctgaa acaatataat ttgtggctgg 12240 attatctgga agcactggaa ctggttgata catccagtgc gccagatatt gaatggccta 12300 cgcctccggc agttcaggcc agatgacatc cggcgcggtg ctggtatctg ttgccgtcac 12360 cgcgtcaatg taatccagca cagcgttaag tctggttgtt tctgcctgcg tcagtttacg 12420 tccggcctgc aatttcagtt gaatcagact aatggaagcc attgcagcat caatcagtga 12480 ctggcgctgt gcttctgccg c gtctactgc ggcgctatgc tgtgcttcag tatcggtcac 12540 ccatttctca ccatcccatt tatcgtatgg agataaaggg gcgatagtgg ttgtattttc 12600 agggtaatca cccggagctt tgatttcttt tgattctcca gttttggtgc tatagaccgt 126 60 ttcaccgcga tggtctggca catattccca tgagttaaaa tctgcagaac ggcagattgc 12720 ataaccagcc ttatgtgtac caggggcatc taaacaggaa catgccggaa tgccgacacc 12780 aacggcaaga tattcatttg aagtggaaat atattcccgt gtttcaccat cgtagttata 12840 aacggtaaca tcccctgcct ttgttgcaat aaggtcacta tttaatattg ctttatgcat 12900 caggctgccc tcacgatata gttaaatgca atattacgcg gacgcgt ttc tgaggctgcg 12960 gcacctaaac catccactga ttgtttatat gttttaaagg ttccataatc cggggctggt 13020 aatccggcat cgtttgtgtt tcctcttttg ataatgtcag tgccactatt tacccatatt 13080 tcatcaaaat agaaattaat cgttgcatca gtcaca atcg tggatcttga cggtaatcca 13140 tgagcatgat cctccgttgc atacccctga atacttaaaa tagagcgacc tgtatcaatc 13200 ccccgcccgt catcccagcc acgaataaac tcaccacgta aatcaggcaa tttatttgtc 13260 ggataagcct ttgccagttc cgggtattct tcagcagaaa aagcggcacc attgcatttc 13320 agccagcctg ttggcggagt ggctgaaggc cacggaaccg ggacaccaac aggtaatgca 13380 gagccttctc ccaaaccaac gtttatgaaa atgaagaaat aacaagcaaa tggcatcatt 13440 cctgctttta ccagggggat ttaacatgct tattggctat gtacgcgtat caacaaatga 13500 ccagaacaca gatctacaac gtaatgcgct gaactgtgca ggatg cgagc tgatttttga 13560 agacaagata agcggcacaa agtccgaaag gccgggactg aaaaaactgc tcaggacatt 13620 atcggcaggt gacactctgg ttgtctggaa gctggatcgg ctggggcgta gtatgcggca 13680 tcttgtcgtg ctggtggagg agttgcgcga acgaggcatc aactttcgta gtctgacgga 13740 ttcaattgat accagcacac caatgggacg ctttttcttt catgtgatgg gtgccctggc 13800 tgaaatggag cgtgaactga ttgttgaacg aacaaaagct ggactggaaa ctgctcgtgc 13860 acagggacga attggtggac gtcgtcccaa acttacacca gaacaatggg cacaagctgg 13920 acgattaatt gcagcaggaa ctcctcgcca ga aggtggcg attatctatg atgttggtgt 13980 gtcaactttg tataagaggt ttcctgcagg ggataaataa agttaaagac actttgtgta 14040 caaaagaaag taaaacaaca gcaacttgtt gcaattttat caataaaagt agtattgtcg 14100 tgaaaaattg attaaagatt aatattatgc atgtttttga taataatgga attgaactga 14160 aagctgagtg ttcgataggt gaagaggatg gtgtttatgg tctaatcctt gagtcgtggg 14220 ggcc gggtga cagaaacaaa gattacaata tcgctcttga ttatatcatt gaacggttgg 14280 ttgattctgg tgtatcccaa gtcgtagtat atctggcgtc atcatcagtc agaaaacata 14340 tgcattcttt ggatgaaaga aaaatccatc ctggtgaata ttttactttg attggtaata 14 400 gcccccgcga tatacgcttg aagatgtgtg gttatcaggc ttattttagt cgtacgggga 14460 gaaaggaaat tccttccggc aatagaacga aacgaatatt gataaatgtt ccaggtattt 14520 atagtgacag tttttgggcg tctataatac gtggagaact atcagagctt tcacagccta 14580 cagatgatga atcgcttctg aatatgaggg ttagtaaatt aattaagaaa acgttgagtc 14640 aacccgaggg ctccaggaaa ccagttgagg tagaaagact acaaaaagtt tatgtccgag 14700 acccgatggt aaaagcttgg attttacagc aaagtaaagg tatatgtgaa aactgtggta 14760 aaaatgctcc gttttattta aatgatggaa acccatattt ggaagtacat catgtaattc 14820 ccctgtcttc a ggtggtgct gatacaacag ataactgtgt tgccctttgt ccgaattgcc 14880 atagagaatt gcactatagt aaaaatgcaa aagaactaat cgagatgctt tacgttaata 14940 taaaccgatt acagaaataa aattatttat taaagtcaca tttaagacgt aataccctac 15000 agggtaaaaa ttttctctga tcttaacttc tgcaaatgtt aactgctatt tttatgctaa 15060 aaatggttat caaaactcaa aaacacatgt ttataat caa tgagttatag aaatgctaag 15120 ggctaatgag ttatatgcaa attagtaaaa ttatgttgct atgtcagata gttacgattt 15180 agtcatctaa ctaatgctgc gcca 15204 <210> 47 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for yeaR <400> 47 atgcttcaaa tcccacagaa ttatattcat acgcgctcaa cgcctttctg gaataaacaa 60 actgcacctg ccggaatatt cgaacgtcat cttgataaag gaacgcgccc gggggtttac 120 ccacgcct tt ccgttatgca tggggcggtc aaatatctcg gctacgctga tgaacacagt 180 gcagagcctg atcaggtgat ccttatcgaa gcggggcagt ttgcggtgtt ccctccagaa 240 aagtggcaca acattgaagc catgactgac gatacttatt tcaacattga cttcttcgtg 300 gctcctgaag tcctgatgga aggtgcgcaa caacggaaag tcattcataa cgggaaatga 360 360 <210> 48 <211> 1206 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for alkB <400> 48 atgctggaaa aacatcgtg t cctggatagt gccccggaat acgtggataa aaagaaatac 60 ctgtggattc tgtctacgct gtggccggcg accccgatga ttggcatctg gctggccaac 120 gaaacgggct ggggtatctt ttatggcctg gtgctgctgg tttggtacgg tgcactgccg 180 ctgctggacg caatgtttgg tgaagatttc aacaatccgc cggaagaagt ggttccgaaa 240 ctggaaaagg aacgtta tta ccgcgtcctg acctatctga cggtgccgat gcattacgcg 300 gccctgattg tttccgcatg gtgggtcggc acccagccga tgtcatggct ggaaattggt 360 gcactggctc tgtcgctggg catcgtgaac ggtctggccc tgaataccgg ccatgaactg 420 ggtcaca aaa aggaaacgtt cgatcgctgg atggcaaaaa ttgttctggc tgtcgtgggc 480 tatggtcatt tctttatcga acacaacaag ggccatcacc gtgacgtcgc aaccccgatg 540 gatccggcta cgagccgtat gggtgaatct atctacaagt tcagtatccg tgaaatcccg 600 ggtgcattta tccgtgcatg gggtctggaa gaacagcgtc tgtctcgtcg cggccaaagc 660 gtgtggtctt ttgacaat ga aattctgcaa ccgatgatta tcaccgtgat cctgtatgca 720 gttctgctgg ctctgtttgg cccgaaaatg ctggttttcc tgccgattca gatggcattt 780 ggttggtggc aactgacgag cgctaactat atcgaacatt acggcctgct gcgtcagaaa 840 atggaaga tg gtcgctacga acatcaaaag ccgcatcaca gttggaactc caatcacatt 900 gtcagcaatc tggtgctgtt ccatctgcaa cgtcactctg accatcacgc acacccgacc 960 cgttcatatc aatcgctgcg tgattttccg ggtctgccgg cactgccgac gggctacccg 1020 ggtgcgttcc tgatggccat gatcccgcag tggtttcgca gtgttatgga tccgaaagtt 1080 gtcgactggg cgggcggtga tctga ataag attcaaatcg atgacagcat gcgtgaaacc 1140 tatctgaaaa agtttggcac ctcatcggcg ggtcattcat caagcacctc ggcagtggcg 1200 tcgtaa 1206 <210> 49 <211> 522 <212> DNA <213> Artificial Sequence < 220> <223> nucleotide sequence for alkG <400> 49 atggcatcct ataagtgtcc ggactgtaac tatgtctatg atgaatctgc gggcaatgtg 60 catgaaggct tttcgccggg tacgccgtgg catctgattc cggaagattg gtgctgtccg 120 gactgcgcag tgcgtga taa actggacttt atgctgatcg aatctggcgt cggtgaaaag 180 ggcgtgacca gcacgcacac ctcaccgaac ctgagcgaag tttctggtac gagtctgacc 240 gcggaagcag tggttgcacc gacgtcgctg gaaaaactgc cgagcgctga tgtcaaaggc 300 cagg acctgt ataagaccca accgccgcgt tccgatgcac agggcggtaa agcgtatctg 360 aagtggattt gcatcacctg tggccatatt tacgatgaag cgctgggtga cgaagccgaa 420 ggctttacgc cgggcacccg tttcgaagat attccggatg actggtgctg cccggattgt 480 ggtgc gacga aagaagatta tgtcctgtat gaagaaaagt aa 522 <210> 50 <211> 1158 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for alkT <400> 50 atggctatcg ttgtggttgg tgctggtacg gctggcgtca atgcggcgtt ttggctgcgt 60 caatacggct ataaaggtga aattcgtatt tttagccgtg aaagcgtggc accgtatcaa 120 cgcccgccgc tgagcaaagc cttcct gacc agcgaaatcg ctgaatctgc ggtgccgctg 180 aagccggaag gtttttacac caacaataac attacgatca gtctgaatac cccgattgtc 240 tccatcgatg tgggtcgtaa aattgttagc tctaaagacg gcaaggaata tgcatacgaa 300 aagctgatcc tggctacccc ggctagtgca cgtcgcctga cgtgcgaagg tagtgaactg 360 tccggcgttt gttatctgcg ttctatggaa gatgcaaaaa atctgcgtcg ca agctggtc 420 gaatcagctt cggtggttgt cctgggcggt ggcgtgattg gcctggaagt tgcaagcgcg 480 gccgtcggtc tgggtaaacg tgtgacggtt atcgaagcaa ccccgcgtgt catggctcgc 540 gtggttaccc cggcagctgc gaacct ggtg cgtgcacgcc tggaagcaga aggtatcgaa 600 tttaaactga acgcaaagct gacgagcatc aaaggtcgca acggccatgt tgaacagtgc 660 gtcctggaat ctggcgaaga aattcaagcc gatctgatcg tcgtgggtat tggcgcgatc 720 ccggaactgg aactggccac cgaagccgca ctggaagttt ccaacggtgt tgtcgtggat 780 gaccagatgt gcacctcaga tacgtcgatt tacgcgatcg gtgactgtgc gatggcg cgt 840 aatccgttct ggggcacgat ggtgcgcctg gaaaccattc ataacgctgt gacgcacgcg 900 cagattgttg ccagttccat ctgtggcacc agcacgccgg caccgacccc gccgcgtttt 960 tggtcggacc tgaagggtat ggcactgcaa ggtctgggtg cactgaaaga ttatgacaag 1020 ctggttgtcg ccattaataa cgaaaccctg gaactggaag tgctggcata caaacaggaa 1080 cgcctgatcg ctacggaaac catcaatctg ccgaagcgtc aaggtgccct ggcgggttca 1140 atcaaactgc cggactaa 1158 <210> 51 <211> 2807 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for dppA(mutation) <400> 51 atgcgtattt cctt gaaaaa gtcagggatg ctgaagcttg gtctcagcct ggtggctatg 60 accgtcgcag caagtgttca ggctaaaact ctggtttatt gctcagaagg atctccggaa 120 gggtttaacc cgcagctgtt tacctccggc accacctatg acgcctcttc cgtcccgctt 180 tataaccgtc tggttgaatt taaaatcggc accaccgaag tgatcccggg cctcgctgaa 24 0 aagtgggaag tcagcgaaga cggtaaaacc tataccttcc atctgcgtaa aggtgtgaag 300 tggcacgaca ataaagaatt caaaccgacg cgtgaactga acgccgatga tgtggtgttc 360 tcgttcgatc gtcagaaaaa cgcgcaaaac ccgtaccata aagtttctgg cggca gctac 420 gaatacttcg aaggcatggg cttgccagag ctgatcagtg aagtgaaaaa ggtggacgac 480 aacaccgttc agtttgtgct gactcgcccg gaagcgccgt tcctcgctga cctggcaatg 540 gacttcgcct ctattctgtc aaaagaatat gctgatgcga tgatgaaagc cggtacaccg 600 gaaaaactgg acctcaaccc aatcggaacc ggtccgttcc agttacagca gtatcaaaaa 660 gattcccg ta tccgctacaa agcgtttgat ggctactggg gcaccaaacc gcagatcgat 720 acgctggttt tctctattac ccctgacgct tccgtgcgtt acgcgaaatt gcagaagaat 780 gaatgccagg tgatgccgta cccgaacccg gcagatatcg ctcgcatgaa gcaggataaa 840 tccatcaatc tgatggaaat gccggggctg aacgtcggtt atctctcgta taacgtgcag 900 aaaaaaccac tcgatgacgt gaaagttcgc caggctctga cctacgcggt gaacaaagac 960 gcgatcatca aagcggttta tcagggcgcg ggcgtatcag cgaaaaacct gatcccgcca 1020 accatgtggg gctataacga cgacgttcag gactacacct acgatcctga aaaagcgaaa 1080 gccttgctga aagaagc ggg tctggaaaaaa ggtttctcca tcgacctgtg ggcgatgccg 1140 gtacaacgtc cgtataaccc gaacgctcgc cgcatggcgg agatgattca ggcagactgg 1200 gcgaaagtcg gcgtgcaggc caaaattgtc acctacgaat ggggtgagta cctcaagcgt 1260 gcgaaagatg gcgagcacca gacggtaatg atgggctgga ctggcgataa cggggatccg 1320 gataacttct tcgccaccct gttcagctgc gccgcctctg aacaaggctc caactactca 1380 aaatggtgct acaaaccgtt tgaagatctg attcaaccgg cgcgtgctac cgacgaccac 1440 aataaacgcg ttgaactgta caaacaagcg caggtggtga tgcacgatca ggctccggca 1500 ctgatcatcg ctcactccac cgtgtt tgaa ccggtacgta aagaagttaa aggctatgtg 1560 gttgatccat tagggaaggt gcgaataagc ggggaaattc ttctcggctg actcagtcat 1620 ttcatttctt catgtttgag ccgatttttt ctcccgtaaa tgccttgaat cagcctattt 1680 agaccgtttc ttcgccattt aaggcgttat ccccagtttt tagtgagatc tctcccactg 1740 acgtatcatt tggtccgccc gaaacaggtt ggccagcgtg aataacatcg ccagttggtt 1800 atcgtttttc agcaacccct tgtatctggc tttcacgaag ccgaactgtc gcttgatgat 1860 gcgaaatggg tgctccaccc tggcccggat gctggctttc atgtattcga tgttgatggc 1920 cgttttgttc ttgcgt ggat gctgtttcaa ggttcttacc ttgccggggc gctcggcgat 1980 cagccagtcc acatccacct cggccagctc ctcgcgctgt ggcgcccctt ggtagccggc 2040 atcggctgag acaaattgct cctctccatg cagcagatta cccagctgat tgaggtcatg 2100 ct cgttggcc gcggtggtga ccaggctgtg ggtcaggcca ctcttggcat cgacaccaat 2160 gtgggccttc atgccaaagt gccactgatt gcctttcttg gtctgatgca tctccggatc 2220 gcgttgctgc tctttgttct tggtcgagct gggtgcctca atgatggtgg catcgaccaa 2280 ggtgccttga gtcatcatga cgcctgcttc ggccagccag cgattgatgg tcttgaacaa 2340 ttggcgggcc agttgatgct gctccagcag gtggcgg aaa ttcatgatgg tggtgcggtc 2400 cggcaaggcg ctatccaggg ataaccgggc aaacagacgc atggaggcga tttcgtacag 2460 agcatcttcc atcgcgccat cgctcaggtt gtaccaatgc tgcatgcagt gaatgcgtag 2520 catggtttcc agcggataag gtcgccggcc attaccagcc ttggggtaaa acggctcgat 2580 gacttccacc atgttttgcc atggcagaat ctgctccatg cgggacaaga aaatctcttt 2640 tctggtctga cggcgcttac tgctgaattc actgtcggcg aaggtaagtt gatgactcat 2700 gatgaaccct gttctatggc tccagatgac aaacatgatc tcatatcagg gacttgttcg 2760 caccttcctt aggcaaacat cacttcgaaa acgtctctat cgaataa 2807 <2 10> 52 <211> 1195 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for IS5 <400> 52 ggaaggtgcg aacaagtccc tgatatgaga tcatgtttgt catctggagc catagaacag 60 ggttcatcat gagtcatcaa cttaccttcg ccgacagtga attcagcagt aagcgccgtc 120 agaccagaaa agagattttc ttgtcccgca tggagcagat tctgccatgg caa aacatgg 180 tggaagtcat cgagccgttt taccccaagg ctggtaatgg ccggcgacct tatccgctgg 240 aaaccatgct acgcattcac tgcatgcagc attggtacaa cctgagcgat ggcgcgatgg 300 aagatgctct gtacgaaatc gcctccatgc gtctgtttgc ccggttatcc ctggatagcg 360 ccttgccgga ccgcaccacc atcatgaatt tccgccacct gctggagcag catcaactgg 420 cccgccaatt gttcaagacc atcaatcgct ggctggccga agcaggcgtc atgatgactc 480 aaggcacctt ggtcgatgcc accatcattg aggcacccag ctcgaccaag aacaaagagc 540 agcaacgcga tccggagatg catcagacca agaaaggcaa tcagtggcac tttggcatga 600 aggcccacat tggtgtcgat gccaagagtg gcctgaccca cagcctggtc accaccgcgg 660 ccaacgagca tgacctcaat cagctgggta atctgctgca tggagaggag caatttgtct 720 cagccgatgc cggctaccaa ggggcgccac agcgcgagga gctggccgag gtggatg tgg 780 actggctgat cgccgagcgc cccggcaagg taagaacctt gaaacagcat ccacgcaaga 840 acaaaacggc catcaacatc gaatacatga aagccagcat ccgggccagg gtggagcacc 900 catttcgcat catcaagcga cagttcggct tcgtgaaagc cagatacaag gggttgctga 960 aaaacgataa ccaactggcg atgttatca cgctggccaa cctgtttcgg gcggaccaaa 1020 tgatacgt ca gtgggagaga tctcactaaa aactggggat aacgccttaa atggcgaaga 1080 aacggtctaa ataggctgat tcaaggcatt tacgggagaa aaaatcggct caaacatgaa 1140 gaaatgaaat gactgagtca gccgagaaga atttccccgc ttattcgcac cttcc 1195 <210> 53 <211> 1709 < 212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for yeaR(mutation) <400> 53 atgcttcaaa tcccacagaa ttatattcat acgcgctcaa cgcctttctg gaataaacaa 60 actgcacctg ccggaatatt cgaacgtcat cttgataaag gaacgcgccc g ggggtttac 120 cccataagcg ctaacttaag ggttgaacca tctgaagaat gcgacgcctc ggtgcctcgt 180 taagacgatg cctcgcgttc ttcaattgcg ttttgtaggc tgtcagggat actgtcccac 240 gaatggccac ctgtaagctc cagatgacca tttttgttat tctccacaac gagttagttc 300 ttcttttcgg atccggcact tctggggggg aaatccagcg atggctggat tatgtcgtca 360 attaaaaatg cggcgagtag attagcaaat atccac gctt tcgcgagttc aggttccttt 420 gcacgcaaag catccaggtg cagcaaactt ttgagccgct taaaagccag ttcaatttgc 480 catcgcagac ggtaacaatc agccacttgc tctgctgaat attcatcttc cggtaatgat 540 gttagcaata gcacatggcc cgctgct tcc agcgtttccg cctgaactac tcgtcctttt 600 cgacgattct cgctgagcag tcgggtttta ctgattaatg ctttttcggg aggaagtgat 660 acggcaatga gacgtgccgg aaagggagct ccggcttttt tattacctga attgcctatc 720 attacagtgg tttcaccgtt cttaccgcaa tccagcccgc gcagaaaacc catcatgtca 780 aagcgcattc cttctgcagt taaccagcgc aatcctc gcc agtgaacccg gacgatataa 840 tcagcttctc caaaagcaag tgagcggata cattcgggac gcgaaccgaa tccccggtca 900 gcaatgcgta tctcgtctgc cgtttgcgca aatcggtcca gccgttcagc gtctctgctg 960 tcggttagct caaaatcagt gaactgacag gtatgaggat catatcccat atgtagtcgc 1020 cattcagcgc tgccgccccc gggcgcactg attgctgttc catcgacaag acgcaatctc 1080 tttccgcttg tacaacccgt aactgcggcg cgtacagcaa gtgtttgtgc ggcaagtatg 1140 ccaaaccagt cggcggcatt ccgcagccgc ttcaggagag ccacgtcaga taatgttgca 1200 acgtcatgga gctgagccca tgcagtgact tc acgtaatg acatcccccc ggggccgtaa 1260 gccagcccca gacgtagcag agttgcagca tcacgaattt cgcggcggcg ggttagagcc 1320 ccggcattac gtgccgaagt atccagttct tcgggcttac caatatgggc cagaattgct 1380 gaccagttat cgtgagagta att catcggc acgttaaatc atatcaggcg taataccaca 1440 acccttaagt tagcgcttat ggggtttacc cacgcctttc cgttatgcat ggggcggtca 1500 aatatctcgg ctacgctgat gaacacagtg cagagcctga tcaggtgatc cttatcgaag 1560 cggggcagtt tgcggtgttc cctccagaaa agtggcacaa cattgaagcc atgactgacg 1620 atacttattt caacattgac ttcttcgtgg ctcctgaagt cctgatggaa ggt gcgcaac 1680 aacggaaagt cattcataac gggaaatga 1709 <210> 54 <211> 1343 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for IS186 <400> 54 cccataagcg ctaacttaag ggttgtggta ttacgcctga tatgatttaa cgtgccgatg 60 aattactctc acgataactg gtcagcaatt ctggcccata ttggtaagcc cgaagaactg 120 gatacttcgg cacgtaatgc cggggctcta acccgccgcc gcga aattcg tgatgctgca 180 actctgctac gtctggggct ggcttacggc cccgggggga tgtcattacg tgaagtcact 240 gcatgggctc agctccatga cgttgcaaca ttatctgacg tggctctcct gaagcggctg 300 cggaatgccg ccgactggtt tggcatactt gccgca caaa cacttgctgt acgcgccgca 360 gttacgggtt gtacaagcgg aaagagattg cgtcttgtcg atggaacagc aatcagtgcg 420 cccgggggcg gcagcgctga atggcgacta catatgggat atgatcctca tacctgtcag 480 ttcactgatt ttgagctaac cgacagcaga gacgctgaac ggctggaccg atttgcgcaa 540 acgg cagacg agatacgcat tgctgaccgg ggattcggtt cgcgtcccga atgtatccgc 600 tcacttgctt ttggagaagc tgattatatc gtccgggttc actggcgagg attgcgctgg 660 ttaactgcag aaggaatgcg ctttgacatg atgggttttc tgcgcgggct gg attgcggt 720 aagaacggtg aaaccactgt aatgataggc aattcaggta ataaaaaaagc cggagctccc 780 tttccggcac gtctcattgc cgtatcactt cctcccgaaa aagcattaat cagtaaaacc 840 cgactgctca gcgagaatcg tcgaaaagga cgagtagttc aggcggaaac gctggaagca 900 gcgggccatg tgctattgct aacatcatta ccggaagatg aatattcagc agagcaagtg 960 gctgattgtt accgtctgcg atgg caaatt gaactggctt ttaagcggct caaaagtttg 1020 ctgcacctgg atgctttgcg tgcaaaggaa cctgaactcg cgaaagcgtg gatatttgct 1080 aatctactcg ccgcattttt aattgacgac ataatccagc catcgctgga tttccccccc 1140 agaagtgcc g gatccgaaaa gaagaactaa ctcgttgtgg agaataacaa aaatggtcat 1200 ctggagctta caggtggcca ttcgtgggac agtatccctg acagcctaca aaacgcaatt 1260 gaagaacgcg aggcatcgtc ttaacgaggc accgaggcgt cgcattcttc agatggttca 1320acccttaagt tagcgcttat ggg 1343

Claims (15)

i) fadR and fadE genes are deleted,
ii) Transformed E. coli in which AcrR, FadD, DppA, Crp, e14 prophage, and YeaR are mutated,
The AcrR mutation is one in which adenine (A), the 567th base of the base sequence shown in SEQ ID NO: 42, is deleted;
The FadD mutation is one in which guanine, the 90th base, and cytosine, the 94th base, of the base sequence shown in SEQ ID NO: 43 are substituted with adenine;
The DppA mutation is one in which the mobile genetic element IS5 is inserted at base 1570 of the base sequence shown in SEQ ID NO: 44;
The Crp mutation is one in which the mobility gene IS5 is inserted at the 125th base of the base sequence shown in SEQ ID NO: 45;
The e14 prophage mutant is one in which the icd-icdC gene of the nucleotide sequence shown in SEQ ID NO: 46 is deleted;
The YeaR mutation is a transformed E. coli in which the mobility gene IS186 is inserted at the 115th base of the base sequence shown in SEQ ID NO: 47. delete delete delete delete delete delete i) cultivating the transformed E. coli of paragraph 1; and
ii) A method for producing nonanedioic acid, comprising the step of obtaining nonanedioic acid produced from the transformed E. coli,
A method for producing nonanedioic acid, wherein the culture is characterized in that transformed E. coli is cultured in the presence of nonanoic acid. delete i) Deleting the fadR and fadE genes of wild-type E. coli;
ii) culturing the E. coli in a minimal medium in which nonanoic acid is the sole carbon source for more than 10 days; and
iii) A method of producing the transformed E. coli of claim 1 that produces nonanedioic acid from nonanoic acid, comprising the step of repeatedly subculturing for more than 90 days. According to clause 10,
In step ii), nonanoic acid is present at a concentration of at least 3 g/L. According to clause 10,
In step ii), the minimal medium is M9 minimal medium to which NaH ₂ PO ₄ , KH ₂ PO ₄ , NaCl, NH ₄ Cl, MgSO _4· 7H ₂ O and CaCl ₂ are added. delete delete delete