CN112301049B - Recombinant plasmid and genetic engineering strain for high yield of heme, construction method thereof and method for high yield of heme - Google Patents

Abstract

The invention relates to the field of metabolic engineering, in particular to a recombinant plasmid and a genetic engineering strain for high yield of heme, a construction method thereof and a method for high yield of heme. The recombinant plasmid comprises the following genes: hemA, hemL, hemB1, hemB2, hemC, hemD, hemE, hemN, hemG1, hemG2, hemG3, hemH1, hemH2, and ccmf. The invention utilizes metabolic engineering and synthetic biology means to reform S.oneidensis MR-1, thereby efficiently synthesizing the heme. Engineering of such a method for electroactive bacteria would facilitate the use of such bacteria in the production of fine chemicals.

Description

Recombinant plasmid and genetic engineering strain for high yield of heme, construction method thereof and method for high yield of heme

Technical Field

The invention relates to the field of metabolic engineering, in particular to a recombinant plasmid and a genetic engineering strain for high yield of heme, a construction method thereof and a method for high yield of heme.

Background

The current production of heme mainly consists in the separation of free heme from biological samples (for example plant tissues and animal blood) by organic extraction or enzymatic hydrolysis, which is not only complex, low in yield, time-consuming but also causes pollution problems. The development of metabolic engineering technology provides a chance for producing heme by utilizing microbial cell engineering.

The electrically active bacteria have evolved unique extracellular electron transfer modes for completing the respiratory metabolic process of cells by utilizing various electron acceptors in an anaerobic environment. The microorganisms also have important roles in energy recovery, chemical production, environmental remediation and the like. To date, a variety of dissimilatory metal-reducing bacteria, such as Shewanella spp, geobactor spp, pseudomonas spp, listeria spp, have been isolated from the environment and characterized for their extracellular electron transport pathways. Shewanella oneidensis MR-1 is widely concerned as a model dissimilatory metal-reducing bacterium. It utilizes a complex extracellular electron transport network to transport electrons produced by intracellular metabolism to an extracellular electron acceptor. The method comprises the following specific steps: the bacterium first converges electrons generated by the oxidative decomposition of intracellular L-lactic acid into an NADH pool, and then the electrons reach various redox terminal enzymes in the inner membrane and the periplasm or a porin-cytochrome complex (OmcA-mtrCAB) on the outer membrane through a quinone pool and are finally transmitted to an extracellular electron acceptor in a direct and indirect manner. The unique extracellular electron transport properties of electroactive bacteria provide the potential for the electrosynthesis of hemoglobin using Microbial Electrochemical Systems (MES).

Disclosure of Invention

In view of the above, the invention provides a recombinant plasmid and a genetic engineering strain for high yield of heme, and a construction method and a method for high yield of heme thereof. The plasmid system and the host system for high yield of heme provided by the invention are used for realizing high-efficiency and amplified production of heme.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a recombinant plasmid for high yield of heme, which comprises the following genes: hemA, hemL, hemB1, hemB2, hemC, hemD, hemE, hemN, hemG1, hemG2, hemG3, hemH1, hemH2, and ccmf.

Preferably, the gene source of hemA, hemL, hemB1, hemB2, hemC, hemD, hemE, hemN, hemG1, hemG2, hemG3, hemH1, hemH2 and ccmf is E.coli MG1655.

Preferably, the plasmid vector of the recombinant plasmid is pBF-PTeto.

Preferably, the sequence of the plasmid vector is as set forth in SEQ ID NO:1 is shown.

Preferably, the sequence of the recombinant plasmid is as shown in SEQ ID NO:2, respectively.

The invention also provides a construction method of the recombinant plasmid, which comprises the following steps:

taking a plasmid vector as a template, and amplifying to obtain a plasmid skeleton;

assembling DNA fragments of genes hemA, hemL, hemB1 and hemB2 with a plasmid framework to obtain a plasmid-HE 1;

assembling DNA fragments of genes hemC, hemD, hemE and hemN with a plasmid framework to obtain a plasmid-HE 2;

assembling DNA fragments of genes hemG1, hemG2, hemG3, hemH1 and hemH2 with a plasmid framework to obtain a plasmid-HE 3;

assembling the gene ccmF with a plasmid framework to obtain a plasmid-HE 4;

on the basis of obtaining the plasmid-HE 1, the plasmid-HE 2, the plasmid-HE 3 and the plasmid-HE 4, assembling HE1, HE2, HE3 and HE4DNA fragments to obtain recombinant plasmids.

The invention also provides a genetic engineering strain for high yield of heme, which comprises the recombinant plasmid.

Preferably, the host strain of the genetically engineered strain is S.oneidensis MR-1.

Preferably, the promoter of the genetically engineered strain is a tetracycline-inducible promoter system.

The invention also provides a construction method of the genetic engineering strain, which comprises the following steps: preparing competent cells by using host bacteria; and mixing the recombinant plasmid with the competent cell, and performing electrotransformation to obtain the genetic engineering strain.

The invention also provides a method for producing heme with high yield, which comprises the following steps:

culturing the above genetic engineering strain, and culturing the OD of the strain solution ₆₀₀ Adding inducer for fermentation culture when the concentration is 0.15-0.25, and collecting supernatant.

The invention provides a recombinant plasmid and a genetic engineering strain for high yield of heme, a construction method thereof and a method for high yield of heme. The recombinant plasmid comprises the following genes: hemA, hemL, hemB1, hemB2, hemC, hemD, hemE, hemN, hemG1, hemG2, hemG3, hemH1, hemH2, and ccmf.

The invention has the technical effects that:

the invention utilizes metabolic engineering and synthetic biology means to reform S.oneidensis MR-1, thereby efficiently synthesizing heme. Engineering of such a method for electroactive bacteria would facilitate the use of such bacteria in the production of fine chemicals.

Drawings

FIG. 1 is a pBF-PTeto map of the plasmid platform for heme synthesis;

FIG. 2 is the measurement of the growth state and heme yield of the engineering bacteria.

Detailed Description

The invention discloses a recombinant plasmid and a genetic engineering strain for high yield of heme, a construction method thereof and a method for high yield of heme. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate variations and combinations of the methods and applications described herein may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides a plasmid system and a host system for high yield of heme, which are used for realizing high-efficiency and scalable production of heme.

In one embodiment of the invention, high levels of extracellular heme are produced by multicopy, incremental expression of genes involved in their heme metabolic pathways. The technology carries out remodeling on related genes endogenous to Shewanella, including hemA, hemL, hemB1, hemB2, hemC, hemD, hemE, hemN, hemG1, hemG2, hemG3, hemH1, hemH2 and ccmf), and the genes are modularly placed in a tetracycline inducible promoter system TetTR-P _TET Control ofThe following steps. The engineered bacteria were cultured in Lysogeny Broth (LB) at 30 ℃ with shaking at 200 rpm. At OD ₆₀₀ When the concentration is about 0.2, 20 μ g/L Anhydrotetracycline (Anhydrotetracycline) is added as an inducer to induce the synthesis of heme. Heme synthetic gene sequences were obtained from the public database of Pubmed (http:// www.pubmed.com /).

The recombinant plasmid and the genetic engineering strain for high yield of heme provided by the invention, the construction method thereof, and biological materials, reagents, instruments and the like used in the method for high yield of heme are all available on the market.

The invention is further illustrated by the following examples:

example 1

1. Strain, plasmid and culture method

All strains and plasmids used in this example are listed in Table 1. Among them, escherichia coli NEB 10. Beta. Was used as a clone strain and for phenotypic verification of gene lines. Coli WM3604 for binding transfer. Coli and s.oneidensis were all cultured in LB medium, and escherichia coli WM3604 required the additional addition of 2-Diaminopimelic Acid (DAP) at a final concentration of 50 μ M. Coli and s. Oneidensis were cultured at 37 ℃ and 30 ℃ respectively. The composition of the 2YT medium was: 10g/L Tryptone (Tryptone), 5g/L Yeast powder (Yeast extract) and 10g/L NaCl. 15g/L Agar powder (Agar) was added to the solid medium.

The above strains are added with antibiotics according to the requirements: chloramphenicol 30. Mu.g/mL (E.coli), 10. Mu.g/mL (S.oneidensis); tetracycline: 10. Mu.g/mL (E. Coli), 5. Mu.g/mL (S. Oneidenesis). Anhydrotetracycline was added to the medium as an inducer at the concentrations noted herein.

The strain is placed in an incubator for static culture or in a shaking table for 200rpm for shaking culture. Bacterial growth was characterized by measuring the light absorption value (OD 600) at a wavelength of 600nm using a BioSpec-1601 ultraviolet spectrophotometer (Shimadzu corporation, japan).

TABLE 1 strains and plasmids used in the invention

2. Sequence analysis

The complete sequence data of the Escherichia coli MG1655 genome was obtained from GenBank (accession number: NC-000913.3).

3. DNA manipulation

The whole genome was extracted by e.z.n.a whole genome DNA extraction kit (OMEGA, beijing, china). Plasmids were extracted by e.z.n.a plasmid miniprep kit (OMEGA, beijing, china). All DNA fragments used for plasmid construction were purified by gel cutting using e.z.n.a gel recovery kit (OMEGA, beijing, china). The enzyme used for PCR amplification, the enzyme used for preparation of Gibson reaction solution, and the restriction enzyme were purchased from NEB corporation (NEB, beijing, china). Q5 High-Fidelity DNA polymerase is used for amplifying the fragment used for plasmid construction, and OneTaq 2X Master Mix is used for PCR verification. The above experimental procedures were all used according to the product specifications.

4. DNA sequencing

The constructed plasmid and the PCR-verified amplified product were both subjected to sequencing verification to determine their correctness. Sequencing was done by general biology, inc. (Anhui, china).

5. Preparation of E.coli electrotransformation competence

1) Coli growth 2YT plate picking single colony, transfer to containing 100mL 2YT liquid medium triangle bottle, shaking table 150rpm to OD ₆₀₀ To reach 0.6, the flask was then taken out of the ice bath for 25min, during which it was gently shaken every 5 min.

2) And subpackaging the bacterial liquid into 50mL centrifuge tubes on an ultraclean workbench, balancing, and centrifuging at 4000rpm at 4 ℃ for 15min to collect thalli.

3) Removing supernatant, suspending the cells with 10% glycerol precooled by an ice bath, gently blowing the suspended cells with a pipette gun, completely suspending and dispersing the precipitated cells, and centrifuging to collect the bacteria.

4) Repeating the step 3) once.

5) Centrifuge, discard the supernatant, resuspend the cells with 1mL of 10% glycerol. Competent cells were dispensed into pre-cooled Eppendorf tube (EP) tubes at 100. Mu.L per tube and frozen at-80 ℃ until use.

6. DNA ligation

The method for constructing the plasmid is mainly by means of Gibson ligation.

1) 6mL of 5X ISO Buffer was prepared, with the following composition:

3mL 1M Tris-HCL(pH 7.5)

+150μL 2M MgCl ₂

+240μL 2M 100mM dNTP mix(25mM each:dGTP,dCTP,dATP,dTTP)

+300μL 1M DTT

+1.5g PEG-8000

+ 300. Mu.L of 100mM NAD

+ddH ₂ O to 6mL, and storing in 160 μ L at-20 deg.C for use.

2) A600. Mu.L Gibson ligation mix system was prepared with the following composition:

160μL 5X ISO Buffer

+0.32μL 10U/μL T5 exonuclease

+10μL 2U/μL Q5 DNA polymerase

+80μL 40U/μL Taq DNA ligase

+ddH ₂ o to 1.2mL, and the mixture was dispensed into 15. Mu.L PCR tubes and stored at-20 ℃ until use.

3) When in use, a 15. Mu.L of Gibson was taken out and mixed with MIX, and dissolved on ice for further use.

4) Plasmid-constructed fragments were prepared and DNA concentration (ng/. Mu.L) was determined using NanoDrop (Thermo).

5) Mixing 100ng of plasmid vector backbone with equimolar amounts of other fragments, pipetting many times with a pipette gun, mixing well with Gibson's ligation MIX to a final volume of 20. Mu.L, and double distilled water (ddH) for the deficient fraction ₂ O) make up.

6) The temperature is kept at 50 ℃ for at least 60 minutes.

7) The MIX reaction was removed, and 1-5. Mu.L of the reaction solution was pipetted into E.coli competence and electroporated.

7. Step of electrotransformation

1) The plasmid or Gibson reaction solution was mixed with competence, transferred to a 2mm cuvette and allowed to stand on ice for 5 minutes.

2) The electric rotator (Bio-rad) was set to 2.5KV for electric shock, and then 1mL of 2YT medium was immediately added, and the mixture was inverted and mixed.

3) Cells suspended in 2YT medium were transferred to 1mL EP tubes, recovered for 1 hour, plated on correspondingly resistant plates, and placed in an incubator for inverted culture.

8. Plasmid construction

The primers used for plasmid construction are listed in Table 2. The different genes in the heme metabolic pathway are divided into four modules, each using P _TET The promoters respectively construct plasmids PTetO-HE1, PTetO-HE2, PTetO-HE3 and PTetO-HE4, and then all modules are assembled in one plasmid PTetO-HEAss.

TABLE 2 primers used in this study

The test steps are as follows:

the starting plasmid pBF-PTeto has the sequence (SEQ ID NO: 1)

CACCTCGCTAACGGATTCACCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGTGGTACCTCTCAGACGCAGAGTTGCCAACCAGATATCGACGTCTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTAATTTTTGTTGACACTCTATCGTTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGAATTCAAAACTCGACCACTCGAGGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCCAGGTGGCACTTTTCGGGGAAATGTGCGCGCCCGCGTTCCTGCTGGCGCTGGGCCTGTTTCTGGCGCTGGACTTCCCGCTGTTCCGTCAGCAGCTTTTCGCCCACGGCCTTGATGATCGCGGCGGCCTTGGCCTGCATATCCCGATTCAACGGCCCCAGGGCGTCCAGAACGGGCTTCAGGCGCTCCCGAAGATCTCGGGCCGTCTCTTGGGCTTGATCGGCCTTCTTGCGCATCTCACGCGCTCCTGCGGCGGCCTGTAGGGCAGGCTCATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCGGCCACGGCTTCCGGCGTCTCAACGCGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGCGGTGCATAGGCGCGTGGCTCGACCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGCCGCTCCAGGGCCTCGTAGAACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGATGCCCCGTTGCAGCCCTAGATCGGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATCTGCGCTTTGTTGCCGATGAACTCCTTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACCACGAACGCGGTCATGTGCGGGCTGGTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGATCCGCCCCGTACTTGTCCGCCAGCCACTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGTTCTTGGCTGGCCGACTTCCACCATTCCGGGCTGGCCGTCATGACGTACTCGACCGCCAACACAGCGTCCTTGCGCCGCTTCTCTGGCAGCAACTCGCGCAGTCGGCCCATCGCTTCATCGGTGCTGCTGGCCGCCCAGTGCTCGTTCTCTGGCGTCCTGCTGGCGTCAGCGTTGGGCGTCTCGCGCTCGCGGTAGGCGTGCTTGAGACTGGCCGCCACGTTGCCCATTTTCGCCAGCTTCTTGCATCGCATGATCGCGTATGCCGCCATGCCTGCCCCTCCCTTTTGGTGTCCAACCGGCTCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGGCCACTCAATGCTTGAGTATACTCACTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGCTGCGGCGCCCTACGGGCTTGCTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCCAGCCCGTGGATATGTGGACGATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCCGTGGACAACCCTGCTGGACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAGGGATTGCCCACCGGCTACCCAGCCTTCGACCACATACCCACCGGCTCCAACTGCGCGGCCTGCGGCCTTGCCCCATCAATTTTTTTAATTTTCTCTGGGGAAAAGCCTCCGGCCTGCGGCCTGCGCGCTTCGCTTGCCGGTTGGACACCAAGTGGAAGGCGGGTCAAGGCTCGCGCAGCGACCGCGCAGCGGCTTGGCCTTGACGCGCCTGGAACGACCCAAGCCTATGCGAGTGGGGGCAGTCGAAGGCGAAGCCCGCCCGCCTGCCCCCCGAGCCTCACGGCGGCGAGTGCGGGGGTTCCAAGGGGGCAGCGCCACCTTGGGCAAGGCCGAAGGCCGCGCAGTCGATCAACAAGCCCCGGAGGGGCCACTTTTTGCCGGAGGGGGAGCCGCGCCGAAGGCGTGGGGGAACCCCGCAGGGGTGCCCTTCTTTGGGCACCAAAGAACTAGATATAGGGCGAAATGCGAAAGACTTAAAAATCAACAACTTAAAAAAGGGGGGTACGCAACAGCTCATTGCGGCACCCCCCGCAATAGCTCATTGCGTAGGTTAAAGAAAATCTGTAATTGACTGCCACTTTTACGCAACGCATAATTGTTGTCGCGCTGCCGAAAAGTTGCAGCTGATTGCGCATGGTGCCGCAACCGTGCGGCACCCTACCGCATGGAGATAAGCATGGCCACGCAGTCCAGAGAAATCGGCATTCAAGCCAAGAACAAGCCCGGTCACTGGGTGCAAACGGAACGCAAAGCGCATGAGGCGTGGGCCGGGCTTATTGCGAGGAAACCCACGGCGGCAATGCTGCTGCATCACCTCGTGGCGCAGATGGGCCACCAGAACGCCGTGGTGGTCAGCCAGAAGACACTTTCCAAGCTCATCGGACGTTCTTTGCGGACGGTCCAATACGCAGTCAAGGACTTGGTGGCCGAGCGCTGGATCTCCGTCGTGAAGCTCAACGGCCCCGGCACCGTGTCGGCCTACGTGGTCAATGACCGCGTGGCGTGGGGCCAGCCCCGCGACCAGTTGCGCCTGTCGGTGTTCAGTGCCGCCGTGGTGGTTGATCACGACGACCAGGACGAATCGCTGTTGGGGCATGGCGACCTGCGCCGCATCCCGACCCTGTATCCGGGCGAGCAGCAACTACCGACCGGCCCCGGCGAGGAGCCGCCCAGCCAGCCCGGCATTCCGGGCATGGAACCAGACCTGCCAGCCTTGACCGAAACGGAGGAATGGGAACGGCGCGGGCAGCAGCGCCTGCCGATGCCCGATGAGCCGTGTTTTCTGGACGATGGCGAGCCGTTGGAGCCGCCGACACGGGTCACGCTGCCGCGCCGGTAGCACTTGGGTTGCGCAGCAACCCGTAAGTGCGCTGTTCCAGACTATCGGCTGTAGCCGCCTCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGG

pBF-PTeto plasmid (SEQ ID NO: 1) is used as a template, and BLT-PTeto F/BLT-PTeto R is used as a primer for amplifying a skeleton. Coli MG1655 genome as template amplification hemA, hemmL, hemB, primer for PTET-HeA F/heL-HeA R, heA-HeL F/HeB1-HeL R, heL-heB F/heB2-heB R. The DNA fragments were assembled to obtain plasmid PTeto-HE1.

Coli MG1655 genome as template to amplify hemC, hemD, hemE, hemN. The primers are PTET-HeCD F/HeE-HeCD R, heD-HeE F/HeN-HeE R and HeE-HeN F/PTET-HeN R respectively. And assembling the DNA fragment and the previously amplified pBF-PTeO plasmid skeleton to obtain a plasmid PTeO-HE 2.

Coli MG1655 genome as template to amplify hemG1, hemG2, hemG3, hemH1, hemH2. The primers are PTET-HeG F/HeG-HeG R, heG1-HeG F/HeG3-HeG R, heG-HeG F/HeH1-HeG R, heG-HeH 1F/HeH 2-HeH 1R, heH1-HeH 2F/T-HeH 2R. And assembling the DNA fragment and the previously amplified pBF-PTeO plasmid skeleton to obtain a plasmid PTeO-HE 3.

And (3) amplifying ccmF by using the S.oneidensis MR-1 genome as a template, using PTET-cmf/PTET-cmf R as a primer, and assembling the primer and the previously amplified pBF-PTeto plasmid skeleton to obtain a plasmid PTeto-HE4.

On the basis of obtaining the plasmid, the PTeto-HE1 is linearized by using a primer L _ D-PTETE 1F/L _ A-PTETE 1R, a corresponding module in the PTeto-HE2 is amplified by using the primer L _ A-HE 2F/L _ B-HE 2R, a corresponding module in the PTeto-HE3 is amplified by using the primer L _ B-HE 3F/L _ C-HE 3R, a corresponding module in the PTeto-HE4 is amplified by using the primer L _ C-HE 4F/L _ D-HE 4R, and the 4DNA fragments are assembled to obtain the PTeto-HEAss (SEQ ID NO: 2).

The sequence of PTeto-HEAss is shown in SEQ ID NO: 2:

CACCTCGCTAACGGATTCACCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGTGGTACCTCTCAGACGCAGAGTTGCCAACCAGATATCGACGTCTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTAATTTTTGTTGACACTCTATCGTTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGAATTCAAAACGCCAAGAATCTAGAAAAGAGTCAGATGAGCCTTGTAGCAATCGGTATTAACCATAAAACAGCCACGGTAGACCTGCGTGAGAAAGTCGCCTTCTCTCCGGACAAAATTCATGATGCCATGAAGAGTCTGGCCAGTCGTACACGCTCGGGTGAAGCCGTTATCGTCTCGACCTGCAATCGCACTGAATTGTATTGTAACAATGGCGATGAGACCGATATCATTGAATGGCTTGAAGAATATCATGGCCTAGAACATCAAGATGTCGCGCCTTGCCTCTACAATTATCACGGGCAAGAGGCGGTAAGGCATTTGATGCGCGTCGCTTCAGGTCTTGACTCGTTGATCCTTGGTGAGCCGCAGATTCTTGGTCAAGTTAAACAAGCCTTTGTCAAAGCCAAAGAAGCCGGCACAGTTGCCTTAACCATTGACCGCTTGTTCCAAAATACCTTCTCAGTTGCTAAAAAAGTGCGTACCGAAACCGAAATCGGTGCCGCTGCTGTATCGGTCGCCTTTGCGGCTGTCAGCATGGCAAAACATATCTTCTCTTCACTGTCGACAACCAAGGTGCTGTTAATTGGTGCTGGTGAAACGATTGAGCTAGTGGCAAAGCATCTAAAAGATAACGGCGTCGCCTCAATGGTGGTGGCAAACCGTACTTTAGAGCGTGCCCAAAGCATGTGTGAGGAATTTAATGCCACGGCAATTACACTGGCACAGATACCAGATTTTCTCCCTAAAGCCGATATCGTGATATCCTCTACCGCCAGCCCGCTGCCAATCCTTGGTAAAGGCATGGTAGAAAAAGCGCTAAAGCAGCGTCGCCATCAACCTATGTTATTGGTTGATATAGCAGTTCCTCGGGATATTGAGCCAGAAGTCGCCGATTTGGACGATGCGTTTCTGTATACAGTGGACGACCTGCATAGCATTATTGAACAGAATAAGGCTTCCCGTAAGGAGGCCGCCGAGCAAGCTGAATTAATTACTGAAGAACAATCTCATCTATTTATGGAGTGGGTGCGTTCTTTAGAGTCGGTCGATAGTATTCGCGAGTATCGCAGCCAGAGCATGGCGATAAAGGATGAGTTGGTGGAACGCGCCCTGAATAAATTAGCGCAGGGGGGCGACACTGAGCAGGTATTGGTTGAATTAGCCAATCGTCTGACCAATAGACTCATTCACGCACCTACCCAAGCCCTCACGGTGGCCAGCCGTCAGGGGGATTTGAATACATTAGGTCAGTTAAGAACAGCGCTCGGATTAGATAAAAACTAATGGCTTCAAACTTTAAGGACTTAACATGACCCGTTCCGAAGCGCTATTTGAACAGGCTAAAAAAACCATCCCCGGCGGTGTTAACTCTCCGGTTCGTGCTTTTAATGGTGTAGGTGGTTCCCCCCTGTTTATTGAAAAAGCCGATGGCGCTTATATCTACGATGCCGATGGCAAAGCCTATATCGACTATGTCGGTTCTTGGGGCCCGATGATCCTCGGCCACAATCATCCGAAGATCCGTGAAGCAGTGCTGGCTGCAGTACACAATGGCCTGTCTTTTGGCGCGCCAACTGAGCTTGAAGTGCAAATGGCCGAAAAAGTGATTGCGATGGTGCCCTCGATTGAGCAAGTCCGTATGGTCAGCTCTGGTACTGAAGCGACCATGAGTGCGATTCGCTTAGCGCGCGGTTTTACTAATCGTGACAAGATCTTAAAGTTTGAAGGTTGCTACCATGGCCACGCTGACTGCCTATTAGTTAAGGCGGGGTCTGGTGCATTAACCTTAGGCCAACCCAGCTCACCCGGCATCCCTGAAGATTTCGCAAAGCACACCTTAACTGCCGTGTATAACGATCTGGATTCTGTTCGTAGCCTATTCGAGCAATATCCAACTGAGATTTCTTGCATCATCATCGAGCCCGTTGCTGGCAACATGAACTGCATCCCACCTATTCCAGGCTTCCTCGAAGGTCTGCGTAGCCTGTGTGATGAGTTTGGTGCGCTGCTGATTATCGACGAAGTGATGACAGGGTTCCGAGTTTCAAAAAGCGGTGCTCAAGGTCACTATGGCGTTACGCCAGACTTAACCACTCTCGGTAAAGTCATCGGTGGCGGTATGCCAGTAGGTGCATTTGGTGGTCGTAAAGATGTGATGCAGTTTATCGCACCAACAGGTCCTGTATACCAAGCAGGTACGCTTTCAGGTAACCCAATTGCGATGTCAGCGGGTCTAGCGCAAATGGAAGCATTGTGTGAAGAAGGACTGTACGAAGCCCTAAGCGCTAAAACCAAGCGCATCGCCGAAGGCTTTAAAGCGGCGGCGGATAAGCACGGCATCCCAATGGCAATCAACTATGTTGGCGGTATGTTCGGCTTCTTTTTTACCGAGCAAGAGCACATCACACGCTTCGACCAAGTGACTAAGTGCAATATTGAGCACTTCCGTACTTTCTACCATGGCATGTTAGATGAAGGCGTTTACTTAGCACCAAGTGCCTATGAAGCAGGCTTCCTGTCGATGGCCCATGGTGAAGAAGAGCTGCGCCTCACACTTGAAGCTGCCGACCGTGTCTTAGCTCGCATGAAAGCGGCAAACTAATGCAAAGTATTAATAAGGAAATTCAATGAGCAGCACACGAGAAAATACCATCGCCCCCCTTCGCCGTTTAAGACGCCTGCGCCGCACCGAGGCCATGCGTGATCTGGTACGTGAAACCCATGTCTCACTCTCCGACTTGATCCACCCACTCTTTATTGAAGAACACATTAACCAAGCTGTGCCAATTTCAACCCTGCCGGGGATTTGCCGTCTGCCCGAAAGCGCACTTGCCGATGAGATCCAAAGGCTCTATGCCTTAGGGATCCGCTATGTGATGCCCTTTGGGATTTCCCATACCAAAGATCCACAGGGCAGCGACACTTGGAATGACAATGGATTGCTCGCACGGATGATCCGTACCATAAAAGCCACCGCGCCAGAGATGATGGTGATCCCCGATATTTGTTTTTGTGAATATACCGACCATGGACACTGCGGCGTGTTGCACCAAGACGAAGTCTGTAACGACCAAACCGTGGCTAACTTAGTCAAACAATCCGTCACCGCCGCTAAAGCGGGAGCTGATATGTTGGCGCCATCAGCAATGATGGATGGTCAAATTAAAGCGATACGACAAGGGCTGGATGAAGCAGGCTTTGAACACGTAGCCATTTTGGCCCACGCAGCCAAGTTCGCCTCCTCTTTCTACGGGCCTTTTAGGGCCGCTGTAGATTGTGAACTCAGTGGTAACCGCAAAGGCTATCAGCTCGATTACGCCAATGGCCGCCAAGCATTGCTCGAAGCACTGCTCGATGAGGAAGAAGGCGCAGATATTTTGATGGTAAAACCCGGCACGCCATATTTGGATGTATTAAGTCGCCTAAGACAAGAAACCCACCTCCCCTTAGCGGCCTATCAAGTGGGTGGCGAATATGCAGGTATTAAATTTGCTGCGCTGGCGGGCGCCCTCGATGAGCGCGCCGTTGTCACTGAAACCTTTATCGGTTTAAAACGGGCTGGTGCCGATTTAATTGTCAGTTATTACACTAAACAATATGCCGAGTGGTTAGCAGAGTTACGCGAATAAAACAGAACCTAATTAGGAGTCTAATGTGAATATCATTACCAGTGCCTTCCCACAGCGCAGAATGCGCCGCATGCGTAAACATGATTTTAGTCGTCGCCTGATGGCTGAGAATCACCTAACAGTGAATGACTTAATCTATCCAATGTTTGTGCTCGAAGGCAGCAATCGCAGCGAAAAAGTGGCTTCAATGCCAGGTGTAGAGCGCTACTCTATCGACCTACTGCTAAAAGAAGCAGAAGAGCTAGTTGAATTAGGTATCCCCTTAATAGCCCTCTTTCCTGTTACGCCTTCCGAGAAAAAGTCGCTTATGGCAGAAGAGGCATACAATGCTGATGCGCTAGTGCAACGCGCAGTGCGTGAACTCAAAAAAGCTTTCCCGCAACTTGGCATTATGACCGACGTAGCTCTAGATCCTTTCACGACCCATGGTCAAGACGGCATTATTGATGAAACAGGCTATATCCTGAATGACATCACCACAGAGATCTTAGTGAAACAAGCACTCTCCCATGCTGAAGCGGGCGCAGACATTGTCGCTCCATCGGATATGATGGACGGCCGCATTGGCGCCATTCGCCAAGCGCTAGAAGCTGCTGGCCATGTTAACACTCAAATCATGGCCTACTCTGCCAAATACTCTTCAAGCTACTACGGCCCATTCCGCGATGCGGTTGGCTCAGCAGGCAACCTAAAAGGTGGCAATAAACATAGCTATCAAATGGACCCAGCAAATAGCGATGAAGCGCTGCACGAAGTCGCACTGGATATCCAAGAGGGCGCAGATATGGTGATGGTAAAACCTGGCATGCCATACCTCGATATCGTTCACCGTGTAAAGACTGAATTAGCAGTCCCCACCTTCGCCTACCAAGTGAGTGGTGAATATGCCATGCACATGGCTGCAATCCAAAATGGCTGGTTAGCAGAAAAGGCAATCGTCATGGAATCGCTACTATGCTTTAAACGCGCTGGCGCCGATGGCATCCTTACCTACTTCGCAAAACGTGCCGCACAGTGGTTAAAAGAAGCTAAATAACTCGACCACTCGAGGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAACCGGTAAACCAGCAATAGACATAGCGGTAGACACCAGTAGTTTATCTTACAATTTTTGTTGACACTCTATCGTTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGAATTCAAAACTAAGATCTCGGATGGAAACTAGGCATGTCTGAAAACCGCATACGTATCGCTACCCGCAAGAGTCCGCTGGCCATGTGGCAAGCAGAATTTGTTAAAGCAGAATTAGAACGCATTCACCCTGGTATTGTGGTGGAGCTATTGCCAATGAGCACTAAGGGCGATGTTATCTTAGACACGCCATTAGCTAAAGTCGGTGGCAAAGGCTTGTTCGTTAAAGAACTCGAAGTCGCCATGCTGGAAGATCAAGCCGACATCGCGGTGCACTCAATGAAAGACGTGCCTGTAGATTTCCCCGAAGGATTAGGACTGGAAGTCATTTGTGAGCGTGAAGATCCTCGCGATGCTTTTGTCTCCAATATTTATAAGTCGATCAGTGAGTTGCCACTTGGTGCCACGGTTGGTACTTCTAGCCTGCGCCGTCAGTGCCAACTGCGTGCTTCCCGTCCCGATCTTATCATCAAAGATCTGCGTGGCAACGTAGGTACTCGCCTCGCGAAACTCGATAACGGTGAATACGATGCGATTATTCTTGCCGCGGCAGGCTTAATCCGCCTGAAGTTATCTGAGCGTATCGCCAGCTTTATCTCTGCCGAAGAGTCCTTACCCGCAAACGGTCAAGGTGCTGTCGGTATCGAATGTCGCACCAATGATGAGCGCGTTAAAGCATTATTAGCGCCATTAGAACACTTAGAGACACGCTACCGCGTGATCGCCGAGCGCGCGATGAATACTCGCCTTGAAGGTGGTTGCCAAGTCCCTATTGGCGCCTTTGCCGAAATTGATGGTGATGAGATGACACTGCGCGGATTAGTGGGTAACCCAGATGGCAGTGAAATCATCGAAGGCGTGATCACCGGTCCTAAGACCGAAGCCACGCAATTAGGTGTTGCCCTTGCGGAAGAACTACTCAGCAAAGGCGCCAAAAGCATCCTCGATGCTGTTTACGCCAAAGCCTAAGTCGATGAAAGTCTTACTGACGCGCCCCGAGGGGCGCAATCAGTCCATGGTCGATGCCTTGAATGAAAGAGGCATCGAACACTGCGTTACGCCACTACTTTGTGTTGAGGCGACACCGGCTCTACCTCCTGAAACCTCACATCCACTCGCTAATGTCGATATGGTTGTCTGTATCAGCGCCAATGCGGTTAGCTTTGCTGACGATACGTTTAAAGCCAATGATGCCAACGTTAAAGGCTGGCCAAAAGTGCCTTATTTCGCTGTAGGGCATGCCACTTGGGAAGCACTACAACAAATCGACATCGATGCCCTTGAGGCGCCCGATGATTGCCAGCAGACTGAAGGGTTACTAACTCTACCTACGCTGCAACACATCCAAGGCAAAAAGATTACCATTATTCGCGGTGTTGGTGGCCGTGAAGCGCTCGCTGAACAACTGACATCTAGAGGCGCACAGGTGCGCTATTGGGAAGTCTATCAACGTGCTTGTCCGCCGCTGGATGGCACTATTATCACCCAACAGTGGATAGACTTAGGGATAGATACTGTCGTGGTGACAAGCGGAGAAGTACTGGATAATCTGATTAATCTAGTGCCAAAAGAGTTATTTGCATGGCTGCGCTCATGTCATATCATAGTCCCCAGTAACCGAGTAGAAGCTCAAGCTCATGCCTTTGGTATAACCCAAGTCACCAATGCCAATGCCGCCAACAGCAAAGCCGTGCTAAACGCCCTTAAACTCTAATCAATAAGTCTAGGAATCAAGCCAAATGGCAGAATTAAAGAATGATCGTTATTTACGCGCCCTACTAAAACAGCCTGTTGATATGACCCCTGTGTGGATGATGCGTCAAGCGGGCCGTTATCTCCCTGAATATAAAGCAACTCGCGCCCAGGCGGGTGATTTTATGTCTTTATGTAAAAACCACGAATTGGCCTGTGAAGTGACGCTACAACCGCTGCGTCGTTACGAGCTAGATGCGGCGATTCTGTTTTCCGACATTCTGACGGTTCCCGATGCTATGGGATTAGGTCTGTATTTTGAGGCGGGTGAAGGCCCACGTTTTGAGCGCCCGACAGACACTATTGATGCCATCAAAAAATTATCAATTCCTGATCCAGAAGATGAGCTGGGTTATGTGATGAAGGCGGTGAGTACAATTCGTCGTGAATTGAATGGTCAAGTGCCATTAATCGGCTTCTCAGGTTCACCTTGGACGCTGGCCACTTATATGGTTGAAGGCGGTTCGAGCAAAACCTTCGAAAAAATCAAAAAAATGGCCTATGCCGAGCCTGCTGCACTGCATATGTTACTCGACAAGTTAGCTGACTCAGTGACTTTATACCTGAATGCGCAGGTCGCTAATGGTGCTCAATCTTTAATGATTTTCGACTCTTGGGGCGGTGCATTGTCGCATACAGCTTACCGTGAGTTTTCACTGCGTTATATGCAGAAGATTGTCGATGGTCTGACTCGCTTTGCCGATGGTCGCCAAGTACCAGTAACCCTATTCACTAAAGGTGGTGGCTTATGGTTAGAAGCCATGGCGGAAACCGGTTGTGACGCCTTAGGTTTAGACTGGACGGTGGATATTGCCGATGCTCGTCGCCGTGTAGGCCATAAAGTTGCGCTACAAGGCAACATGGACCCATCTATGTTATATGCACCTATCCCACGCATTGAAGAGGAAGTTGGTCAAATCCTTGCGGGATACGGTGAAGGTACAGGCCACGTATTTAACTTAGGCCATGGTATTCACCAGCATGTCGATCCAGAGCATGCAGGCGCCTTTATTAAGGCGGTCCACGCTCAATCTAAGCAATACCATAAGTAACTAATTTACCCTCGGAGGACACGCCTTGAAGCAGCCCACTCAGATAAGCTGGGATCAGTCGATGATCGAAAAATATAACTACAGCGGTCCCCGTTATACTTCTTATCCAACGGCGCTAGAGTTCGATGATTCATTCACTGAACAAAACCTGTTAACTGCGATTGAAAACAGCAAGAGTGACAAACTGTCGCTGTATATTCACATCCCCTTCTGCGCCAAACTTTGCTATTACTGTGGCTGCAATAAAGTCATCACTCGTCACGCCCATAAGGCCGACCAATACATTGAGTATTTAAGCCACGAAATTATTAAGCGCGCCCCACTGTTTAAGCATTACTCGGTCACCCAAATGCACTGGGGCGGCGGCACACCAACCTTCTTAAATCCTGAGCAAATTATTAAACTTACCGCATTAATTAAGGCTAACTTTAACTTTGCCGATGAAGGTGAGTTCTCTATCGAAGTTGACCCGCGTGAAATTGAACTTTCCATGCTCGACACCCTTAAAGAAGCCGGCTTTAACCGTATTTCGATTGGTGTTCAGGACTTCAACAAAGAAGTACAAGTCGCGGTTAACCGTGAGCAGGACGAGCAATTTATTTTCGATTTAATGGCCAAAGCCAAGGCTATGGGATTCGTCTCAACCAATATCGATTTAATCTACGGCTTGCCACACCAGACTCCAGAGACATTCGCCGCCACCATGCAGCGCGTGTTAGATCTGTCGCCCGATCGTCTATCTGTATTCAACTATGCTCACTTACCCGCGCGTTTTGCCGCGCAGCGTAAAATTAAAGATGAGCATTTACCTTCGCCAAAACAAAAGCTTGAGATGCTGCATCAAACTATCGAGACCTTAACTGGCGCGGGTTATCAATACATTGGTATGGACCACTTCGCGAAGCCTGACGATGAGCTGGCTAAGTTACAACGCGAAGGCAAACTGCACCGCAACTTCCAGGGTTATACCACCCAAGAAGAATGCGATTTGCTTGGTCTTGGCGTGTCGTCTATCAGCCAAATTGGCGATTGCTATGCGCAAAACCAAAAGGACATTCGCCCTTACTACGAAGCCATCGATAAAGATGGCCATGCGCTCTGGAAAGGCTGCAGCTTGAATCGTGACGACGAAATCCGCCGCGTAGTGATCAAACAATTGATCTGCCACTTCGACTTAGATATGGCCAAAATAGATGAAAAACTGGGAATTAAGTTTGAGGAATACTTCGCCGAAGACTTAAAACTACTGCAAACCTTTATCGATGATAAATTAGTTGAAGTCGCCGACAGAAAGATCACCATCAGCCCCACTGGCCGCCTGTTGATCCGCAATATCTGTATGTGCTTCGACCTTTACTACCGTCAAAAAGCGCGTCAACAACAGTTCTCTCGTGTGATCTAACTCGACCACTCGAGGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAACCGGTAAACCAGCAATAGACGGCGAACAGAACTCTATTGAGATTTTATCCAATTTTTGTTGACACTCTATCGTTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGAATTCAAAAACGCCAACTTTCTGGAAAGATTAATATGCAAACATTAATAATCTACTCAACCATCGACGGTCAAACGCTAGAAATTTGTCGTAAGATTAAAGCATTTGCTGAGCGGGCGGGTGAAAAGGTCTCCCTCTTTAGCTTAGAACAGGCTGAAGCTATAAACCTTGCAGATGTTGATAAGGTATTGATTGGTGCGAGTATCCGCTATGGTAAACATAGACCTGAGCTTTACCAATTTGTGAATCGCAACCATGCGGTGTTAAGCGCGAAAGTTAATGGTTTTTTTACGGTAAATGTGGTGGCGCGGAAGCCGTTAAAGAACACCCCAGAAACCAATCCTTATATGCAGAAATTCCTCAAATTATCCCTTTGGCAACCACAACACTTGGCGGTATTTGCTGGTAAGATTGATTACCCTAAATATGGCTTATTCGACCGCACAATGATCTGTTTTATCATGTGGATGACTAAGGGGCCAACGGATCTTAAGGGCACGTTTGAGTTTACCGATTGGGCTAAAGTCGAAGCTTTTGGCACTCATTTTAGTAAGCTATAATACTTTTACTGAACGAGAATACCCCATGACACGCATCCTGGTGCTTTATTTTACCCGAGGAGGCCATACGGCCAAGATTGCCAATGCAATAGCGGAGCAATTAACCCTGCGTGGCGCAAAGGTTGACTTAGTCGATATCAATAGTGCGGCTGCAACACGAATCAACTGGTCCGATTATCAGCTGGTGGCTTTGGGTGCCTGTGTACTCTATGGCACTTATGATAAGAGTGTATTTCAATTTATTGAGCAACATGTGCAGGCTTTGAGTTTTTTGCCAAACAGTTTTTTCTGCGTCAACGTGGTCGCACGTAATCCGGAGAAACGTATTCCTGAAAACAATAAATACCTGCAAAAGTTTATCACTCTATCTCCTTGGACTCCCGCAGATTTGAAGATTATTGCTGGTAAAGTCGATTATCCATCTTGGCCTTGGTACGACAGATTAATGATCCAGTTAATTATGAAAATCACTAAAGGCCCAACGGATCCAAAAGCAGTTATTGATTATACCGATTGGGAAGATGTGAAAGTCTATGCCGACCATCTGCTGACCTTGGTCGAGGTTGCTGAACCTGCCTAATTTTATTGAGATAATCGAGGCTTAAGTGAAAAAAATTCTGATCATCTTTTCCAGTGTTCATGGCCACACTCGTAAAATCACCAATCAGTTAGCTCAGCAACTCAAAGAGTTGGGTAATTCGGTGGTGATTGCGGATATTAAGGCTGTCCCTGCGATGGAGTCTTTCGATAAGATCATTATTGGCGCGAGTATTCGTCACGGAAAACATAATCCTGCGTTGTACGAATTTATCCAAAAGCATCAACAGATCCTCACGCAAAAGGTCAGTGGCTTTTTCTCCGTCAGTTTAGTGGCTCGTAAACCCGAGAAAAATACTCCAGAAACCAACCCATACATGCAAGCATTTTTAAGCAAAACGACTTGGCGCCCTAAGTTGCTACAAGTGTTTGGCGGTAATCTGAATTACCAAGGTTATAATGCTTTCGATAGAAATATTATTCGTTTTATCATGTGGTTAACTAAGGGGCCAACGGATCCTGTCACCAATGTTGAATACACAGACTGGCAAAAAGTGCAAGAGTTTGGTTTACAAATCCATCAAGCCTAACTAGGTTTTCAGGATTATCAAAAGAAATGGGTCACGCTGCGCGTGGCAAAGTTGGAGTGCTGTTATTAAACCTTGGCACTCCTGATGCGCCAACAGCATCGGCAGTAAGGCGTTATCTTGCCGAGTTTTTATCTGATCCACGGGTGGTGGAAATCCCAAAACTCCTCTGGATGCTGATTTTGTATGGCATAGTACTTAGGGTACGCCCTGCAAAGTCTGCAGCACTTTATCAAAAGGTGTGGACTGAGGCGGGGTCGCCACTGATGGATATCAGTTTGCGACAAACGGCTAAGCTATCCGATAAATTAACCGCGGATGGTCATCAAGTTTCGGTTCACTTAGCTATGCGTTATGGTAATCCTTCTGTTGCCAGCACTTTACGAGAGATGCACAAACAAGGGATTGATAAGCTGGTGGTTTTACCGCTCTATCCGCAATATGCTGCGCCGACAACTGGCTCGGCTTTTGACGCTATCGCTAAAGAGTTATCCCAATGGCGCTACCTGCCATCGCTGCATTTTATTAATACTTATCACGATAACCCTGATTTTATTGCTGCATTAGTCAATTCGATTCGTGACGATTTTGATAAACATGGCAAGCCGCAAAAGTTAGTGCTGTCTTACCATGGAATGCCTGAGCGCAATCTTCATCTGGGTGATCCATATTATTGTTTTTGCATGAAGACTACTCGTCTTGTGGCTGAGCAATTAGGTTTGAGCAAAGATGAATTTGCGATCACATTCCAATCTCGCTTTGGTAAGGCCAAGTGGTTGCAACCCTATACGGATGCAACGATGGCGGCTCTACCGAGTCAAGGTGTGCGTGATGTAGCGATTGTGTGCCCAGCATTTAGCGCCGATTGTTTAGAGACGCTAGAAGAGATTGTCGGCGAAAATGGCCATATCTTTACTCATGCGGGGGGAGAGAAGTTCCGATATATTCCTGCGCTCAATGATAATGATGACCACATCGCCATGATGGCGAATTTGGTGAAGCCGTATCTGTAATATTTGGCTGATAAAAGGTAGCACATTGACTTCTCCCTCTCCTGCGTTTGGCGTGTTATTAGTAAATCTTGGTACGCCTGATGAACCCACTCCGAAAGCGGTTAAGCGATTTCTCAAGCAGTTTTTAAGTGATCCTCGGGTCGTCGATTTATCCCCTTGGTTGTGGCAACCCATTTTGCAGGGGATTATCCTGAACACCCGTCCTAAGAAAGTCGCTAAACTTTATCAAAGTGTGTGGACGGAGCAAGGTTCGCCGTTAATGGTGATTAGTCAGTGCCAAGCCCAAAAGTTGGCAACGGATTTAAGCGCCACCTTTAATCAGACCATTCCGGTGGAACTGGGTATGAGCTATGGCAATCCTTCGATTGAGAGTGGCTTTGCCAAACTCAAAGCCCAAGGCGCCGAACGTATCGTGGTGCTGCCGCTGTATCCGCAGTATTCCTGCTCAACCGTCGCCAGTGTGTTTGATGCGGTAGCGCATTATTTGACTCGCGTGCGTGATATACCTGAGCTGCGTTTTAACAAGCAGTATTTCGCCCATGAAGCCTATATTGCGGCGCTGGCGCATTCGGTAAAGCGCCATTGGAAAACCCATGGTCAGGCCGAGAAGCTGATTTTATCTTTCCACGGGATCCCGCTGCGCTACGCGACCGAAGGCGACCCATACCCAGAGCAGTGCCGCACCACCGCCAAGTTATTAGCGCAGGCCTTAGGCTTGACCGACGGACAATGGCAAGTATGTTTCCAATCCCGCTTCGGTAAAGAAGAGTGGTTGACGCCCTATGCCGATGAGTTGCTGGCGGATTTACCCCGCCAAGGCGTGAAAAGTGTCGATGTGATTTGCCCTGCCTTTGCTACCGATTGCCTTGAAACCCTAGAAGAAATCTCGATTGGCGCGAAAGAGACCTTCCTCCATGCAGGTGGCGAAGCCTATCATTTTATTCCTTGTTTGAATGATGATGAGCTACATATAGAGCTACTCAGGTTATTAGTACAAGAACAAACGCAGTCTTGGATAAGTGCAGAATAACTCGACCACTCGAGGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAACCGGTAAACCAGCAATAGACTGTCAGGAAAATACTGCGTTTACTATCAGCAATTTTTGTTGACACTCTATCGTTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGAATTCAAAATATTATTAGAGACTGAGGAAAACCCATGATCCCAGAACTTGGACACTTTTCGCTGATAATAGGAGTGGCTTTTGCCTTCTTATTAACCAGCGTTCCCCTTATAGGTGTTGCCCGTAAAGACCAATATCTAGTGAGGTATGCTTGGCCGTTAGCCTACGGAATGTTCTTTTTTATCGCTTTATCCGTAGTATCACTTGGTTACAGCTTCGCCGTTGATGATTTCTCCGTGGCCTATGTGGCACATCACTCTAATTCTCAGTTACCTATCTTCTTTAAGATTGCGGCCGTATGGGGCGGTCATGAAGGGTCGCTGCTATTCTGGGTGTTCGCGCTATCCACTTGGGCTGCCTCTGTTGCTTTATTCAGTAAAGGCTTAGAAGAAGTCTTCACCGCTCGCGTGTTAGCGGTATTGGCATTGATTGTGATTGGCTTCAGCTTGTTTATGTTACTCACTTCTAGCCCATTTGAACGTATATTCCCAATGCCTGCTGAAGGCCGCGACTTAAATCCAATGCTGCAGGATGTGGGCTTAATCTTCCACCCTCCGATGTTATATTTGGGTTATGTGGGTTTCTCAGTCAGTTTTGCATTTGCGATTGCAGCTTTGATGAGTGGTCACCTTGATTCTGCTTGGGCTCGTTGGTCGCGTCCTTGGACCTTAGCCGCTTGGGTTTTCCTCACTGGTGGTATTGCATTAGGTTCTTGGTGGGCGTATTACGAATTAGGCTGGGGTGGATGGTGGTTCTGGGATCCAGTAGAAAACGCTTCATTTATGCCTTGGCTAGTGGGTACGGCTTTAGTGCATTCACTGATCGTGACGGAAAAGCGCGGTGCATTCCGTAACTGGACGGTATTGCTGTCTATCTTCGCATTCTCGCTAAGTTTACTCGGTACCTTTATCGTACGCTCTGGTGTGTTGACTTCAGTGCATTCATTTGCAGCCGATCCAAGCCGCGGTATGTTTATTCTATTATTGCTTGGTCTCGCTATCGGTGGTTCGCTCACTTTGTTTGCCTTCCGTGCAAGTGAAATGAGCAGCCCTGCACGCTTCGAGCTGAAATCAAAAGAAACCATGCTGTTAGTTTGTAACGTGTTACTGACGGTTGCAGCGGGTACTGTTCTGTTAGGCACTTTATACCCACTGCTGATCGATGCGTTAGGCATGGGTAAGATTTCTGTTGGACCTCCATACTTTAACGCTGTGTTTGTACCCATAGTACTTGTACTGTTTGCCTTTATGGGCGTCGGTCCAATCATTCGTTGGAAAAAATCGAAAGCTGGTGAATTGAAGCGACAGTTACTCGTACCTGCATTAGTATCTCTGGTGATTGGTATTGTCACGCCGTTTATCGTCGATGGGGCCTTTAACGCTTGGGTTGCTTGCGGTATCGCCGCTGCAGCTTGGATTATTTTGGCAACCGCAAAAGCAGCTTACAGCATCGTTAAGCCTAAAGATGGTGAAGTAAGCATCGCACGTATGGGCCGTAGCCAACTCGGTATGATCATCGCGCACTTAGGTATTGCTGTGTCTGTTATTGGTGCCACTATGGTGTCTAACTACTCAGTGGAGAAAAGTGTGCGTATGGGGCCTGGCGTAAGCCAAGAGCTAGCGGGTTACACCTTTAAATATCTCGAAACTAAAAACGTTGTTGGTCCTAATTACACTGCGCAGCAAGGTCAAATTGAGATTTACAAAGGCGATAAGTTACTGACACTGCTCAAACCCGATCGTCGCCAATACAATGTGCGTACTATGGATATGACCGAAGCCGGTATCGATTGGGGTCTGTTCCGCGATCTGTATGTGACGATGGGCGATCCGATTAGTAGTACTGAATTTGCTGTGCGTTTGAACTACAAGCCGTTTGTGCGTTGGTTATGGTTCGGAGCAATATTCATGATGGTCGGCGGTTTCTTTGCCGCATCGGATAAACGCTATCGCTCAAAAGTCGCGGCTACCGTTAAACCGCAAGCTGAAAAAGCGAAATTAGCTACCGCTCAATAACTCGACCACTCGAGGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAACCGGTAAACCAGCAATAGACACTATCGGAATAAAGGAGTTCGCTTACTTCCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCCAGGTGGCACTTTTCGGGGAAATGTGCGCGCCCGCGTTCCTGCTGGCGCTGGGCCTGTTTCTGGCGCTGGACTTCCCGCTGTTCCGTCAGCAGCTTTTCGCCCACGGCCTTGATGATCGCGGCGGCCTTGGCCTGCATATCCCGATTCAACGGCCCCAGGGCGTCCAGAACGGGCTTCAGGCGCTCCCGAAGATCTCGGGCCGTCTCTTGGGCTTGATCGGCCTTCTTGCGCATCTCACGCGCTCCTGCGGCGGCCTGTAGGGCAGGCTCATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCGGCCACGGCTTCCGGCGTCTCAACGCGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGCGGTGCATAGGCGCGTGGCTCGACCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGCCGCTCCAGGGCCTCGTAGAACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGATGCCCCGTTGCAGCCCTAGATCGGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATCTGCGCTTTGTTGCCGATGAACTCCTTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACCACGAACGCGGTCATGTGCGGGCTGGTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGATCCGCCCCGTACTTGTCCGCCAGCCACTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGTTCTTGGCTGGCCGACTTCCACCATTCCGGGCTGGCCGTCATGACGTACTCGACCGCCAACACAGCGTCCTTGCGCCGCTTCTCTGGCAGCAACTCGCGCAGTCGGCCCATCGCTTCATCGGTGCTGCTGGCCGCCCAGTGCTCGTTCTCTGGCGTCCTGCTGGCGTCAGCGTTGGGCGTCTCGCGCTCGCGGTAGGCGTGCTTGAGACTGGCCGCCACGTTGCCCATTTTCGCCAGCTTCTTGCATCGCATGATCGCGTATGCCGCCATGCCTGCCCCTCCCTTTTGGTGTCCAACCGGCTCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGGCCACTCAATGCTTGAGTATACTCACTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGCTGCGGCGCCCTACGGGCTTGCTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCCAGCCCGTGGATATGTGGACGATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCCGTGGACAACCCTGCTGGACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAGGGATTGCCCACCGGCTACCCAGCCTTCGACCACATACCCACCGGCTCCAACTGCGCGGCCTGCGGCCTTGCCCCATCAATTTTTTTAATTTTCTCTGGGGAAAAGCCTCCGGCCTGCGGCCTGCGCGCTTCGCTTGCCGGTTGGACACCAAGTGGAAGGCGGGTCAAGGCTCGCGCAGCGACCGCGCAGCGGCTTGGCCTTGACGCGCCTGGAACGACCCAAGCCTATGCGAGTGGGGGCAGTCGAAGGCGAAGCCCGCCCGCCTGCCCCCCGAGCCTCACGGCGGCGAGTGCGGGGGTTCCAAGGGGGCAGCGCCACCTTGGGCAAGGCCGAAGGCCGCGCAGTCGATCAACAAGCCCCGGAGGGGCCACTTTTTGCCGGAGGGGGAGCCGCGCCGAAGGCGTGGGGGAACCCCGCAGGGGTGCCCTTCTTTGGGCACCAAAGAACTAGATATAGGGCGAAATGCGAAAGACTTAAAAATCAACAACTTAAAAAAGGGGGGTACGCAACAGCTCATTGCGGCACCCCCCGCAATAGCTCATTGCGTAGGTTAAAGAAAATCTGTAATTGACTGCCACTTTTACGCAACGCATAATTGTTGTCGCGCTGCCGAAAAGTTGCAGCTGATTGCGCATGGTGCCGCAACCGTGCGGCACCCTACCGCATGGAGATAAGCATGGCCACGCAGTCCAGAGAAATCGGCATTCAAGCCAAGAACAAGCCCGGTCACTGGGTGCAAACGGAACGCAAAGCGCATGAGGCGTGGGCCGGGCTTATTGCGAGGAAACCCACGGCGGCAATGCTGCTGCATCACCTCGTGGCGCAGATGGGCCACCAGAACGCCGTGGTGGTCAGCCAGAAGACACTTTCCAAGCTCATCGGACGTTCTTTGCGGACGGTCCAATACGCAGTCAAGGACTTGGTGGCCGAGCGCTGGATCTCCGTCGTGAAGCTCAACGGCCCCGGCACCGTGTCGGCCTACGTGGTCAATGACCGCGTGGCGTGGGGCCAGCCCCGCGACCAGTTGCGCCTGTCGGTGTTCAGTGCCGCCGTGGTGGTTGATCACGACGACCAGGACGAATCGCTGTTGGGGCATGGCGACCTGCGCCGCATCCCGACCCTGTATCCGGGCGAGCAGCAACTACCGACCGGCCCCGGCGAGGAGCCGCCCAGCCAGCCCGGCATTCCGGGCATGGAACCAGACCTGCCAGCCTTGACCGAAACGGAGGAATGGGAACGGCGCGGGCAGCAGCGCCTGCCGATGCCCGATGAGCCGTGTTTTCTGGACGATGGCGAGCCGTTGGAGCCGCCGACACGGGTCACGCTGCCGCGCCGGTAGCACTTGGGTTGCGCAGCAACCCGTAAGTGCGCTGTTCCAGACTATCGGCTGTAGCCGCCTCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGG

9. induction of heme production

The engineering strain is inoculated into LB liquid culture medium (5 mL) from a-80 ℃ bacteria-preserving tube according to 1% (v/v), and is used as seed liquid for standby after shaking culture overnight. Then, the seed solution was inoculated at 1% into LB liquid medium for scale-up culture (500 mL). To be OD ₆₀₀ When the concentration was about 0.2, 20. Mu.g/L of aTc inducer was added thereto, and the fermentation was continued for 3 days. The supernatant was collected for measurement of hemoglobin.

10. Measurement of hemoglobin

The hemoglobin was measured by High Performance Liquid Chromatography (HPLC) using an Agilent 1290 II HPLC column (2.1x 150mm,1.8 um), an Extend-C18 liquid chromatography column (2.1X 150mm,1.8 um) with a mobile phase of methanol: 0.1% formic acid =80, a flow rate of 0.5mL/min, and a photodiode array detector (DAD) at 399 nM.

11. Results of the experiment

The engineering bacteria constructed by the technology have higher heme yield, cell breaking extraction is not needed, and extracellular free heme can reach 250mg/L after 3 days of culture.

The method can produce large amount of heme in short time, save cost for cell disruption, simplify extraction process, and reduce loss. Thus, this strategy provides a sustainable method for high yield of heme.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of science and technology in China

<120> recombinant plasmid and genetic engineering strain for high yield of heme, construction method thereof and method for high yield of heme

<130> MP2007875

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5051

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cacctcgcta acggattcac cgtttttatc aggctctggg aggcagaata aatgatcata 60

tcgtcaatta ttacctccac ggggagagcc tgagcaaact ggcctcaggc atttgagaag 120

cacacggtca cactgcttcc ggtagtcaat aaaccggtca gaatttcaga taaaaaaaat 180

ccttagcttt cgctaaggat gatttctgtg gtacctctca gacgcagagt tgccaaccag 240

atatcgacgt cttaagaccc actttcacat ttaagttgtt tttctaatcc gcatatgatc 300

aattcaaggc cgaataagaa ggctggctct gcaccttggt gatcaaataa ttcgatagct 360

tgtcgtaata atggcggcat actatcagta gtaggtgttt ccctttcttc tttagcgact 420

tgatgctctt gatcttccaa tacgcaacct aaagtaaaat gccccacagc gctgagtgca 480

tataatgcat tctctagtga aaaaccttgt tggcataaaa aggctaattg attttcgaga 540

gtttcatact gtttttctgt aggccgtgta cctaaatgta cttttgctcc atcgcgatga 600

cttagtaaag cacatctaaa acttttagcg ttattacgta aaaaatcttg ccagctttcc 660

ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca tctcaatggc taaggcgtcg 720

agcaaagccc gcttattttt tacatgccaa tacaatgtag gctgctctac acctagcttc 780

tgggcgagtt tacgggttgt taaaccttcg attccgacct cattaagcag ctctaatgcg 840

ctgttaatca ctttactttt atctaatcta gacatcatta attcctaatt tttgttgaca 900

ctctatcgtt gatagagtta ttttaccact ccctatcagt gatagagaaa agaattcaaa 960

actcgaccac tcgaggccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc 1020

tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct 1080

tcgggtgggc ctttctgcgt ttataaccgg taaaccagca atagacataa gcggctattt 1140

aacgaccctg ccctgaaccg acgaccgggt cgaatttgct ttcgaacccc agagtcccgc 1200

tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat 1260

accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg 1320

ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa 1380

tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac 1440

gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc 1500

gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt 1560

acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag 1620

cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg 1680

agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc 1740

agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg 1800

cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac 1860

cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg 1920

tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc 1980

atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc ttgatcccct 2040

gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac 2100

cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca 2160

gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt 2220

ttcccttgtc cagatagccc agtagctgac attcatccca ggtggcactt ttcggggaaa 2280

tgtgcgcgcc cgcgttcctg ctggcgctgg gcctgtttct ggcgctggac ttcccgctgt 2340

tccgtcagca gcttttcgcc cacggccttg atgatcgcgg cggccttggc ctgcatatcc 2400

cgattcaacg gccccagggc gtccagaacg ggcttcaggc gctcccgaag atctcgggcc 2460

gtctcttggg cttgatcggc cttcttgcgc atctcacgcg ctcctgcggc ggcctgtagg 2520

gcaggctcat acccctgccg aaccgctttt gtcagccggt cggccacggc ttccggcgtc 2580

tcaacgcgct ttgagattcc cagcttttcg gccaatccct gcggtgcata ggcgcgtggc 2640

tcgaccgctt gcgggctgat ggtgacgtgg cccactggtg gccgctccag ggcctcgtag 2700

aacgcctgaa tgcgcgtgtg acgtgccttg ctgccctcga tgccccgttg cagccctaga 2760

tcggccacag cggccgcaaa cgtggtctgg tcgcgggtca tctgcgcttt gttgccgatg 2820

aactccttgg ccgacagcct gccgtcctgc gtcagcggca ccacgaacgc ggtcatgtgc 2880

gggctggttt cgtcacggtg gatgctggcc gtcacgatgc gatccgcccc gtacttgtcc 2940

gccagccact tgtgcgcctt ctcgaagaac gccgcctgct gttcttggct ggccgacttc 3000

caccattccg ggctggccgt catgacgtac tcgaccgcca acacagcgtc cttgcgccgc 3060

ttctctggca gcaactcgcg cagtcggccc atcgcttcat cggtgctgct ggccgcccag 3120

tgctcgttct ctggcgtcct gctggcgtca gcgttgggcg tctcgcgctc gcggtaggcg 3180

tgcttgagac tggccgccac gttgcccatt ttcgccagct tcttgcatcg catgatcgcg 3240

tatgccgcca tgcctgcccc tcccttttgg tgtccaaccg gctcgacggg ggcagcgcaa 3300

ggcggtgcct ccggcgggcc actcaatgct tgagtatact cactagactt tgcttcgcaa 3360

agtcgtgacc gcctacggcg gctgcggcgc cctacgggct tgctctccgg gcttcgccct 3420

gcgcggtcgc tgcgctccct tgccagcccg tggatatgtg gacgatggcc gcgagcggcc 3480

accggctggc tcgcttcgct cggcccgtgg acaaccctgc tggacaagct gatggacagg 3540

ctgcgcctgc ccacgagctt gaccacaggg attgcccacc ggctacccag ccttcgacca 3600

catacccacc ggctccaact gcgcggcctg cggccttgcc ccatcaattt ttttaatttt 3660

ctctggggaa aagcctccgg cctgcggcct gcgcgcttcg cttgccggtt ggacaccaag 3720

tggaaggcgg gtcaaggctc gcgcagcgac cgcgcagcgg cttggccttg acgcgcctgg 3780

aacgacccaa gcctatgcga gtgggggcag tcgaaggcga agcccgcccg cctgcccccc 3840

gagcctcacg gcggcgagtg cgggggttcc aagggggcag cgccaccttg ggcaaggccg 3900

aaggccgcgc agtcgatcaa caagccccgg aggggccact ttttgccgga gggggagccg 3960

cgccgaaggc gtgggggaac cccgcagggg tgcccttctt tgggcaccaa agaactagat 4020

atagggcgaa atgcgaaaga cttaaaaatc aacaacttaa aaaagggggg tacgcaacag 4080

ctcattgcgg caccccccgc aatagctcat tgcgtaggtt aaagaaaatc tgtaattgac 4140

tgccactttt acgcaacgca taattgttgt cgcgctgccg aaaagttgca gctgattgcg 4200

catggtgccg caaccgtgcg gcaccctacc gcatggagat aagcatggcc acgcagtcca 4260

gagaaatcgg cattcaagcc aagaacaagc ccggtcactg ggtgcaaacg gaacgcaaag 4320

cgcatgaggc gtgggccggg cttattgcga ggaaacccac ggcggcaatg ctgctgcatc 4380

acctcgtggc gcagatgggc caccagaacg ccgtggtggt cagccagaag acactttcca 4440

agctcatcgg acgttctttg cggacggtcc aatacgcagt caaggacttg gtggccgagc 4500

gctggatctc cgtcgtgaag ctcaacggcc ccggcaccgt gtcggcctac gtggtcaatg 4560

accgcgtggc gtggggccag ccccgcgacc agttgcgcct gtcggtgttc agtgccgccg 4620

tggtggttga tcacgacgac caggacgaat cgctgttggg gcatggcgac ctgcgccgca 4680

tcccgaccct gtatccgggc gagcagcaac taccgaccgg ccccggcgag gagccgccca 4740

gccagcccgg cattccgggc atggaaccag acctgccagc cttgaccgaa acggaggaat 4800

gggaacggcg cgggcagcag cgcctgccga tgcccgatga gccgtgtttt ctggacgatg 4860

gcgagccgtt ggagccgccg acacgggtca cgctgccgcg ccggtagcac ttgggttgcg 4920

cagcaacccg taagtgcgct gttccagact atcggctgta gccgcctcgc cgccctatac 4980

cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg 5040

gaagccggcg g 5051

<210> 2

<211> 20457

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cacctcgcta acggattcac cgtttttatc aggctctggg aggcagaata aatgatcata 60

tcgtcaatta ttacctccac ggggagagcc tgagcaaact ggcctcaggc atttgagaag 120

cacacggtca cactgcttcc ggtagtcaat aaaccggtca gaatttcaga taaaaaaaat 180

ccttagcttt cgctaaggat gatttctgtg gtacctctca gacgcagagt tgccaaccag 240

atatcgacgt cttaagaccc actttcacat ttaagttgtt tttctaatcc gcatatgatc 300

aattcaaggc cgaataagaa ggctggctct gcaccttggt gatcaaataa ttcgatagct 360

tgtcgtaata atggcggcat actatcagta gtaggtgttt ccctttcttc tttagcgact 420

tgatgctctt gatcttccaa tacgcaacct aaagtaaaat gccccacagc gctgagtgca 480

tataatgcat tctctagtga aaaaccttgt tggcataaaa aggctaattg attttcgaga 540

gtttcatact gtttttctgt aggccgtgta cctaaatgta cttttgctcc atcgcgatga 600

cttagtaaag cacatctaaa acttttagcg ttattacgta aaaaatcttg ccagctttcc 660

ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca tctcaatggc taaggcgtcg 720

agcaaagccc gcttattttt tacatgccaa tacaatgtag gctgctctac acctagcttc 780

tgggcgagtt tacgggttgt taaaccttcg attccgacct cattaagcag ctctaatgcg 840

ctgttaatca ctttactttt atctaatcta gacatcatta attcctaatt tttgttgaca 900

ctctatcgtt gatagagtta ttttaccact ccctatcagt gatagagaaa agaattcaaa 960

acgccaagaa tctagaaaag agtcagatga gccttgtagc aatcggtatt aaccataaaa 1020

cagccacggt agacctgcgt gagaaagtcg ccttctctcc ggacaaaatt catgatgcca 1080

tgaagagtct ggccagtcgt acacgctcgg gtgaagccgt tatcgtctcg acctgcaatc 1140

gcactgaatt gtattgtaac aatggcgatg agaccgatat cattgaatgg cttgaagaat 1200

atcatggcct agaacatcaa gatgtcgcgc cttgcctcta caattatcac gggcaagagg 1260

cggtaaggca tttgatgcgc gtcgcttcag gtcttgactc gttgatcctt ggtgagccgc 1320

agattcttgg tcaagttaaa caagcctttg tcaaagccaa agaagccggc acagttgcct 1380

taaccattga ccgcttgttc caaaatacct tctcagttgc taaaaaagtg cgtaccgaaa 1440

ccgaaatcgg tgccgctgct gtatcggtcg cctttgcggc tgtcagcatg gcaaaacata 1500

tcttctcttc actgtcgaca accaaggtgc tgttaattgg tgctggtgaa acgattgagc 1560

tagtggcaaa gcatctaaaa gataacggcg tcgcctcaat ggtggtggca aaccgtactt 1620

tagagcgtgc ccaaagcatg tgtgaggaat ttaatgccac ggcaattaca ctggcacaga 1680

taccagattt tctccctaaa gccgatatcg tgatatcctc taccgccagc ccgctgccaa 1740

tccttggtaa aggcatggta gaaaaagcgc taaagcagcg tcgccatcaa cctatgttat 1800

tggttgatat agcagttcct cgggatattg agccagaagt cgccgatttg gacgatgcgt 1860

ttctgtatac agtggacgac ctgcatagca ttattgaaca gaataaggct tcccgtaagg 1920

aggccgccga gcaagctgaa ttaattactg aagaacaatc tcatctattt atggagtggg 1980

tgcgttcttt agagtcggtc gatagtattc gcgagtatcg cagccagagc atggcgataa 2040

aggatgagtt ggtggaacgc gccctgaata aattagcgca ggggggcgac actgagcagg 2100

tattggttga attagccaat cgtctgacca atagactcat tcacgcacct acccaagccc 2160

tcacggtggc cagccgtcag ggggatttga atacattagg tcagttaaga acagcgctcg 2220

gattagataa aaactaatgg cttcaaactt taaggactta acatgacccg ttccgaagcg 2280

ctatttgaac aggctaaaaa aaccatcccc ggcggtgtta actctccggt tcgtgctttt 2340

aatggtgtag gtggttcccc cctgtttatt gaaaaagccg atggcgctta tatctacgat 2400

gccgatggca aagcctatat cgactatgtc ggttcttggg gcccgatgat cctcggccac 2460

aatcatccga agatccgtga agcagtgctg gctgcagtac acaatggcct gtcttttggc 2520

gcgccaactg agcttgaagt gcaaatggcc gaaaaagtga ttgcgatggt gccctcgatt 2580

gagcaagtcc gtatggtcag ctctggtact gaagcgacca tgagtgcgat tcgcttagcg 2640

cgcggtttta ctaatcgtga caagatctta aagtttgaag gttgctacca tggccacgct 2700

gactgcctat tagttaaggc ggggtctggt gcattaacct taggccaacc cagctcaccc 2760

ggcatccctg aagatttcgc aaagcacacc ttaactgccg tgtataacga tctggattct 2820

gttcgtagcc tattcgagca atatccaact gagatttctt gcatcatcat cgagcccgtt 2880

gctggcaaca tgaactgcat cccacctatt ccaggcttcc tcgaaggtct gcgtagcctg 2940

tgtgatgagt ttggtgcgct gctgattatc gacgaagtga tgacagggtt ccgagtttca 3000

aaaagcggtg ctcaaggtca ctatggcgtt acgccagact taaccactct cggtaaagtc 3060

atcggtggcg gtatgccagt aggtgcattt ggtggtcgta aagatgtgat gcagtttatc 3120

gcaccaacag gtcctgtata ccaagcaggt acgctttcag gtaacccaat tgcgatgtca 3180

gcgggtctag cgcaaatgga agcattgtgt gaagaaggac tgtacgaagc cctaagcgct 3240

aaaaccaagc gcatcgccga aggctttaaa gcggcggcgg ataagcacgg catcccaatg 3300

gcaatcaact atgttggcgg tatgttcggc ttctttttta ccgagcaaga gcacatcaca 3360

cgcttcgacc aagtgactaa gtgcaatatt gagcacttcc gtactttcta ccatggcatg 3420

ttagatgaag gcgtttactt agcaccaagt gcctatgaag caggcttcct gtcgatggcc 3480

catggtgaag aagagctgcg cctcacactt gaagctgccg accgtgtctt agctcgcatg 3540

aaagcggcaa actaatgcaa agtattaata aggaaattca atgagcagca cacgagaaaa 3600

taccatcgcc ccccttcgcc gtttaagacg cctgcgccgc accgaggcca tgcgtgatct 3660

ggtacgtgaa acccatgtct cactctccga cttgatccac ccactcttta ttgaagaaca 3720

cattaaccaa gctgtgccaa tttcaaccct gccggggatt tgccgtctgc ccgaaagcgc 3780

acttgccgat gagatccaaa ggctctatgc cttagggatc cgctatgtga tgccctttgg 3840

gatttcccat accaaagatc cacagggcag cgacacttgg aatgacaatg gattgctcgc 3900

acggatgatc cgtaccataa aagccaccgc gccagagatg atggtgatcc ccgatatttg 3960

tttttgtgaa tataccgacc atggacactg cggcgtgttg caccaagacg aagtctgtaa 4020

cgaccaaacc gtggctaact tagtcaaaca atccgtcacc gccgctaaag cgggagctga 4080

tatgttggcg ccatcagcaa tgatggatgg tcaaattaaa gcgatacgac aagggctgga 4140

tgaagcaggc tttgaacacg tagccatttt ggcccacgca gccaagttcg cctcctcttt 4200

ctacgggcct tttagggccg ctgtagattg tgaactcagt ggtaaccgca aaggctatca 4260

gctcgattac gccaatggcc gccaagcatt gctcgaagca ctgctcgatg aggaagaagg 4320

cgcagatatt ttgatggtaa aacccggcac gccatatttg gatgtattaa gtcgcctaag 4380

acaagaaacc cacctcccct tagcggccta tcaagtgggt ggcgaatatg caggtattaa 4440

atttgctgcg ctggcgggcg ccctcgatga gcgcgccgtt gtcactgaaa cctttatcgg 4500

tttaaaacgg gctggtgccg atttaattgt cagttattac actaaacaat atgccgagtg 4560

gttagcagag ttacgcgaat aaaacagaac ctaattagga gtctaatgtg aatatcatta 4620

ccagtgcctt cccacagcgc agaatgcgcc gcatgcgtaa acatgatttt agtcgtcgcc 4680

tgatggctga gaatcaccta acagtgaatg acttaatcta tccaatgttt gtgctcgaag 4740

gcagcaatcg cagcgaaaaa gtggcttcaa tgccaggtgt agagcgctac tctatcgacc 4800

tactgctaaa agaagcagaa gagctagttg aattaggtat ccccttaata gccctctttc 4860

ctgttacgcc ttccgagaaa aagtcgctta tggcagaaga ggcatacaat gctgatgcgc 4920

tagtgcaacg cgcagtgcgt gaactcaaaa aagctttccc gcaacttggc attatgaccg 4980

acgtagctct agatcctttc acgacccatg gtcaagacgg cattattgat gaaacaggct 5040

atatcctgaa tgacatcacc acagagatct tagtgaaaca agcactctcc catgctgaag 5100

cgggcgcaga cattgtcgct ccatcggata tgatggacgg ccgcattggc gccattcgcc 5160

aagcgctaga agctgctggc catgttaaca ctcaaatcat ggcctactct gccaaatact 5220

cttcaagcta ctacggccca ttccgcgatg cggttggctc agcaggcaac ctaaaaggtg 5280

gcaataaaca tagctatcaa atggacccag caaatagcga tgaagcgctg cacgaagtcg 5340

cactggatat ccaagagggc gcagatatgg tgatggtaaa acctggcatg ccatacctcg 5400

atatcgttca ccgtgtaaag actgaattag cagtccccac cttcgcctac caagtgagtg 5460

gtgaatatgc catgcacatg gctgcaatcc aaaatggctg gttagcagaa aaggcaatcg 5520

tcatggaatc gctactatgc tttaaacgcg ctggcgccga tggcatcctt acctacttcg 5580

caaaacgtgc cgcacagtgg ttaaaagaag ctaaataact cgaccactcg aggccaggca 5640

tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc 5700

ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta 5760

taaccggtaa accagcaata gacatagcgg tagacaccag tagtttatct tacaattttt 5820

gttgacactc tatcgttgat agagttattt taccactccc tatcagtgat agagaaaaga 5880

attcaaaact aagatctcgg atggaaacta ggcatgtctg aaaaccgcat acgtatcgct 5940

acccgcaaga gtccgctggc catgtggcaa gcagaatttg ttaaagcaga attagaacgc 6000

attcaccctg gtattgtggt ggagctattg ccaatgagca ctaagggcga tgttatctta 6060

gacacgccat tagctaaagt cggtggcaaa ggcttgttcg ttaaagaact cgaagtcgcc 6120

atgctggaag atcaagccga catcgcggtg cactcaatga aagacgtgcc tgtagatttc 6180

cccgaaggat taggactgga agtcatttgt gagcgtgaag atcctcgcga tgcttttgtc 6240

tccaatattt ataagtcgat cagtgagttg ccacttggtg ccacggttgg tacttctagc 6300

ctgcgccgtc agtgccaact gcgtgcttcc cgtcccgatc ttatcatcaa agatctgcgt 6360

ggcaacgtag gtactcgcct cgcgaaactc gataacggtg aatacgatgc gattattctt 6420

gccgcggcag gcttaatccg cctgaagtta tctgagcgta tcgccagctt tatctctgcc 6480

gaagagtcct tacccgcaaa cggtcaaggt gctgtcggta tcgaatgtcg caccaatgat 6540

gagcgcgtta aagcattatt agcgccatta gaacacttag agacacgcta ccgcgtgatc 6600

gccgagcgcg cgatgaatac tcgccttgaa ggtggttgcc aagtccctat tggcgccttt 6660

gccgaaattg atggtgatga gatgacactg cgcggattag tgggtaaccc agatggcagt 6720

gaaatcatcg aaggcgtgat caccggtcct aagaccgaag ccacgcaatt aggtgttgcc 6780

cttgcggaag aactactcag caaaggcgcc aaaagcatcc tcgatgctgt ttacgccaaa 6840

gcctaagtcg atgaaagtct tactgacgcg ccccgagggg cgcaatcagt ccatggtcga 6900

tgccttgaat gaaagaggca tcgaacactg cgttacgcca ctactttgtg ttgaggcgac 6960

accggctcta cctcctgaaa cctcacatcc actcgctaat gtcgatatgg ttgtctgtat 7020

cagcgccaat gcggttagct ttgctgacga tacgtttaaa gccaatgatg ccaacgttaa 7080

aggctggcca aaagtgcctt atttcgctgt agggcatgcc acttgggaag cactacaaca 7140

aatcgacatc gatgcccttg aggcgcccga tgattgccag cagactgaag ggttactaac 7200

tctacctacg ctgcaacaca tccaaggcaa aaagattacc attattcgcg gtgttggtgg 7260

ccgtgaagcg ctcgctgaac aactgacatc tagaggcgca caggtgcgct attgggaagt 7320

ctatcaacgt gcttgtccgc cgctggatgg cactattatc acccaacagt ggatagactt 7380

agggatagat actgtcgtgg tgacaagcgg agaagtactg gataatctga ttaatctagt 7440

gccaaaagag ttatttgcat ggctgcgctc atgtcatatc atagtcccca gtaaccgagt 7500

agaagctcaa gctcatgcct ttggtataac ccaagtcacc aatgccaatg ccgccaacag 7560

caaagccgtg ctaaacgccc ttaaactcta atcaataagt ctaggaatca agccaaatgg 7620

cagaattaaa gaatgatcgt tatttacgcg ccctactaaa acagcctgtt gatatgaccc 7680

ctgtgtggat gatgcgtcaa gcgggccgtt atctccctga atataaagca actcgcgccc 7740

aggcgggtga ttttatgtct ttatgtaaaa accacgaatt ggcctgtgaa gtgacgctac 7800

aaccgctgcg tcgttacgag ctagatgcgg cgattctgtt ttccgacatt ctgacggttc 7860

ccgatgctat gggattaggt ctgtattttg aggcgggtga aggcccacgt tttgagcgcc 7920

cgacagacac tattgatgcc atcaaaaaat tatcaattcc tgatccagaa gatgagctgg 7980

gttatgtgat gaaggcggtg agtacaattc gtcgtgaatt gaatggtcaa gtgccattaa 8040

tcggcttctc aggttcacct tggacgctgg ccacttatat ggttgaaggc ggttcgagca 8100

aaaccttcga aaaaatcaaa aaaatggcct atgccgagcc tgctgcactg catatgttac 8160

tcgacaagtt agctgactca gtgactttat acctgaatgc gcaggtcgct aatggtgctc 8220

aatctttaat gattttcgac tcttggggcg gtgcattgtc gcatacagct taccgtgagt 8280

tttcactgcg ttatatgcag aagattgtcg atggtctgac tcgctttgcc gatggtcgcc 8340

aagtaccagt aaccctattc actaaaggtg gtggcttatg gttagaagcc atggcggaaa 8400

ccggttgtga cgccttaggt ttagactgga cggtggatat tgccgatgct cgtcgccgtg 8460

taggccataa agttgcgcta caaggcaaca tggacccatc tatgttatat gcacctatcc 8520

cacgcattga agaggaagtt ggtcaaatcc ttgcgggata cggtgaaggt acaggccacg 8580

tatttaactt aggccatggt attcaccagc atgtcgatcc agagcatgca ggcgccttta 8640

ttaaggcggt ccacgctcaa tctaagcaat accataagta actaatttac cctcggagga 8700

cacgccttga agcagcccac tcagataagc tgggatcagt cgatgatcga aaaatataac 8760

tacagcggtc cccgttatac ttcttatcca acggcgctag agttcgatga ttcattcact 8820

gaacaaaacc tgttaactgc gattgaaaac agcaagagtg acaaactgtc gctgtatatt 8880

cacatcccct tctgcgccaa actttgctat tactgtggct gcaataaagt catcactcgt 8940

cacgcccata aggccgacca atacattgag tatttaagcc acgaaattat taagcgcgcc 9000

ccactgttta agcattactc ggtcacccaa atgcactggg gcggcggcac accaaccttc 9060

ttaaatcctg agcaaattat taaacttacc gcattaatta aggctaactt taactttgcc 9120

gatgaaggtg agttctctat cgaagttgac ccgcgtgaaa ttgaactttc catgctcgac 9180

acccttaaag aagccggctt taaccgtatt tcgattggtg ttcaggactt caacaaagaa 9240

gtacaagtcg cggttaaccg tgagcaggac gagcaattta ttttcgattt aatggccaaa 9300

gccaaggcta tgggattcgt ctcaaccaat atcgatttaa tctacggctt gccacaccag 9360

actccagaga cattcgccgc caccatgcag cgcgtgttag atctgtcgcc cgatcgtcta 9420

tctgtattca actatgctca cttacccgcg cgttttgccg cgcagcgtaa aattaaagat 9480

gagcatttac cttcgccaaa acaaaagctt gagatgctgc atcaaactat cgagacctta 9540

actggcgcgg gttatcaata cattggtatg gaccacttcg cgaagcctga cgatgagctg 9600

gctaagttac aacgcgaagg caaactgcac cgcaacttcc agggttatac cacccaagaa 9660

gaatgcgatt tgcttggtct tggcgtgtcg tctatcagcc aaattggcga ttgctatgcg 9720

caaaaccaaa aggacattcg cccttactac gaagccatcg ataaagatgg ccatgcgctc 9780

tggaaaggct gcagcttgaa tcgtgacgac gaaatccgcc gcgtagtgat caaacaattg 9840

atctgccact tcgacttaga tatggccaaa atagatgaaa aactgggaat taagtttgag 9900

gaatacttcg ccgaagactt aaaactactg caaaccttta tcgatgataa attagttgaa 9960

gtcgccgaca gaaagatcac catcagcccc actggccgcc tgttgatccg caatatctgt 10020

atgtgcttcg acctttacta ccgtcaaaaa gcgcgtcaac aacagttctc tcgtgtgatc 10080

taactcgacc actcgaggcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg 10140

cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac 10200

cttcgggtgg gcctttctgc gtttataacc ggtaaaccag caatagacgg cgaacagaac 10260

tctattgaga ttttatccaa tttttgttga cactctatcg ttgatagagt tattttacca 10320

ctccctatca gtgatagaga aaagaattca aaaacgccaa ctttctggaa agattaatat 10380

gcaaacatta ataatctact caaccatcga cggtcaaacg ctagaaattt gtcgtaagat 10440

taaagcattt gctgagcggg cgggtgaaaa ggtctccctc tttagcttag aacaggctga 10500

agctataaac cttgcagatg ttgataaggt attgattggt gcgagtatcc gctatggtaa 10560

acatagacct gagctttacc aatttgtgaa tcgcaaccat gcggtgttaa gcgcgaaagt 10620

taatggtttt tttacggtaa atgtggtggc gcggaagccg ttaaagaaca ccccagaaac 10680

caatccttat atgcagaaat tcctcaaatt atccctttgg caaccacaac acttggcggt 10740

atttgctggt aagattgatt accctaaata tggcttattc gaccgcacaa tgatctgttt 10800

tatcatgtgg atgactaagg ggccaacgga tcttaagggc acgtttgagt ttaccgattg 10860

ggctaaagtc gaagcttttg gcactcattt tagtaagcta taatactttt actgaacgag 10920

aataccccat gacacgcatc ctggtgcttt attttacccg aggaggccat acggccaaga 10980

ttgccaatgc aatagcggag caattaaccc tgcgtggcgc aaaggttgac ttagtcgata 11040

tcaatagtgc ggctgcaaca cgaatcaact ggtccgatta tcagctggtg gctttgggtg 11100

cctgtgtact ctatggcact tatgataaga gtgtatttca atttattgag caacatgtgc 11160

aggctttgag ttttttgcca aacagttttt tctgcgtcaa cgtggtcgca cgtaatccgg 11220

agaaacgtat tcctgaaaac aataaatacc tgcaaaagtt tatcactcta tctccttgga 11280

ctcccgcaga tttgaagatt attgctggta aagtcgatta tccatcttgg ccttggtacg 11340

acagattaat gatccagtta attatgaaaa tcactaaagg cccaacggat ccaaaagcag 11400

ttattgatta taccgattgg gaagatgtga aagtctatgc cgaccatctg ctgaccttgg 11460

tcgaggttgc tgaacctgcc taattttatt gagataatcg aggcttaagt gaaaaaaatt 11520

ctgatcatct tttccagtgt tcatggccac actcgtaaaa tcaccaatca gttagctcag 11580

caactcaaag agttgggtaa ttcggtggtg attgcggata ttaaggctgt ccctgcgatg 11640

gagtctttcg ataagatcat tattggcgcg agtattcgtc acggaaaaca taatcctgcg 11700

ttgtacgaat ttatccaaaa gcatcaacag atcctcacgc aaaaggtcag tggctttttc 11760

tccgtcagtt tagtggctcg taaacccgag aaaaatactc cagaaaccaa cccatacatg 11820

caagcatttt taagcaaaac gacttggcgc cctaagttgc tacaagtgtt tggcggtaat 11880

ctgaattacc aaggttataa tgctttcgat agaaatatta ttcgttttat catgtggtta 11940

actaaggggc caacggatcc tgtcaccaat gttgaataca cagactggca aaaagtgcaa 12000

gagtttggtt tacaaatcca tcaagcctaa ctaggttttc aggattatca aaagaaatgg 12060

gtcacgctgc gcgtggcaaa gttggagtgc tgttattaaa ccttggcact cctgatgcgc 12120

caacagcatc ggcagtaagg cgttatcttg ccgagttttt atctgatcca cgggtggtgg 12180

aaatcccaaa actcctctgg atgctgattt tgtatggcat agtacttagg gtacgccctg 12240

caaagtctgc agcactttat caaaaggtgt ggactgaggc ggggtcgcca ctgatggata 12300

tcagtttgcg acaaacggct aagctatccg ataaattaac cgcggatggt catcaagttt 12360

cggttcactt agctatgcgt tatggtaatc cttctgttgc cagcacttta cgagagatgc 12420

acaaacaagg gattgataag ctggtggttt taccgctcta tccgcaatat gctgcgccga 12480

caactggctc ggcttttgac gctatcgcta aagagttatc ccaatggcgc tacctgccat 12540

cgctgcattt tattaatact tatcacgata accctgattt tattgctgca ttagtcaatt 12600

cgattcgtga cgattttgat aaacatggca agccgcaaaa gttagtgctg tcttaccatg 12660

gaatgcctga gcgcaatctt catctgggtg atccatatta ttgtttttgc atgaagacta 12720

ctcgtcttgt ggctgagcaa ttaggtttga gcaaagatga atttgcgatc acattccaat 12780

ctcgctttgg taaggccaag tggttgcaac cctatacgga tgcaacgatg gcggctctac 12840

cgagtcaagg tgtgcgtgat gtagcgattg tgtgcccagc atttagcgcc gattgtttag 12900

agacgctaga agagattgtc ggcgaaaatg gccatatctt tactcatgcg gggggagaga 12960

agttccgata tattcctgcg ctcaatgata atgatgacca catcgccatg atggcgaatt 13020

tggtgaagcc gtatctgtaa tatttggctg ataaaaggta gcacattgac ttctccctct 13080

cctgcgtttg gcgtgttatt agtaaatctt ggtacgcctg atgaacccac tccgaaagcg 13140

gttaagcgat ttctcaagca gtttttaagt gatcctcggg tcgtcgattt atccccttgg 13200

ttgtggcaac ccattttgca ggggattatc ctgaacaccc gtcctaagaa agtcgctaaa 13260

ctttatcaaa gtgtgtggac ggagcaaggt tcgccgttaa tggtgattag tcagtgccaa 13320

gcccaaaagt tggcaacgga tttaagcgcc acctttaatc agaccattcc ggtggaactg 13380

ggtatgagct atggcaatcc ttcgattgag agtggctttg ccaaactcaa agcccaaggc 13440

gccgaacgta tcgtggtgct gccgctgtat ccgcagtatt cctgctcaac cgtcgccagt 13500

gtgtttgatg cggtagcgca ttatttgact cgcgtgcgtg atatacctga gctgcgtttt 13560

aacaagcagt atttcgccca tgaagcctat attgcggcgc tggcgcattc ggtaaagcgc 13620

cattggaaaa cccatggtca ggccgagaag ctgattttat ctttccacgg gatcccgctg 13680

cgctacgcga ccgaaggcga cccataccca gagcagtgcc gcaccaccgc caagttatta 13740

gcgcaggcct taggcttgac cgacggacaa tggcaagtat gtttccaatc ccgcttcggt 13800

aaagaagagt ggttgacgcc ctatgccgat gagttgctgg cggatttacc ccgccaaggc 13860

gtgaaaagtg tcgatgtgat ttgccctgcc tttgctaccg attgccttga aaccctagaa 13920

gaaatctcga ttggcgcgaa agagaccttc ctccatgcag gtggcgaagc ctatcatttt 13980

attccttgtt tgaatgatga tgagctacat atagagctac tcaggttatt agtacaagaa 14040

caaacgcagt cttggataag tgcagaataa ctcgaccact cgaggccagg catcaaataa 14100

aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg 14160

ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataaccggt 14220

aaaccagcaa tagactgtca ggaaaatact gcgtttacta tcagcaattt ttgttgacac 14280

tctatcgttg atagagttat tttaccactc cctatcagtg atagagaaaa gaattcaaaa 14340

tattattaga gactgaggaa aacccatgat cccagaactt ggacactttt cgctgataat 14400

aggagtggct tttgccttct tattaaccag cgttcccctt ataggtgttg cccgtaaaga 14460

ccaatatcta gtgaggtatg cttggccgtt agcctacgga atgttctttt ttatcgcttt 14520

atccgtagta tcacttggtt acagcttcgc cgttgatgat ttctccgtgg cctatgtggc 14580

acatcactct aattctcagt tacctatctt ctttaagatt gcggccgtat ggggcggtca 14640

tgaagggtcg ctgctattct gggtgttcgc gctatccact tgggctgcct ctgttgcttt 14700

attcagtaaa ggcttagaag aagtcttcac cgctcgcgtg ttagcggtat tggcattgat 14760

tgtgattggc ttcagcttgt ttatgttact cacttctagc ccatttgaac gtatattccc 14820

aatgcctgct gaaggccgcg acttaaatcc aatgctgcag gatgtgggct taatcttcca 14880

ccctccgatg ttatatttgg gttatgtggg tttctcagtc agttttgcat ttgcgattgc 14940

agctttgatg agtggtcacc ttgattctgc ttgggctcgt tggtcgcgtc cttggacctt 15000

agccgcttgg gttttcctca ctggtggtat tgcattaggt tcttggtggg cgtattacga 15060

attaggctgg ggtggatggt ggttctggga tccagtagaa aacgcttcat ttatgccttg 15120

gctagtgggt acggctttag tgcattcact gatcgtgacg gaaaagcgcg gtgcattccg 15180

taactggacg gtattgctgt ctatcttcgc attctcgcta agtttactcg gtacctttat 15240

cgtacgctct ggtgtgttga cttcagtgca ttcatttgca gccgatccaa gccgcggtat 15300

gtttattcta ttattgcttg gtctcgctat cggtggttcg ctcactttgt ttgccttccg 15360

tgcaagtgaa atgagcagcc ctgcacgctt cgagctgaaa tcaaaagaaa ccatgctgtt 15420

agtttgtaac gtgttactga cggttgcagc gggtactgtt ctgttaggca ctttataccc 15480

actgctgatc gatgcgttag gcatgggtaa gatttctgtt ggacctccat actttaacgc 15540

tgtgtttgta cccatagtac ttgtactgtt tgcctttatg ggcgtcggtc caatcattcg 15600

ttggaaaaaa tcgaaagctg gtgaattgaa gcgacagtta ctcgtacctg cattagtatc 15660

tctggtgatt ggtattgtca cgccgtttat cgtcgatggg gcctttaacg cttgggttgc 15720

ttgcggtatc gccgctgcag cttggattat tttggcaacc gcaaaagcag cttacagcat 15780

cgttaagcct aaagatggtg aagtaagcat cgcacgtatg ggccgtagcc aactcggtat 15840

gatcatcgcg cacttaggta ttgctgtgtc tgttattggt gccactatgg tgtctaacta 15900

ctcagtggag aaaagtgtgc gtatggggcc tggcgtaagc caagagctag cgggttacac 15960

ctttaaatat ctcgaaacta aaaacgttgt tggtcctaat tacactgcgc agcaaggtca 16020

aattgagatt tacaaaggcg ataagttact gacactgctc aaacccgatc gtcgccaata 16080

caatgtgcgt actatggata tgaccgaagc cggtatcgat tggggtctgt tccgcgatct 16140

gtatgtgacg atgggcgatc cgattagtag tactgaattt gctgtgcgtt tgaactacaa 16200

gccgtttgtg cgttggttat ggttcggagc aatattcatg atggtcggcg gtttctttgc 16260

cgcatcggat aaacgctatc gctcaaaagt cgcggctacc gttaaaccgc aagctgaaaa 16320

agcgaaatta gctaccgctc aataactcga ccactcgagg ccaggcatca aataaaacga 16380

aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 16440

tactagagtc acactggctc accttcgggt gggcctttct gcgtttataa ccggtaaacc 16500

agcaatagac actatcggaa taaaggagtt cgcttacttc ctatttaacg accctgccct 16560

gaaccgacga ccgggtcgaa tttgctttcg aaccccagag tcccgctcag aagaactcgt 16620

caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga 16680

ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta 16740

tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc 16800

cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc 16860

cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct 16920

cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga 16980

tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc 17040

gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat 17100

cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga 17160

gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct 17220

gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg 17280

ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc 17340

cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca 17400

tgcgaaacga tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc 17460

ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg 17520

ccccagctgg caattccggt tcgcttgctg tccataaaac cgcccagtct agctatcgcc 17580

atgtaagccc actgcaagct acctgctttc tctttgcgct tgcgttttcc cttgtccaga 17640

tagcccagta gctgacattc atcccaggtg gcacttttcg gggaaatgtg cgcgcccgcg 17700

ttcctgctgg cgctgggcct gtttctggcg ctggacttcc cgctgttccg tcagcagctt 17760

ttcgcccacg gccttgatga tcgcggcggc cttggcctgc atatcccgat tcaacggccc 17820

cagggcgtcc agaacgggct tcaggcgctc ccgaagatct cgggccgtct cttgggcttg 17880

atcggccttc ttgcgcatct cacgcgctcc tgcggcggcc tgtagggcag gctcataccc 17940

ctgccgaacc gcttttgtca gccggtcggc cacggcttcc ggcgtctcaa cgcgctttga 18000

gattcccagc ttttcggcca atccctgcgg tgcataggcg cgtggctcga ccgcttgcgg 18060

gctgatggtg acgtggccca ctggtggccg ctccagggcc tcgtagaacg cctgaatgcg 18120

cgtgtgacgt gccttgctgc cctcgatgcc ccgttgcagc cctagatcgg ccacagcggc 18180

cgcaaacgtg gtctggtcgc gggtcatctg cgctttgttg ccgatgaact ccttggccga 18240

cagcctgccg tcctgcgtca gcggcaccac gaacgcggtc atgtgcgggc tggtttcgtc 18300

acggtggatg ctggccgtca cgatgcgatc cgccccgtac ttgtccgcca gccacttgtg 18360

cgccttctcg aagaacgccg cctgctgttc ttggctggcc gacttccacc attccgggct 18420

ggccgtcatg acgtactcga ccgccaacac agcgtccttg cgccgcttct ctggcagcaa 18480

ctcgcgcagt cggcccatcg cttcatcggt gctgctggcc gcccagtgct cgttctctgg 18540

cgtcctgctg gcgtcagcgt tgggcgtctc gcgctcgcgg taggcgtgct tgagactggc 18600

cgccacgttg cccattttcg ccagcttctt gcatcgcatg atcgcgtatg ccgccatgcc 18660

tgcccctccc ttttggtgtc caaccggctc gacgggggca gcgcaaggcg gtgcctccgg 18720

cgggccactc aatgcttgag tatactcact agactttgct tcgcaaagtc gtgaccgcct 18780

acggcggctg cggcgcccta cgggcttgct ctccgggctt cgccctgcgc ggtcgctgcg 18840

ctcccttgcc agcccgtgga tatgtggacg atggccgcga gcggccaccg gctggctcgc 18900

ttcgctcggc ccgtggacaa ccctgctgga caagctgatg gacaggctgc gcctgcccac 18960

gagcttgacc acagggattg cccaccggct acccagcctt cgaccacata cccaccggct 19020

ccaactgcgc ggcctgcggc cttgccccat caattttttt aattttctct ggggaaaagc 19080

ctccggcctg cggcctgcgc gcttcgcttg ccggttggac accaagtgga aggcgggtca 19140

aggctcgcgc agcgaccgcg cagcggcttg gccttgacgc gcctggaacg acccaagcct 19200

atgcgagtgg gggcagtcga aggcgaagcc cgcccgcctg ccccccgagc ctcacggcgg 19260

cgagtgcggg ggttccaagg gggcagcgcc accttgggca aggccgaagg ccgcgcagtc 19320

gatcaacaag ccccggaggg gccacttttt gccggagggg gagccgcgcc gaaggcgtgg 19380

gggaaccccg caggggtgcc cttctttggg caccaaagaa ctagatatag ggcgaaatgc 19440

gaaagactta aaaatcaaca acttaaaaaa ggggggtacg caacagctca ttgcggcacc 19500

ccccgcaata gctcattgcg taggttaaag aaaatctgta attgactgcc acttttacgc 19560

aacgcataat tgttgtcgcg ctgccgaaaa gttgcagctg attgcgcatg gtgccgcaac 19620

cgtgcggcac cctaccgcat ggagataagc atggccacgc agtccagaga aatcggcatt 19680

caagccaaga acaagcccgg tcactgggtg caaacggaac gcaaagcgca tgaggcgtgg 19740

gccgggctta ttgcgaggaa acccacggcg gcaatgctgc tgcatcacct cgtggcgcag 19800

atgggccacc agaacgccgt ggtggtcagc cagaagacac tttccaagct catcggacgt 19860

tctttgcgga cggtccaata cgcagtcaag gacttggtgg ccgagcgctg gatctccgtc 19920

gtgaagctca acggccccgg caccgtgtcg gcctacgtgg tcaatgaccg cgtggcgtgg 19980

ggccagcccc gcgaccagtt gcgcctgtcg gtgttcagtg ccgccgtggt ggttgatcac 20040

gacgaccagg acgaatcgct gttggggcat ggcgacctgc gccgcatccc gaccctgtat 20100

ccgggcgagc agcaactacc gaccggcccc ggcgaggagc cgcccagcca gcccggcatt 20160

ccgggcatgg aaccagacct gccagccttg accgaaacgg aggaatggga acggcgcggg 20220

cagcagcgcc tgccgatgcc cgatgagccg tgttttctgg acgatggcga gccgttggag 20280

ccgccgacac gggtcacgct gccgcgccgg tagcacttgg gttgcgcagc aacccgtaag 20340

tgcgctgttc cagactatcg gctgtagccg cctcgccgcc ctataccttg tctgcctccc 20400

cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcgg 20457

Claims (6)

1. A recombinant plasmid for highly producing heme, which is characterized by comprising the following genes: hemA, hemL, hemB1, hemB2, hemC, hemD, hemE, hemN, hemG1, hemG2, hemG3, hemH1, hemH2, and ccmf;

the source of hemA, hemL, hemB1, hemB2, hemC, hemD, hemE, hemN, hemG1, hemG2, hemG3, hemH1, hemH2 is E.coli MG1655;

the source of the ccmf is S. oneidensis MR-1.

2. The recombinant plasmid of claim 1, wherein the plasmid vector of the recombinant plasmid is pBF-ptoto;

the sequence of the plasmid vector is shown as SEQ ID NO:1 is shown.

3. The recombinant plasmid according to any one of claims 1 to 2, wherein the sequence of the recombinant plasmid is as set forth in SEQ ID NO:2, respectively.

4. A method for constructing the recombinant plasmid according to any one of claims 1 to 3, comprising the steps of:

taking a plasmid vector as a template, and amplifying to obtain a plasmid skeleton;

using E, coli MG1655 genome as template to amplify hemA, hemL, hemB, assembling the obtained DNA fragment and plasmid skeleton to obtain plasmid-HE 1;

using E, coli MG1655 genome as template to amplify hemC, hemD, hemE, hemN, assembling the obtained DNA fragment and plasmid skeleton to obtain plasmid-HE 2;

using E, coli MG1655 genome as template to amplify hemG1, hemG2, hemG3, hemH1, hemH2, and assembling the obtained DNA fragment and plasmid skeleton to obtain plasmid-HE 3;

amplifying ccmF by using an S, oneidensis MR-1 genome as a template, and assembling the obtained DNA fragment and a plasmid skeleton to obtain a plasmid-HE 4;

on the basis of obtaining the plasmid-HE 1, the plasmid-HE 2, the plasmid-HE 3 and the plasmid-HE 4, assembling HE1, HE2, HE3 and HE4DNA fragments to obtain recombinant plasmids.

5. A genetically engineered strain for high production of heme comprising the recombinant plasmid of any one of claims 1 to 3; the host bacterium of the genetic engineering strain is S. oneidensis MR-1; the promoter of the genetic engineering strain is a tetracycline inducible promoter system.

6. A method for producing heme with high yield is characterized by comprising the following steps:

culturing the genetically engineered strain of claim 5, wherein the OD of the bacterial liquid is ₆₀₀ Adding an inducer for fermentation culture when the concentration is 0.15 to 0.25, and collecting supernatant.

Images