CN113151129B

CN113151129B - Construction method of recombinant escherichia coli for high-yield p-aminophenylalanine

Info

Publication number: CN113151129B
Application number: CN202110271292.2A
Authority: CN
Inventors: 欧一新; 胡晓婧; 康前进
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2022-10-18
Anticipated expiration: 2041-03-12
Also published as: CN113151129A

Abstract

The invention discloses a construction method of recombinant escherichia coli for high-yield p-aminophenylalanine. The invention screens PAPA biosynthesis pathways from different strains, and determines the pathway source of high-yield PAPA through combinatorial optimization. The combined approach is transferred into escherichia coli, and host strains are subjected to genetic engineering transformation, so that the metabolic approach of the chorismic acid is weakened while the synthetic approach of the chorismic acid is strengthened. To obtain the recombinant Escherichia coli which can produce PAPA with high yield. The yield of the strain PAPA can reach 752.63mg/L. The invention lays a foundation for the industrial production and application of the PAPA.

Description

Construction method of recombinant escherichia coli for high-yield p-aminophenylalanine

Technical Field

The invention belongs to the field of synthetic biology or metabolic engineering, and relates to a construction method of a p-aminophenylalanine (PAPA) high-yield strain, in particular to a construction method of recombinant escherichia coli for high-yield p-aminophenylalanine.

Background

P-aminophenylalanine (PAPA) is used as a non-protein amino acid and is a rare natural product structural block. In nature, PAPAs are present in a variety of different microorganisms. For example, in precious actinomyces fascicularis a. Prediction, PAPA is a backbone source of a secondary metabolite dnacin B1 thereof, and the compound has excellent antitumor activity; in streptomyces venezuelae s.venezuelae, PAPA is a structural unit of its secondary metabolite chloramphenicol, a widely used antibacterial drug; in streptomyces pristinaespiralis, PAPA is a building block for the secondary metabolite plinamycin pristinamycin a, a commonly used antibiotic; in pseudomonas fluorescens, PAPA can form secondary metabolites pyrazine compounds DMBAP and medbap. In addition, PAPA is also a synthetic building block of melphalan, an anticancer drug, and the drug has obvious curative effect on multiple myeloma. Therefore, the PAPA obtained in large quantity has certain industrial value and medicinal value.

In microorganisms, the biosynthesis of PAPA starts with the branching acid. Firstly, the chorismic acid forms 4-amino deoxy chorismic acid under the action of 4-amino deoxy chorismate synthetase PapA, then forms 4-amino deoxy prephenate under the catalysis of 4-amino deoxy chorismate mutase PapB, and forms p-aminophenylpyruvic acid under the action of 4-amino deoxy prephenate dehydratase PapC. Finally, PAPA is formed by the action of nonspecific transaminase. Previous experiments show that the tyrosine aminotransferase TyrB in the Escherichia coli can convert p-aminophenylpyruvate into PAPA. It is therefore feasible to construct a recombinant strain that produces PAPA in high yield using E.coli as a host. Provides a research foundation for subsequent large-scale industrial production.

Disclosure of Invention

The invention aims to provide a construction method of recombinant escherichia coli with high yield of p-aminophenylalanine. The escherichia coli engineering bacteria with high yield of p-aminophenylalanine is obtained, the yield of the bacterial strain is further improved through genetic engineering modification, and a foundation is laid for industrial production of the bacterial strain.

The invention screens PAPA biosynthesis pathways from different strains, and determines the pathway source of high-yield PAPA through combinatorial optimization. Transferring the combined approach into escherichia coli, carrying out genetic engineering transformation on a host strain, strengthening a branched acid synthesis approach and weakening a metabolic approach of the branched acid synthesis approach; finally, the recombinant escherichia coli with high PAPA yield is obtained.

The purpose of the invention is realized by the following technical means.

In a first aspect, the invention relates to a construction method of an escherichia coli engineering bacterium for synthesizing p-aminophenylalanine (PAPA), wherein key enzyme required in a PAPA synthesis path is expressed in escherichia coli; the key enzymes include 4-aminodeoxychorismate synthetase PapA, 4-aminodeoxychorismate mutase PapB and 4-aminodeoxyprephenate dehydratase PapC.

As an embodiment, the key enzymes are selected from the PAPA synthesis pathways from different hosts, including Actinosynnema Presotium, streptomyces venezuelae, streptomyces pristinaespiralis, pseudomonas fluorescens papA, papB, papC genes, and Corynebacterium glutamicum papA genes.

As one embodiment, the PAPA synthetic pathway is selected from the papABC combination of two of apppa, appbc, svpapA, svpapBC, sppapA, sppapBC, pfpappa, pfpapBC, cgpapA; the nucleotide sequences of Apapa, apapBC, sppapA, sppapBC, svpappa, pfpapa, pfpappaB C and CgpA are respectively shown in SEQ ID No.1-9 in sequence.

As an embodiment, the PAPA synthetic pathway is selected from one of appabc, sppapab bc, svpapab bc, pfpapab, cgpapA and appbc, cgpapA and SppapBC, cgpapA and SvpapBC, cgpapA and PfpapBC.

As an embodiment, the PAPA synthetic pathway is preferably a combination of CgpapA and SppapBC.

As an embodiment, the method comprises the steps of:

s1, carrying out codon optimization and total synthesis on the key enzyme genes; obtaining Apapa, apapBC, sppapA, sppapBC, svpapa, svpapaBC, pfpapa, pfpappa, cgppA genes with nucleotide sequences shown as SEQ ID No.1-9 in sequence;

s2, respectively constructing plasmids containing a papABC combination consisting of key enzyme genes, and introducing the plasmids into escherichia coli MG1655 (DE 3) to obtain a series of recombinant strains;

and S3, respectively detecting fermentation products of the recombinant strains, and screening the recombinant strains with high PAPA yield.

In one embodiment, the papABC combination is selected from the group consisting of appabc gene, sppapABC gene, svpapABC gene, pfpapABC gene, cgpapA-appbc gene, cgpapA-SppapBC gene, cgpapA-SvpapBC gene, cgpapA-PfpapBC gene in step S2.

As an embodiment, in step S2, a plasmid containing the papABC combination is constructed by inserting the corresponding papA and papBC genes into the first and second multiple cloning sites of pETDuet vector with BamHI/HindIII and NdeI/XhoI cleavage sites, respectively.

As an embodiment, the step S2 includes the steps of:

s2-1, constructing a plasmid containing ApapaABC gene, and introducing the plasmid into Escherichia coli MG1655 (DE 3) to obtain a recombinant strain HXJE61;

s2-2, constructing a plasmid containing SppapaBC genes, and introducing the plasmid into escherichia coli MG1655 (DE 3) to obtain a recombinant strain HXJE62;

s2-3, constructing a plasmid containing an SvpapABC gene, and introducing the plasmid into Escherichia coli MG1655 (DE 3) to obtain a recombinant strain HXJE63;

s2-4, constructing a plasmid containing a PfpapaBC gene, and introducing the plasmid into escherichia coli MG1655 (DE 3) to obtain a recombinant strain HXJE64;

s2-5, constructing a plasmid containing CgpA and ApapBC genes, and introducing the plasmid into escherichia coli MG1655 (DE 3) to obtain a recombinant strain HXJE65;

s2-6, constructing a plasmid containing CgppA and SppapbC genes, and introducing the plasmid into Escherichia coli MG1655 (DE 3) to obtain a recombinant strain HXJE66;

s2-7, constructing a plasmid containing CgppA and SvppBC genes, and introducing the plasmid into Escherichia coli MG1655 (DE 3) to obtain a recombinant strain HXJE67;

s2-8, constructing a plasmid containing CgppA and PfpappBC genes, and introducing the plasmid into Escherichia coli MG1655 (DE 3) to obtain a recombinant strain HXJE68.

As one embodiment, in step S3, the fermentation is to inoculate the recombinant strain to 100mL MM medium and culture to OD at 37 ℃ and 220rpm ₆₀₀ When the concentration is about 0.6, IPTG is added to the medium at a final concentration of 0.5mM, and the culture is continued at 220rpm at 25 ℃ for 60 hours.

Specifically, the recombinant strains HXJE61, HXJE62, HXJE63, HXJE64, HXJE65, HXJE66, HXJE67, and HXJE68 were inoculated into 100mL of MM medium, and cultured at 37 ℃ and 220rpm until OD is reached ₆₀₀ When the concentration is about 0.6, IPTG is added to the medium at a final concentration of 0.5mM, and the culture is continued at 220rpm at 25 ℃ for 60 hours. And (3) detecting the fermentation product of the 8 recombinant bacteria, wherein the HXJE66 recombinant bacteria PAPA yield is optimal.

The recombinant strain with high (optimal) PAPA yield is subjected to genetic engineering transformation, so that the synthesis pathway of chorismate is enhanced, the metabolic pathway of chorismate is weakened, and more chorismate flows to the synthesis of PAPA.

As an embodiment, the method further comprises the steps of:

s4, carrying out genetic engineering transformation on the screened recombinant strain with high PAPA yield, and knocking out chorismate mutase/prephenate dehydratase gene pheA, chorismate mutase/prephenate dehydrogenase gene tyrA, anthranilate synthase gene trpE and 4-aminodeoxychorismate lyase gene pabC.

The purpose of the gene modification is as follows: the biosynthesis of tyrosine, phenylalanine, tryptophan and p-aminobenzoic acid in escherichia coli is blocked.

The step S4 specifically comprises the following steps: knocking out chorismate mutase/prephenate dehydratase gene pheA, chorismate mutase/prephenate dehydrogenase gene tyrA, anthranilate synthase gene trpE and 4-aminodeoxychorismate lyase gene pabC, and constructing recombinant Escherichia coli (HXJE 70); and (3) transferring the plasmid corresponding to the recombinant strain with high PAPA yield screened out in the step (S2) into the recombinant escherichia coli to obtain a genetic engineering strain (HXJE 71).

S4, knocking out a target gene by adopting a lambda-red homologous recombination technology; the knockout specifically comprises the following steps:

s4-1, pheA/tyrA knock-out:

a. carrying out PCR by using a linearized pIJ773 carrier fragment as a template and pheA-tyrA-del-F/R to obtain a fragment containing a pheA-tyrA homologous arm, an FRT site and a Ai Bola resistance gene, and electrically transferring the fragment into escherichia coli MG1655 (DE 3) competence containing a pIJ790 plasmid;

b. b, transferring the BT340 plasmid containing FLP recombinase into the escherichia coli obtained in the step a, coating the escherichia coli on a LA plate without resistance for culture, and screening out a mutant strain with pheA/tyrA successfully knocked out;

s4-2, knock-out of pabC:

a. carrying out PCR by using the linearized pIJ773 vector fragment as a template and the FRT3-apr-F/R as a primer to obtain a fragment containing an FTR3 site and a Ai Bola resistance gene;

b. carrying out PCR by using the obtained fragment as a template and utilizing pabC-del-F/R to obtain a fragment containing a pabC homologous arm, an FRT3 site and a Ai Bola resistance gene, and electrically transferring the fragment into an escherichia coli MG1655 (DE 3)/delta pheA-tyrA competence;

c. b, transferring the BT340 plasmid containing FLP recombinase into the escherichia coli obtained in the step b, coating the escherichia coli on a LA plate without resistance for culture, and screening out a mutant strain with successful pabC knockout;

s4-3, trpE knockout:

a. carrying out PCR by using the linearized pIJ773 vector fragment as a template and FRT5-apr-F/R as a primer to obtain a fragment containing an FTR5 site and a Ai Bola resistance gene;

b. carrying out PCR by using the obtained fragment as a template and utilizing trpE-del-F/R to obtain a fragment containing a trpE homologous arm, an FRT5 site and a Ai Bola resistance gene, and electrically transferring the fragment into escherichia coli MG1655 (DE 3)/delta pheA-tyrA/delta pabC competence;

c. and (c) electrotransfering the BT340 plasmid containing FLP recombinase into the escherichia coli obtained in the step (b), coating the escherichia coli on a LA plate without resistance for culture, and screening out a mutant strain with successful trpE knockout.

As an embodiment, the method further comprises the steps of:

s5, overexpressing 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthetase encoding gene aroG in the genetically engineered strain obtained in the step S4 ^fbr ；aroG ^fbr The nucleotide sequence of the gene is shown as SEQ ID No. 10.

As an embodiment, step S5 specifically includes: construction of a plasmid containing aroG ^fbr The plasmid of the gene is transferred into the genetically engineered strain (HXJE 71) obtained in step S4, and a final recombinant strain (HXJE 72) is obtained.

As an embodiment, the construct is: will aroG ^fbr The gene is inserted into the BamHI/HindIII enzyme cutting site of the pACYCDuet vector to construct over-expressed aroG ^fbr The recombinant plasmid of (1).

In a second aspect, the invention relates to an escherichia coli engineering bacterium obtained by the construction method of the escherichia coli engineering bacterium for synthesizing the p-aminophenylalanine.

In a third aspect, the invention relates to an engineering bacterium of Escherichia coli for synthesizing p-aminophenylalanine, wherein the engineering bacterium is Escherichia coli HXJE72 with the preservation number of CGMCC No.21810.

In a fourth aspect, the invention relates to a recombinant plasmid for constructing escherichia coli engineering bacteria for synthesizing p-aminophenylalanine, wherein the recombinant plasmid is a recombinant plasmid containing genes CgpaA and SppapbC, or a recombinant plasmid containing genes CgpaA and SppapbC.

The Escherichia coli HXJE72 related by the invention is preserved in China general microbiological culture Collection center (CGMCC), and the preservation addresses are as follows: the institute of microbiology, national academy of sciences No. 3, xilu No.1, beijing, chaoyang, beijing; the preservation number is CGMCC No.21810, and the preservation date is 2021.2.4.

Compared with the prior art, the invention has the following beneficial effects:

the invention selects PAPA synthetic pathways from different species, and randomly combines the pathways, and systematically screens the high-efficiency synthetic pathway of PAPA. Finally, the combination with the optimal yield is obtained, and a good platform is provided for the subsequent industrial synthesis of the PAPA.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1: a construction schematic diagram of high-yield p-aminophenylalanine recombinant escherichia coli;

FIG. 2: comparing the yield of different PAPA biosynthesis routes;

FIG. 3: verifying an agarose gel electrophoresis picture by using a recombinant strain HXJE70 PCR;

FIG. 4: and (3) liquid phase detection of the recombinant strain synthesized PAPA.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

Example 1 construction and screening of an efficient synthetic pathway for PAPA

In order to impart the ability of Escherichia coli to synthesize PAPA, the present invention introduces the gene papABC required for PAPA synthesis into Escherichia coli (FIG. 1). The invention selects PAPA synthesis routes from different hosts, including Actinosynnema preservatum, streptomyces venezuelae, streptomyces pristinaespiralis, papABC gene of Pseudomonas fluorescens and papA gene of Corynebacterium glutamicum. And (3) carrying out escherichia coli codon optimization and total synthesis on the genes. In synthesis, the papB and papC genes from the same host are fused with a nucleotide sequence including a ribosome binding site sequence, and the fused gene is referred to as a papBC gene.

The Apapa and ApapBC genes were inserted into the first and second multiple cloning sites of pETDuet vector with BamHI/HindIII and NdeI/XhoI sites, respectively, to obtain recombinant plasmids containing ApapaABC gene. Recombinant plasmids containing SppapaBC, svppapaBC, pfppapaBC, cgpapaA-ApppaBC, cgpapaA-SppapBC, cgpapaA-SvppapBC, and CgpapaA-PvppBC were constructed in the same manner, respectively. The constructed series of plasmids are respectively transferred into the competence of Escherichia coli MG1655 (DE 3) to obtain eight recombinant Escherichia coli strains which are respectively named as HXJE61-68.

Inoculating the above engineering bacteria HXJE61-68 into 100mL MM culture medium, and culturing at 37 deg.C and 220rpm to OD ₆₀₀ When the concentration is about 0.6, IPTG is added to the medium at a final concentration of 0.5mM, and the culture is continued at 220rpm at 25 ℃ for 60 hours. Taking 1mL fermentation liquid, carrying out ultrasonic lysis in an ultrasonic cleaner for 20min, centrifuging at 12000rpm for 10min, and taking supernatant for HPLC detection. Elution was carried out using an Agilent TC C18 reverse phase column of 4.6 μm.times.250 mm, 0.1% formic acid as phase A and pure methanol as phase B at a flow rate of 1mL/min under the following conditions: 0-10min,2% by weight; 10-12min,2% -50% by weight B;12-17min,50% by weight B. The detection wavelength was 245nm. The results show that the eight recombinant Escherichia coli strains have the ability of producing PAPA (FIG. 2), wherein the yield of HXJE66 is the highest and reaches 19.17mg/L. It was shown that the combination of CgppA and SppapBC allows for efficient synthesis of PAPA.

Wherein, the formula of the MM culture medium is as follows: glucose 10g/L, yeast extract 1g/L, na ₂ HPO ₄ ·6g/L，KH ₂ PO ₄ 3g/L，NaCl 0.5g/L，NH ₄ Cl 2g/L，MgSO ₄ ·7H ₂ O 0.5g/L，CaCl ₂ 15mg/L, thiamine-HCl 50mg/L and trace elements 1mL/L.

Example 2 construction of recombinant Strain HXJE71

The synthesis of PAPA starts with chorismate, which is a compound that is a point of divergence in the basal metabolism of escherichia coli, and is a precursor in the synthesis of aromatic amino acids as well as certain aromatic compounds. Generating phenylpyruvic acid by chorismate mutase/prephenate dehydratase PheA, and transamination to form phenylalanine; and forming p-hydroxyphenylpyruvate under the action of chorismate mutase/prephenate dehydrogenase TyrA so as to form tyrosine. In the synthetic route of tryptophan, anthranilic acid is formed under the action of anthranilate synthase TrpE, and tryptophan is generated through a series of enzyme catalytic reactions. In addition, the aminodeoxychorismate can be catalyzed by 4-aminodeoxychorismate lyase PabC to form p-aminobenzoic acid. Therefore, in order to reduce the consumption of chorismate, it is necessary to interrupt the synthesis of aromatic amino acids (phenylalanine, tyrosine, tryptophan) and p-aminobenzoic acid in E.coli, and thus, the deletion of pheA, tyrA, trpE and pabC genes in E.coli was selected (FIG. 1).

The invention adopts a lambda-red homologous recombination technology to knock out a target gene, and takes the knock-out of pheA/tyrA as an example. The linearized pIJ773 vector fragment is used as a template, and the pheA-tyrA-del-F/R is used for PCR to obtain a fragment containing an FRT site and a Ai Bola resistance gene, and the two ends of the fragment contain sequences of pheA/tyrR at the two ends of a genome. The fragment was electroporated into E.coli MG1655 (DE 3) competent containing pIJ790 plasmid and plated on plates containing 50. Mu.g/mL Ai Bola resistance. PCR verification is carried out on the grown single clone by using a primer pheA-tyrA-val-F/R, and a correct clone is screened out. To further erase the Ai Bola resistance gene on the genome, the BT340 plasmid containing FLP recombinase was electroporated into the correctly verified strain, plated on LA plates without resistance, and incubated at 42 ℃. The grown monoclonals were screened by replica plating, i.e., the monoclonals were first plated on plates containing 50. Mu.g/mL Ai Bola and then on plates without resistance, and both plates were incubated overnight at 37 ℃. Colonies which do not grow on the resistant plates but grow on the non-resistant plates are selected, PCR verification is carried out by using the primer pheA-tyrA-val-F/R (figure 3), and finally, mutant strains with target genes and resistant genes knocked out successfully are screened. The trpE and pabC genes were deleted in the same manner, and the information of the primers is shown in Table 1. The resulting mutant was designated HXJE70.

The recombinant plasmid containing the genes CgpA and SppapbC constructed in example 1 was electrotransferred into HXJE70 to obtain a recombinant strain HXJE71 capable of synthesizing PAPA. By using the fermentation method and the detection method described in example 1, the PAPA yield of the recombinant strain HXJE71 was calculated to be 68.68mg/L, which is about 3.5 times the HXJE66 yield.

TABLE 1 primers and sequences used in the present invention

Example 3 construction of recombinant Strain HXJE72

In the chorismate synthesis pathway, the first rate-limiting enzyme is DAHP synthase. The enzyme has three isoenzymes in E.coli: aroG, aroF and AroH. The enzyme activity accounts for 80%, 20% and 1% respectively. In order to enhance the chorismate synthesis pathway, the present invention selects overexpression of aroG gene. Whereas the enzyme activity of AroG is feedback-inhibited by phenylalanine, so that a mutant AroG resistant to feedback inhibition is finally selected ^fbr . The gene is inserted into a BamHI/HindIII enzyme cutting site of a pACYCDuet vector to construct over-expressed aroG ^fbr The recombinant plasmid of (1). The plasmid is electrically transferred into a recombinant strain HXJE71 to obtain a strain HXJE72. The results of the liquid phase assay using the fermentation and assay methods described in example 1 are shown in FIG. 4. The PAPA yield of the recombinant strain HXJE72 was calculated to be 752.63mg/L, which is about 11 times the HXJE71 yield (Table 2).

TABLE 2 PAPA production by different recombinant strains

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

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gtgcgcaccg tgcgaaccct gctgatcgac aactacgact cgttcaccta caacctcttc 60

cagatgctgg ccgaggtgaa cggcgccgct ccgctcgtcg tccgcaacga cgacacccgc 120

acctggcagg ccctggcgcc gggcgacttc gacaacgtcg tcgtctcacc cggccccggc 180

caccccgcca ccgacaccga cctgggcctc agccgccggg tgatcaccga atgggacctg 240

ccgctgctcg gggtgtgcct gggccaccag gccctgtgcc tgctcgccgg cgccgccgtc 300

gtccacgcac ccgaaccctt tcacggccgc accagcgaca tccgccacga cgggcagggc 360

ctgttcgcga acatcccctc cccgctgacc gtggtccgct accactcgct gaccgtccgg 420

caactgcccg ccgacctgcg cgccaccgcc cacaccgccg acgggcagct gatggccgtc 480

gcccaccgcc acctgccccg cttcggcgtg cagttccacc ccgaatcgat cagcagcgaa 540

cacggccacc ggatgctcgc caacttccgc gacctgtccc tgcgcgcggc cggccaccgc 600

cccccgcaca ccgaacgcat acccgcaccc gcacccgccc ccgcccccgc ccccgcaccg 660

gcaccgcccg cgtccgcgcc ggtgggggag taccggctgc atgtgcgcga ggtcgcctgc 720

gtgcccgacg cggacgccgc gttcaccgcc ctgttcgccg acgccccggc ccggttctgg 780

ctcgacagca gccgcgtcga gccgggcctc gcccgcttca ccttcctcgg cgcccccgcc 840

ggcccgctcg gcgaacagat cacctacgac gtcgccgacc gggccgtgcg cgtcaaggac 900

ggttcaggcg gcgagacccg ccggcccggc accctcttcg accacctgga acacgaactg 960

gccgcccgcg ccctgcccgc caccggcctg cccttcgagt tcaacctcgg ctacgtcggc 1020

tacctcggct acgagaccaa ggccgacagc ggcggcgagg acgcccaccg cggcgaactg 1080

cccgacggcg ccttcatgtt cgccgaccgg atgctcgccc tcgaccacga acagggccgg 1140

gcctggctcc tggcactgag cagcacccga cggcccgcca ccgcacccgc cgccgaacgc 1200

tggctcaccg acgccgcccg gaccctcgcc accaccgccc cccgcccgcc cttcaccctg 1260

ctgcccgacg accaactgcc cgccctggac gtccactacc gccacagcct gccccgctac 1320

cgggaactgg tcgaggaatg ccgccgcctg atcaccgacg gcgagaccta cgaggtgtgc 1380

ctgacgaaca tgctccgggt gcccggccgg atcgacccgc tcaccgccta ccgcgccctg 1440

cgcaccgtca gccccgcccc ctacgccgcc tacctgcagt tccccggggc caccgtgctc 1500

agctcctcac ccgaacggtt cctgcgcatc ggcgcggacg gctgggcgga gtccaaaccc 1560

atcaagggca cccgcccccg cggcgccggc cccgcccagg acgccgccgt caaggcctcc 1620

ctcgccgcgg ccgagaagga ccgcagcgag aacctgatga tcgtcgacct ggtccgcaac 1680

gacctcggcc aggtctgcga catcggctcc gtccacgtac cgggcctgtt cgaggtggag 1740

acctacgcca ccgtccacca gctcgtcagc acggtccgcg gccgcctggc ggccgacgtc 1800

tcccgccccc gcgcggtacg ggccgccttc cccggcgggt cgatgaccgg cgcgcccaag 1860

gtccgcacca tgcagttcat cgaccggctc gagaagggcc cgcgcggcgt gtactcgggc 1920

gcgctgggct acttcgccct cagcggcgcg gccgacctca gcatcgtcat ccgcaccatc 1980

gtcgccaccg aggaggccgc caccatcggc gtgggcggcg ccgtcgtcgc cctgtccgac 2040

cccgacgacg aggtccgcga aatgctcctc aaggcgcaga ccaccctcgc cgccctgcgc 2100

caggcacacg cggccgccac cgcctcggac cgtgaactcc tggccggcag cctgcggtga 2160

<210> 4

<211> 1301

<212> DNA

<213> Streptomyces pristinaespiralis

<400> 4

atgaccccgc ccgccatccc cgccgccccg cccgccaccg ggcccgcccc cgccaccgac 60

cccctcgacg cgctgcgcgc ccgcctggac gccgcggacg ccgccctgct ggacgccgtc 120

cgcacacgcc tggacatctg cctgcgcatc ggcgagtaca agcgcctcca ccaggtgccg 180

atgatgcagc cccaccggat cgcccaggtc cacgccaacg ccgcccgcta cgccgccgac 240

cacggcatcg accccgcctt cctgcgcacc ctgtacgaca cgatcatcac cgagacctgc 300

cgcctcgagg acgagtggat cgcctccggc ggcgcccccg tccccacgcc cgtgcacgcg 360

tccgcgtccg cgcggggggc cgtgtcgtga gtcgacgaag gagatatacc atgaggggtg 420

gttcggtgtt cgggcgttgt gtggtggtgg gcggggccgg tgcggtgggc cgcatgttca 480

gccactggct ggtgcgttcg ggggtggcgg tgacctggct ggacgtggcc ggggccggtg 540

cggcggacgg ggtgcgggtg gtggccggtg atgtgcggcg gccggggccg gaggcggtcg 600

cggcgctggc ggcggcggac gtggtggtgc tggcggtgcc ggagccggtg gcgtgggagg 660

cggtggaggt gctggcgggg gtgatgcggc ccggtgcggt gctcgcggac accttgtcgg 720

tcaagagccg gatcgccggg cggctgcgtg aggcggcgcc ggggctgcag gcggtggggc 780

tgaacccgat gttcgccccc tcgctgggtc ttcaggggcg gccggtggcg gcggtggtgg 840

tcaccgacgg gcccggtgtg cgggccctgg tggagctggt ggccgggtgg ggggcccggg 900

tggtggagat gccggcgcgg cggcacgacg agctgaccgc cgcgcagcag gccgccacgc 960

atgccgcggt gctggccttc gggctgggcc tgggtgagct gtcggtggac gtgggggcgc 1020

tgcgggacag tgccccgccg ccgcatctgg cgatgctggc gctgctggcc cggatcgccg 1080

gcgggacgcc ggaggtgtat ttcgacatcc aggccgccaa ccccggcgcg ccggccgcgc 1140

ggcaggcgct gggccgcggc ctggtgcggc tggggcaggc cgtcgagagg ggcgacgagg 1200

agacgttcgc cgccctgttc gccgaactgc gcggtgtgct gggcgagcac ggtgcggagc 1260

tggaacggct gtgcgcgcgg atgttcaccg ccctgcactg a 1301

<210> 5

<211> 2061

<212> DNA

<213> Streptomyces venezuelae (Streptomyces venezuelae)

<400> 5

atgcgcacgc ttctgatcga caactacgac tcgttcaccc acaacctgtt ccagtacatc 60

ggcgaggcca ccgggcagcc ccccgtcgtc gtgcccaacg acgccgactg gtcgcggctg 120

cccctcgagg acttcgacgc gatcgtcgtg tccccgggcc ccggcagccc cgaccgggaa 180

cgggacttcg ggatcagccg ccgggcgatc accgacagcg gcctgcccgt cctcggcgtc 240

tgcctcggcc accagggcat cgcccagctc ttcggcggaa ccgtcggcct cgccccggaa 300

cccatgcacg gccgggtctc cgaggtgcgg cacaccggcg aggacgtctt ccggggcctc 360

ccctcgccgt tcaccgccgt gcgctaccac tccctggccg ccaccgacct ccccgacgag 420

ctcgaacccc tcgcctggag cgacgacggc gtcgtcatgg gcctgcggca ccgcgagaag 480

ccgctgtggg gcgtccagtt ccacccggag tccatcggca gcgacttcgg ccgggagatc 540

atggccaact tccgcgacct cgccctcgcc caccaccggg cacgtcgcga cgcggccgac 600

tccccgtacg aactccacgt gcgccgcgtc gacgtgctgc cggacgccga agaggtacgc 660

cgcggctgcc tgcccggcga gggcgccacg ttctggctgg acagcagctc cgtcctcgaa 720

ggcgcctcgc gcttctcctt cctcggcgac gaccgcggcc cgctcgccga gtacctcacc 780

taccgcgtcg ccgacggcgt cgtctccgtc cgcggctccg acggcaccac gacccggacg 840

cggcgaccct tcttcagcta cctggaggag cagctcgaac gccggcgggt ccccgtcgcc 900

cccgacctgc ccttcgagtt caacctcggc tacgtcggct acctcggcta cgagctgaag 960

gcggagacca ccggcgaccc cgcgcaccgg tccccgcacc ccgacgccgc gttcctcttc 1020

gccgaccgcg ccatcgccct cgaccaccag gaaggctgct gctacctgct ggccctcgac 1080

cgccggggcc acgacgacgg cgcccgcgcc tggctgcggg agacggccga gaccctcacc 1140

ggcctggccg tccgcgtccc ggccgagccg acccccgcca tggtcttcgg ggtccccgag 1200

gcggcggccg gcttcggccc cctggcccgc gcacgccacg acaaggacgc ctacctcaag 1260

cgcatcgacg agtgcctcaa ggagatccgc aacggcgagt cgtacgagat ctgcctgacc 1320

aacatggtca ccgcgccgac cgaggcgacg gccctgccgc tctactccgc gctgcgcgcc 1380

atcagccccg tcccgtacgg cgccctgctc gagttccccg agctgtcggt gctcagcgcc 1440

tcgcccgagc ggttcctcac gatcggcgcc gacggcggcg tcgagtccaa gcccatcaag 1500

gggacccgcc cccggggcgg caccgcggag gaggacgagc ggctccgcgc cgacctggcc 1560

ggccgggaga aggaccgggc cgagaacctg atgatcgtcg acctggtccg caacgacctc 1620

aacagcgtct gcgcgatcgg ctccgtccac gtgccccggc tcttcgaggt ggagacctac 1680

gcgcccgtgc accagctggt gtcgaccatc cggggacggc tgcggcccgg caccagcacc 1740

gccgcctgcg tacgcgccgc cttccccggc ggctccatga ccggcgcgcc caagaagcgc 1800

accatggaga tcatcgaccg cctggaggaa ggcccccggg gcgtctactc cggggcgctc 1860

ggatggttcg ccctcagcgg cgccgccgac ctcagcatcg tcatccgcac catcgtgctg 1920

gccgacggcc gggccgagtt cggcgtcggc ggggcgatcg tgtccctctc cgaccaggag 1980

gaggagttca ccgagaccgt ggtcaaggcc cgcgccatgg tcaccgccct cgacggcagc 2040

gcagtggcgg gcgcccgatg a 2061

<210> 6

<211> 1301

<212> DNA

<213> Streptomyces venezuelae (Streptomyces venezuelae)

<400> 6

atgaccgaac agaatgaact gcagcgtctg cgtgcagaac tggatgcact ggatggcacc 60

ctgctggata ccgttcgtcg tcgtattgat ctgggtgttc gtattgcacg ttataaaagc 120

cgtcatggtg ttccgatgat gcagcctggt cgtgttagcc tggttaaaga tcgtgcagca 180

cgttatgcag cagatcatgg tctggatgaa agttttctgg tgaatctgta tgatgtgatc 240

atcaccgaaa tgtgtcgtgt tgaggatctg gttatgagcc gtgaaagcct gaccgcagaa 300

gatcgtcgtt aagtcgacga aggagatata ccatgagcgg ttttccgcgt agcgttgttg 360

ttggtggtag cggtgcagtg ggtggtatgt ttgcaggtct gctgcgtgaa gcaggtagcc 420

gtaccctggt tgttgatctg gttccgcctc cgggtcgtcc ggatgcatgt ctggttggtg 480

atgttaccgc accgggtccg gaactggcag cagcactgcg tgatgccgat ctggtgctgc 540

tggccgttca tgaagatgtt gcactgaaag cagttgcacc ggttacccgt ctgatgcgtc 600

cgggtgcact gctggcagat accctgagcg ttcgtaccgg tatggcagca gagctggcag 660

cacatgcacc gggtgttcag catgttggtc tgaatccgat gtttgcaccg gcagcaggta 720

tgacaggccg tccggttgca gcagttgtta cccgtgatgg tcctggtgtg accgcactgc 780

tgcgtctggt tgaaggtggt ggtggtcgtc ctgttcgtct gacagccgaa gaacatgatc 840

gtaccaccgc agcaacccag gcactgaccc atgcagttct gctgagcttt ggtctggcac 900

tggcacgtct gggtgtggat gttcgtgcac tggcagccac cgcaccgcct ccgcatcaag 960

tactgctggc cctgctggca cgtgttctgg gtggtagtcc ggaagtttat ggtgatattc 1020

agcgtagcaa tccgcgtgca gcaagcgcac gtcgtgccct ggctgaagcc ctgcgtagct 1080

ttgcagcact ggttggagat gatcctgatc gtgccgatgc accaggtcgc gcagatgccc 1140

ctggtcatcc gggtggttgt gatggtgcag gtaatctgga tggtgttttt ggtgaactgc 1200

gtcgcctgat gggtcctgag ctggctgcag gccaggatca ttgtcaagaa ctgtttcgta 1260

ccctgcatcg taccgatgat gaaggtgaaa aagatcgcta a 1301

<210> 7

<211> 2070

<212> DNA

<213> Pseudomonas fluorescens (Pseudomonas fluorescens)

<400> 7

atgaaaattc tgctgattga caactttgat tcctttaccc aaaacatcgc tcagtatctg 60

tacgaagtga cgggcatctg cgccgacatt gtgaccaaca cggttaccta tgaacatctg 120

cagattgaac aatacgatgc cgtggttctg tccccgggtc cgggtcaccc gggcgaatat 180

ctggactttg gcgtctgcgg tcaggtgatc ctgcattcac cggtgccgct gctgggtatt 240

tgtctgggcc accaaggtat cgcccagttc ctgggcggta ccgttggtca tgcaccgacc 300

ccggtccacg gttatcgtag caaaattacc catagtggct ccggtctgtt tcgtgatctg 360

ccggaacaat tcgaagtcgt gcgctaccat tccctgatgt gcacccacct gccgcaggaa 420

ctgcgttgta cggcctggac cgaagaaggc gttgtcatgg caattgaaca cgaaagccgc 480

ccgatctggg gcgttcagtt tcatccggaa tctatcgata gtgaatatgg tcacgctctg 540

ctgtcgaact tcattggcat ggcgatcgaa cataacggta atcaccgtac gagcgcgacc 600

cagaacccgg atgcatcagc ttcggcgaat gaacattatc gtgctgtggg cggtctgctg 660

aatatgcagc tggcgtatcg cacctatccg ggtccgtttg acccgctggc cctgttcacc 720

caacgctacg cccaggatca tcacgcattt tggctggact ccgaaaaatc agaacgtccg 780

aacgcccgct attcgattat gggcagcggt caggcacaag gctctatccg tctgacgtac 840

gatgtgaata gcgaatctct gaccctggcg ggcccgaaag gtagtcgcat tgtcacgggt 900

gactttttca ccctgttttc ccaaatcgtg gaatcagtga acgtggccgt cccgcagtat 960

ctgccgtttg aattcaaagg cggtttcgtt ggctatatgg gttacgaact gaaagcactg 1020

accggcggta ataaagtgta tcgtagcggc cagccggatg ctggttttat gttcgcgccg 1080

catttctttg tttttgatca tcacgaccag acggtttacg aatgcatgat ttcggcaacc 1140

ggtcagagcc cgcaatggcc gcagctgctg accagcatga ccacgctgaa caatgctacc 1200

gatcgtcgtc cgtttgtgcc gggtgccgtc gatgaactgg aactgagtct ggaagacggt 1260

ccggatgact acatccgtaa agttaaacaa tccctgcagt atattacgga tggcgaatca 1320

tacgaaatct gcctgaccaa tcgtgcgcgc atgagttatt ccggtgaacc gctggccgca 1380

taccgtcgca tgcgtgaagc tagcccggtt ccgtatggcg cgtacctgtg ctttgattca 1440

ttctcggtcc tgagcgcgtc tccggaaacc tttctgcgta ttgacgaagg cggtctgatt 1500

gaatctcgcc cgatcaaagg tacccgtgcg cgctctaaag atccgagtga agaccaacgt 1560

ctgcgctctg atctgcaggc cagtaccaaa gaccgcgcag aaaacctgat gattgtcgat 1620

ctggtgcgtc atgacctgaa tcaggtgtgc cgcagtggtt ccgtgcatgt tccgcacatc 1680

tttgccgtcg aatcgttcag ctctgtgcat cagctggtta gcacggtccg tggccacctg 1740

cgcaacgata tttctaccat ggaagccatc cgtgcatgct ttccgggcgg tagtatgacg 1800

ggtgccccga aaaaacgtac catggaaatt atcgacggcc tggaaacctg tgcccgcggt 1860

gtttattccg gcgcactggg ttggatttca ttttcgggca gcgcagaact gtcaattgtg 1920

atccgcaccg ctgttctgca taaacagcaa gcggaattcg gtattggcgg tgctatcgtg 1980

gcgcacagcg atccgaatga agaactggaa gaaaccctgg tcaaagcaag cgtgccgtat 2040

tattcgttct atgccggtag tgaaaaatga 2070

<210> 8

<211> 1235

<212> DNA

<213> Pseudomonas fluorescens (Pseudomonas fluorescens)

<400> 8

atgaatatga ccgaacaccg ccacatgagc ccgaccacgc cgtctgccat cctgcaaccg 60

caacgcgacc aactggaccg tatcaacaac catctggttg atctgctggg cgaacgtatg 120

agtgtctgca tggatattgc ggaactgaaa gcggcccacg acattccgat gatgcagccg 180

caacgtatcg tgcaggttct ggatcaactg aaagacaaaa gctctaccgt gggtctgcgc 240

ccggactatg tccagagcgt gtttaaactg attatcgaag aaacgtgtat ccaggaagaa 300

caactgattc aacgccgtcg taaccagggt caacgctcgt gagtcgacga aggagatata 360

ccatgaacac gaacacggtg gtggtgctgg gcggcgctgg tctgattggc tccatgatct 420

ctcgcatcct gaaacagtac ggctactttg tgcgtgtggt tgatcgtcgc ccggccgaat 480

tcgaatgcga atatcatgaa atggatgtca ccaaaccgtt taacgacacc ggtgccgtgt 540

tccgtaatgc taccgccgtc gtgtttgcac tgccggaaag cgtggccgtt tctgcaattc 600

cgtgggttac cacgttcctg agctctgaag ttgtcctgat cccgacgtgt tcagtgcagg 660

gtccgtttta caaagctctg aaagccgcgg caccgcgtca accgtttgtc ggtgtgaacc 720

cgatgttcag tccgaaactg tccgttcagg gtcgttcagt tgcggtctgc gtggaagata 780

cccaggctgc gcagaccttt attgaacgcc atctgatgga agctggcatg aaaatccgtc 840

gcatgacccc gtcggcgcat gacgaactga tggctctgtg ccaggcgctg ccgcatgcag 900

caattctggg ctttggtatg gccctggcaa aaagttccgt ggatatggac atcgttgccg 960

aagtcatgcc gccgccaatg cgtaccatga tggcactgct gagccgcatt ctggtgaacc 1020

cgccggaagt ttattgggat atccagctgg aaaatgacca ggctacggcg caacgtgatg 1080

ccctggttca cggtctggaa cgcctgcagg aaaatattgt cgaacaagat tacgaacgct 1140

ttaaatctga cctgcaatca gtgtcgaccg cactgggtaa acgcctgaac gctggtgccg 1200

tggattgtca acacctgttt tccctgctga actaa 1235

<210> 9

<211> 1884

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 9

gtggttttgt cagaggatgt catgcgcgtt ttaattattg ataattatga ttctttcacg 60

tttaatctcg ccacctatgt ggaagaggtt acgggtcagg cacctgtggt ggtgcctaat 120

gatcaagaaa tagatgagat gcttttcgac gccgtcatcc tctcacctgg cccgggccac 180

gccggcgttg cggctgattt tggtatctgt gcaggcgtca ttgagcgtgc acgcgttccg 240

attttgggtg tgtgtttagg ccaccagggc attgcgttgg cctatggcgg tgatgttgat 300

ttggcgccca ggccggtcca cggtgaggtt tcgcagatca cccatgatgg ttcaggttta 360

tttgcaggca tccctgaaac gtttgaggcg gtgcgttatc actcgatggt ggcaacccgc 420

ttgccggagt cattgaaagc tacagctacc agcgatgatg gtttgatcat ggcattggca 480

catgaagtgc ttccgcagtg gggtgtgcaa tttcatccgg aatctattgg tggacaattc 540

ggccatcaga tcattaagaa cttccttaat ttagcgcgca catatcgctg gcaactcacg 600

gagaaaacta ttccgctcag cgttgattca gcagcggttt ttgaaacatt ctttgcccat 660

tcctcccatg ctttttggct cgatgatgcc caaggaacca gctatcttgg tgatgccagc 720

ggtcctctcg cacgcacaaa aacccataat gtcggcgagg gggatttctt cacctggcta 780

aaggaggatc tcgccgccaa ctcagttgcg cccggtcaag gttttcgtct tggctgggtt 840

ggttacgttg gttatgagct taaagcggaa gctggcgcac gggctgcgca cacttcgagt 900

cttccggatg cgcacctcat ttttgccgat cgcgccatcg cagtggaatc ggatcaggtt 960

cggttgctgg cgttggggga gcaggacgag tggtttgaag aaaccatcaa gaagctgcat 1020

aatcttgtcg ccccgcggat acctgcgtcc ggacacctcg ctttgcaggt tcgagattcc 1080

aaagatgagt atctcgacaa aattcgcaga gcccaggagc tgattactcg cggcgaatcg 1140

tatgaaatct gcctgaccac aaaacttcag ggcaccactg atgtggcccc tctggctgcc 1200

tatctagcac tgcgtggggc caatcccacc gcttatggtg cgtatcttca gctgggggat 1260

acctctattt tgagttcctc gccggagcgg ttcatcacca ttgattcggc agggtatgtg 1320

gaatcaaagc ccattaaagg caccaggccg cgtgggcgaa cagcgcaaga agaccaagaa 1380

atcattgctg agctgcgcag taatcctaaa gatcgtgcag aaaacttgat gatcgtggat 1440

ttggtccgca acgacttagc ccgcggcgct ttgcccacca cagttaaaac atccaagctc 1500

ttcgacgtcg aaacctacgc cacagtccac caacttgtca gcaccgtctc tgcagagttg 1560

gggccacgca gtccgattga gtgcgtgcgc gcagcattcc ccggtggttc gatgactggt 1620

gccccaaagc tgcgcaccat ggagatcatc gatgagctgg aggcagctcc tcgcggtatt 1680

tactcaggtg gcttgggata tttttccctc gacggcgcag ttgatctctc catggtgatc 1740

agaactctcg tcatccagaa caatcacgtg gagtacggag tgggcggtgc acttcttgct 1800

ctgtctgatc cggaggctga gtgggaggaa atccgcgtta aatcacggcc tctgctgaat 1860

ttgtttgggg ttgaattccc atga 1884

<210> 10

<211> 1053

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 10

atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60

gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120

aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180

tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240

gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300

acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360

gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420

gcaggtgagt ttctcgatat gatcacccta caatatctcg ctgacctgat gagctggggc 480

gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540

tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600

aatgccgccg gtgcgccgca ctgcttcctg tccgtaacga aatgggggca ttcggcgatt 660

gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720

tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780

caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840

gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900

gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960

ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020

ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053

<210> 11

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcaggatctg aacgggcagc tgacggctcg cgtggcttaa gaggtttatt attccgggga 60

tccgtcgacc 70

<210> 12

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aggcctccca aatcgggggg ccttttttat tgataacaaa aaggcaacac ttgtaggctg 60

gagctgcttc 70

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtatccgtaa ccgatgcctg ca 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgaatgggag gcgtttcgtc gt 22

<210> 15

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gaagttccta ttcttcaaat agtataggaa cttcgaagtt cccg 44

<210> 16

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gaagttccta tactatttga agaataggaa cttcggaata ggaa 44

<210> 17

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gataaggagc cggtatgttc ttaattaacg gtcataagca ggaatcgctg gaagttccta 60

ttcttcaaat a 71

<210> 18

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

acactttttt catgactaat tcgggcgctc acaaagtggg gctaaatatt cgaagttcct 60

atactatttg aa 72

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tagtgaacat gctgccacac 20

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atacccagta ccaccagcaa t 21

<210> 21

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gaagttccta ttcttcaaaa ggtataggaa cttcgaagtt cccg 44

<210> 22

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gaagttccta taccttttga agaataggaa cttcggaata ggaa 44

<210> 23

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

agaataacaa tgcaaacaca aaaaccgact ctcgaacagg aagttcctat tcttcaaaag 60

<210> 24

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tgtcagccat cagaaagtct cctgtgcatg atgcgcggtg aagttcctat acctttt 57

<210> 25

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gacaattaat catcgaacta gttaact 27

<210> 26

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

caagcgggtg aggagttccg gcatac 26

Claims

1. A construction method of Escherichia coli engineering bacteria for synthesizing p-aminophenylalanine is characterized in that key enzyme required in a PAPA synthesis path is expressed in Escherichia coli; the key enzymes comprise 4-aminodeoxy-chorismate synthetase PapA, 4-aminodeoxy-chorismate mutase PapB and 4-aminodeoxy prephenate dehydratase PapC;

the key enzymes select the synthetic pathway of PAPA from different hosts, including Streptomyces pristinaespiralisStreptomyces pristinaespiralisIs/are as followspapB、papCGene and Corynebacterium glutamicumCorynebacterium glutamicumIspapAA gene;

the method comprises the following steps:

s1, carrying out codon optimization and total synthesis on the key enzyme genes; obtaining nucleotide sequences respectively shown as SEQ ID No.4 in sequenceSppapBCGene, shown as SEQ ID No.9CgpapAA gene;

s2, construction of a peptide containingCgpapA-SppapBCIntroducing the plasmid of the gene into Escherichia coli MG1655 (DE 3) to obtain a recombinant strain;

s3, carrying out genetic engineering transformation on the recombinant strain to knock out the chorismate mutase/prephenate dehydratase genepheAChorismate mutase/prephenate dehydrogenase genetyrAAnthranilic acid synthase genetrpEAnd 4-aminodeoxychorismate lyase genepabC；

S4、Overexpressing a 3-deoxy-D-arabinoheptulose-7-phosphate synthase-encoding gene in the genetically engineered strain obtained in step S3aroG ^fbr ；aroG ^fbr The nucleotide sequence of the gene is shown as SEQ ID No. 10.

2. The escherichia coli engineering bacteria constructed according to the construction method of the escherichia coli engineering bacteria for synthesizing p-aminophenylalanine in claim 1.

3. An Escherichia coli engineering bacterium for synthesizing p-aminophenylalanine, wherein the engineering bacterium is Escherichia coli (Escherichia coli) HXJE72 with the preservation number of CGMCC No.21810.

4. The recombinant plasmid for constructing escherichia coli engineering bacteria for synthesizing p-aminophenylalanine is characterized by containingCgpapAAndSppapBCrecombinant plasmids of the genes;CgpapAthe gene sequence is shown as SEQ ID No.9,SppapBCthe gene sequence is shown in SEQ ID No. 4.