CN117051028A - Method for constructing recombinant strain for stably synthesizing heme by using T7 expression system - Google Patents

Method for constructing recombinant strain for stably synthesizing heme by using T7 expression system Download PDF

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CN117051028A
CN117051028A CN202311053028.7A CN202311053028A CN117051028A CN 117051028 A CN117051028 A CN 117051028A CN 202311053028 A CN202311053028 A CN 202311053028A CN 117051028 A CN117051028 A CN 117051028A
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heme
t7rnap
plasmid
gene
yfex
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王文雅
史珂欣
黄建
唐艳斌
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Nutrition And Health Institute Chinese Center For Disease Control And Prevention
Beijing University of Chemical Technology
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Nutrition And Health Institute Chinese Center For Disease Control And Prevention
Beijing University of Chemical Technology
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Abstract

The invention belongs to the field of biological medicine, and relates to a method for constructing a recombinant strain for stably synthesizing heme by using a T7 expression system. Specifically, the present invention improves the stability of recombinant strains by modulating the activity of T7RNAP in host cells, thereby synthesizing heme in high yield.

Description

Method for constructing recombinant strain for stably synthesizing heme by using T7 expression system
Technical Field
The invention belongs to the field of biological medicine, relates to a microorganism with an RNA polymerase (namely T7 RNAP) transcription system of escherichia coli T7 phage, and in particular relates to a genetic engineering strain for producing heme by utilizing the T7RNAP system and a construction method thereof.
Background
Iron is a trace element essential for human body, and participates in physiological activities such as oxygen transportation, active oxygen quenching, electron transfer and the like, and is also a constituent of many biological macromolecules, which must be taken in from the outside, so that iron supplement is required for human body to strengthen iron nutrition. Common iron supplements can be divided into two types, heme iron supplements and non-heme iron supplements. Compared with non-heme iron supplements, heme iron supplements have high utilization efficiency, little interference by food components, and the heme iron supplement enhanced iron nutrition has no side effects of other iron supplements, such as constipation, gastrointestinal disorders, and the like. At present, heme iron supplements are mainly extracted from blood of slaughtered animals, and heme iron prepared by the method has risks of infecting zoonotic diseases, taking growth hormone and antibiotics for livestock and the like.
5-aminolevulinic acid (5-Aminolevulinic acid, ALA) is a precursor for heme biosynthesis, and studies have shown that ALA can promote heme iron accumulation, and that ALA is synthesized in organisms in two ways, namely C4 and C5. In recent decades, researchers have been trying to engineer microorganisms using genetic engineering techniques to ferment heme. In 2003, kwon et al constructed a recombinant escherichia coli strain producing hemE for the first time by overexpressing 7 genes in escherichia coli, which strain produced ALA using ALAS (hemA) from rhodobacter capsulatus (Rhodobacter capsulatus) to construct C4 pathway, while overexpressing PBGS (hemB), PBGD (hemC), UROS (hemD) and CPO (hemF) from escherichia coli, and FECH (hemH) from UROD (hemE) from synechocyssp) and bacillus subtilis (Bacillus subtilis), enhanced the metabolic pathway of ALA to hemE (see, non-patent document 1). In one study hereafter, the cells were isolated by over-expression from Rhodobacter sphaeroides (Rhodobacter sphaeroidesAloides), the maeB gene and the dctA gene endogenous to escherichia coli, using a complex medium supplemented with succinic acid, L-glycine and FeCl 3 The fermentation method is used for producing heme. ALA synthesis in the previous studies was all via the C4 pathway (see, non-patent document 2).
The system formed by the T7RNAP of the escherichia coli and a transcription unit started by the T7RNAP (transcription unit started by a T7 promoter) has high transcription activity which is 5 times of that of the escherichia coli RNA polymerase, has strong transcription persistence, and can be independently transcribed in any host in principle. Thus, the T7RNAP transcription system is widely applied to prokaryotes, eukaryotes and cell-free lines for transcription of synthetic RNA and expression of proteins. In E.coli, it has been reported that the protein synthesized by the T7RNAP system occupies more than 50% of the total intracellular protein. In 2018, korean Sang Yup Lee et al used a BL21 (DE 3) escherichia coli strain with a T7 expression system as a host strain, introduced a C5 pathway to synthesize ALA, deleted genes in escherichia coli that might degrade heme, attempted to secrete heme outside the cell, and produced heme by fermentation (see, non-patent document 3).
The high transcriptional activity of the T7 expression system also has the disadvantage that large amounts of synthetic RNA and proteins consume intracellular resources dramatically, causing biotic stress on host cells and, in severe cases, causing death of the host. The host also gives feedback to the biotic stress of the T7 expression system, such as adjusting its physiological metabolism, generating genetic mutation (mutation occurring for the production of the target product), etc., resulting in a large change in genetically engineered bacteria constructed based on the T7 expression system and progeny strains produced therefrom, for example, unstable strain properties, etc., affecting the yield of the target product (see non-patent document 4).
Prior art literature:
non-patent document 1: kwon S J, de Boe AL, petri R, et AL high-level production of porphyrins in metabolically engineered Escherichia coli: systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis.applied and Environmental Microbiology,2003,69 (8): 4875-4883.
Non-patent document 2: kwon O H, kim S, hahm D H, et al potential application of the recombinant Escherichia coli-synthesized heme as a bioavailable iron source journal of Microbiology and Biotechnology,2009,19 (6): 604-609.
Non-patent document 3: zhao X R, choi K R, lee S Y.Metabolic engineering of Escherichia coli for secretory production of free haem.Nature Catalysis,2018,1 (9): 720-728.
Non-patent document 4: wang, w., li, y, wang, y, shi, c, li, q, linhardt, r.j. Bacteriophage T7transcription system: an enabling tool in synthetic biotechnology advance.2018, 36 (8): 2129-2137.
Disclosure of Invention
Problems to be solved by the invention
As is clear from the above-described studies in the art, T7 expression systems have the characteristics of high transcriptional activity and high transcriptional persistence, but have the problem that recombinant strains are unstable and heme cannot be stably synthesized in high yield.
Solution for solving the problem
The invention improves the stability of the recombinant strain by regulating the activity of T7RNAP in the host cell, thereby achieving the purpose of improving the yield of heme.
In particular, the method of the invention comprises the following scheme.
[1] A method for constructing a recombinant strain for stably synthesizing heme using a T7 expression system, comprising the steps of:
1) Constructing an integration vector for expressing T7RNAP,
2) Constructing recombinant strain expressing T7RNAP to synthesize heme,
wherein the recombinant strain of stable synthetic heme is obtained by modulating T7RNAP activity.
[2] The method according to [1], wherein the activity of T7RNAP is regulated by altering the sequence of the ribosome binding site in the DNA sequence of T7 RNAP.
[3] The method according to [1] or [2], wherein a DNA sequence expressing T7RNAP is inserted into the position where the yfeX gene is located, while deleting the yfeX gene.
[4] The method according to [3], wherein in step 2), the DNA sequence expressing T7RNAP for insertion is amplified, a plasmid vector for deleting the target yfeX gene is constructed, an electrotransformation competent cell is prepared, the DNA sequence expressing T7RNAP is inserted and the target yfeX gene is deleted, the recombinant strain is obtained, and the plasmid in the recombinant strain is eliminated.
[5] The method according to [3] or [4], wherein the DNA sequence for inserting and expressing T7RNAP while deleting the yfeX gene comprises a sequence selected from the group consisting of SEQ ID No: 2. SEQ ID No: 3. SEQ ID No:4, and any one of the following.
[6] The method according to [4], wherein the sequence of the plasmid vector constructed to delete the target yfeX gene is SEQ ID NO: 10.
[7] The method according to any one of [1] to [6], wherein the method comprises constructing a plasmid vector that enhances the heme synthesis pathway.
[8] The method according to [7], wherein the heme synthesis pathway is constructed by dividing into three plasmids:
a first plasmid, from alpha-ketoglutarate, into which aminolevulinic acid is synthesized, and in which this pathway is constructed;
a second plasmid from which uroporphyrinogen III is formed, this pathway being constructed in the second plasmid;
a third plasmid for converting uroporphyrinogen III into protoporphyrin IX via successive decarboxylation and oxidation reactions via coproporphyrinogen III and protoporphyrinogen IX, allowing the ferrochelatase to catalyze Fe 2+ Chelate protoporphyrin IX to form heme, and this pathway is constructed in the third plasmid.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention utilizes a T7 expression system to construct a recombinant strain, and synthesizes heme stably and in high yield by regulating the activity of T7RNAP in a host cell. The method is suitable for large-scale industrialized production of heme.
Drawings
FIG. 1 is a map of LT7P plasmid.
FIG. 2 is a map of MT7P plasmid.
FIG. 3 is a map of HT7P plasmid.
FIG. 4 is a map of pEcgRNA-yfeX plasmid.
FIG. 5 shows T7RNAP activity of different strains.
FIG. 6 is a map of the p1 plasmid.
FIG. 7 is a map of the p2 plasmid.
FIG. 8 is a map of the p3 plasmid.
FIG. 9 shows heme production and T7RNAP activity of different recombinant strains of synthetic heme constructed using the T7 expression system.
Detailed Description
The present invention will be described in detail below.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the nomenclature used herein and the laboratory procedures are well known and commonly used in the art.
The term "instability" refers to a rapid increase in the proportion of cells having a mutation in a DNA sequence and cells having a large negative change in physiological/biochemical state in a host population in a short period of time by using a T7 expression system to synthesize a target product, and a decrease in the ability of the cell population to synthesize the target product, or even an inability to synthesize the target product.
"mutation" refers to a change in the DNA base associated with the T7 expression system and the synthesis of the target product, or other mutation due to the high transcriptional activity of T7 RNAP. This mutation reduces the ability of the cell to synthesize the target product, even without synthesis.
By "negative change in physiological/biochemical state" is meant a change in physiological/biochemical state that results in a decrease in the ability of the cell to synthesize the target product, or even in the inability to synthesize the target product, which negative change is caused by a biotic stress induced by high transcriptional activity of T7 RNAP.
The invention discloses a method for improving the stability of a recombinant strain for synthesizing heme by regulating the activity of T7RNAP, thereby improving the yield of heme. The method comprises two steps, 1) constructing an integration vector expressing T7 RNAP; 2) Recombinant strains expressing T7RNAP were constructed to synthesize heme. Preferably, the T7RNAP protein expression level is regulated by changing the sequence of the ribosome binding site (riboname-binding sequence), so as to achieve the purpose of regulating the activity of T7 RNAP.
The present invention may use a biological source capable of producing heme from a carbon source, but is not limited thereto. Biological sources that may be used in the present invention include bacteria, archaebacteria, yeasts, fungi, animal cells and plant cells, preferably, but not limited to, escherichia coli (Escherichia coli), bacillus species (Bacillus sp.), corynebacterium species (Corynebacterium sp.), lactobacillus species (Lactobacillus sp.), and the like.
The recombinant strain of the invention utilizes the C5 pathway through L-glutamic acid to synthesize heme, but the invention is also applicable to the C4 heme synthesis pathway involving L-glycine and succinic acid.
Genes involved in the C5 biosynthetic pathway may be selected from gltX encoding glutamyl-tRNA synthase (GluRS), hemA encoding glutamyl-tRNA reductase (GluTR), and hemL encoding glutamate-1-semialdehyde 2, 1-aminomutase (GSAM). Genes involved in the pathway of hemE synthesis from ALA may be selected from hemB encoding porphobilinogen synthase (PBGS), hemC encoding porphobilinogen deaminase (PBGD), hemD encoding uroporphyrinogen III synthase (UROS), hemE encoding uroporphyrinogen III decarboxylase (UROD), hemF encoding coproporphyrinogen III oxidase (CPO), hemG encoding protoporphyrinogen oxidase (PPO), and hemH encoding Ferrochelatase (FECH).
The recombinant strain of the present invention deletes the gene encoding heme degrading enzyme or heme intermediate degrading enzyme, preferably, the gene is yfeX gene having the sequence shown in SEQ ID No: 1. The protein expressed by yfeX gene is the removal of Fe from heme 2+ Ionic heme dechelating enzymes or porphyrinsPeroxidase converted to porphyrin. Overexpression of the yfeX gene disrupts heme homeostasis, and therefore deletion of the yfeX gene increases heme production. In the invention, when the integrated vector for expressing the activity of the T7RNAP is constructed in the step 1), the DNA sequence for expressing the T7RNAP can be inserted into the position of the yfeX gene, and the yfeX gene can be deleted. The DNA sequence expressing T7RNAP may comprise a sequence identical to SEQ ID No: 2. SEQ ID No:3 or SEQ ID No:4, preferably, the homology is 95% or more, more preferably 99% or more, most preferably, the sequence having 90% or more homology, most preferably, the sequence comprising SEQ ID No: 2. SEQ ID No:3 or SEQ ID No: 4.
SEQ ID No：1：
ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGG
GAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCTGAAG
AGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACCACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTGGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACAGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCCGGCGGCAGCTATGTGTTTGTGCAGCGTTGGGAGCACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGATGATCGGGCGCACCAAAGAGGCCAACGAAGAGATTGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACCGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA
SEQ ID No：2：
GCCCCTGCAGCCGAATTATATTATTTTTGCCAAATAATTTTTAACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATGATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATCACGCTGTGAGTAAGTTCTAAACCATTTTTTTATTGTTGTATTATCTCTAATCTTACTACTCGATGAGTTTTCGGTATTATCTCTATTTTTAACTTGGAGCAGGTTCCATTCATTGTTTTTTTCATCATAGTGAATAAAATCAACTGCTTTAACACTTGTGCCTGAACACCATATCCATCCGGCGTAATACGACTCACTATAGGGAGAGCGGCCGCCAGATCTTCCGGATGGCTCGAGTTTTTCAGCAAGATGCACAGTGTATTACGCAGTCAGGTACGCGGGGCGATTATTCCCGGCCTGCTGGGCGTTGGCGAACCGTTGATTTATGGCGTCACCCTGCCCCGCATGAAACCGTTTATTACCGCCTGTTTAGGCGGCGCGGCGGGCGGTTTGTTTATCGGATTGATAGCCTGGTGGGGTCTGCCGATGGGCTTAAACAGCGCCTTTGGGCCGTCTGGTCTGGTGGCGCTGCCGCTGATGACTTCCGCACAAGGCATCCTTCCGGCAATGGCGGTTTATGCTGGCGGGATTCTGGTGGCATGGGTTAGTGGGTTTATTTTCACCACGCTCTTTGGCTGCCGTAACGTCAATCTGGACTGATATCATGAAACGGACAATGCTCTATCTTTCTCTGCTGGCTGTTAGTTGTAGCGTCAGCGCCGCAAAATACCCTGTTCTGATAGAAGGTAGTTGATCAAGTATATGCGGCGTTAAAGCAGTAATAAAAAGGCCAGTCAGTAATGTTACTGCCTGGCCTTTAATCGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGACATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTAACATGAGAATTACAACTTATATCGTATGGGGCTGACTTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAACAGGTTTCATTAAAAACGCGCATCAAAAACCTAAATGAACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTCAACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGTTGGCCCTTGAGCATGAGTCTTACGAGATGGGTGAAGCACGCTTCCGCAAGATGTTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACGCTGCCGCCAAGCCTCTCATCACTACCCTACTCCCTAAGATGATTGCACGCATCAACGACTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTCCAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATTAAGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAGCGCAATCGGTCGGGCCATTGAGGACGAGGCTCGCTTCGGTCGTATCCGTGACCTTGAAGCTAAGCACTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTACAAGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTGGCGAGGCGTGGTCTTCGTGGCATAAGGAAGACTCTATTCATGTAGGAGTACGCTGCATCGAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGCTGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATACGCTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGATGTTCCAACCTTGCGTAGTTCCTCCTAAGCCGTGGACTGGCATTACTGGTGGTGGCTATTGGGCTAACGGTCGTCGTCCTCTGGCGCTGGTGCGTACTCACAGTAAGAAAGCACTGATGCGCTACGAAGACGTTTACATGCCTGAGGTGTACAAAGCGATTAACATTGCGCAAAACACCGCATGGAAAATCAACAAGAAAGTCCTAGCGGTCGCCAACGTAATCACCAAGTGGAAGCATTGTCCGGTCGAGGACATCCCTGCGATTGAGCGTGAAGAACTCCCGATGAAACCGGAAGACATCGACATGAATCCTGAGGCTCTCACCGCGTGGAAACGTGCTGCCGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTCTCGCCGTATCAGCCTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCATAAGGCCATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGTGTCAATGTTCAACCCGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGGCGAAAGGTAAACCAATCGGTAAGGAAGGTTACTACTGGCTGAAAATCCACGGTGCAAACTGTGCGGGTGTCGATAAGGTTCCGTTCCCTGAGCGCATCAAGTTCATTGAGGAAAACCACGAGAACATCATGGCTTGCGCTAAGTCTCCACTGGAGAACACTTGGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCTTGCGTTCTGCTTTGAGTACGCTGGGGTACAGCACCACGGCCTGAGCTATAACTGCTCCCTTCCGCTGGCGTTTGACGGGTCTTGCTCTGGCATCCAGCACTTCTCCGCGATGCTCCGAGATGAGGTAGGTGGTCGCGCGGTTAACTTGCTTCCTAGTGAAACCGTTCAGGACATCTACGGGATTGTTGCTAAGAAAGTCAACGAGATTCTACAAGCAGACGCAATCAATGGGACCGATAACGAAGTAGTTACCGTGACCGATGAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAGAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACCATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCAATCCGTGGCATTGTTGAGCGCTATTTTACGTGTTTTTTCGCGGCGGCGGTTTGTTCAGACGCAAATTAACGCCGCCAGATAATTTTACTGACTTTCCAGTTTTTCAGGGTATCGTCCGAAGGCATTAACCCTTCAGGACCACGCCATTCCCCGCTGAACTGGTAGCTAATATGTTGACTGCCTTCGGCTTTACATTCGACGGCACGATTATCATCACCATCAGCTTTTTGGCAATTGCCAAATGCTTTGCTGTAAAGGTCGCTAAACGGTGTACCGATTTTAACACCGGTGTCAGCAGGAATATCGCTATCCAGCACGTCAATGCGGCTGATCGTGCCTTGCTCGCCGTTAATCACCATCGCCACATTGTCGTCTTTCATCACTTCAAAAAAGCGCACCACGTTGCCGTTCGCGGTTTTCATTCCGCTGCGCAGGCGATAATCGCCATCAAGCGCATCAGCAATGGCTTGTTCTTGCAGTGGTGTGGACGCCGTTAATTCACATCTTTCTAGAAGATCTCCTACAATATTCTCAGCTGCCATGGAAAATCGATGTTCTTCTTTTATTCTCTCAAGATTTTCAGGCTGTATATTAAAACTTATATTAAGAACTATGCTAACCACCTCATCAGGAACCGTTGTAGGTGGCGTGGGTTTTCTTGGCAATCGACTCTCATGAAAACTACGAGCTAAATATTCAATATGTTCCTCTTGACCAACTTTATTCTGCATTTTTTTTGAACGAGGTTTAGAGCAAGCTTCAGGAAACTGAGACAGGAATTTTATTAAAAATTTAAATTTTGAAGAAAGTTCAGGGTTAATAGCATCCATTTTTTGCTTTGCAAGTTCCTCAGCATTCTTAACAAAAGACGTCTCTTTTGACATGTTTAAAGTTTAAACCTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCAATTGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCC
SEQ ID No：3
GCCCCTGCAGCCGAATTATATTATTTTTGCCAAATAATTTTTAACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATGATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATCACGCTGTGAGTAAGTTCTAAACCATTTTTTTATTGTTGTATTATCTCTAATCTTACTACTCGATGAGTTTTCGGTATTATCTCTATTTTTAACTTGGAGCAGGTTCCATTCATTGTTTTTTTCATCATAGTGAATAAAATCAACTGCTTTAACACTTGTGCCTGAACACCATATCCATCCGGCGTAATACGACTCACTATAGGGAGAGCGGCCGCCAGATCTTCCGGATGGCTCGAGTTTTTCAGCAAGATGCACAGTGTATTACGCAGTCAGGTACGCGGGGCGATTATTCCCGGCCTGCTGGGCGTTGGCGAACCGTTGATTTATGGCGTCACCCTGCCCCGCATGAAACCGTTTATTACCGCCTGTTTAGGCGGCGCGGCGGGCGGTTTGTTTATCGGATTGATAGCCTGGTGGGGTCTGCCGATGGGCTTAAACAGCGCCTTTGGGCCGTCTGGTCTGGTGGCGCTGCCGCTGATGACTTCCGCACAAGGCATCCTTCCGGCAATGGCGGTTTATGCTGGCGGGATTCTGGTGGCATGGGTTAGTGGGTTTATTTTCACCACGCTCTTTGGCTGCCGTAACGTCAATCTGGACTGATATCATGAAACGGACAATGCTCTATCTTTCTCTGCTGGCTGTTAGTTGTAGCGTCAGCGCCGCAAAATACCCTGTTCTGATAGAAGGTAGTTGATCAAGTATATGCGGCGTTAAAGCAGTAATAAAAAGGCCAGTCAGTAATGTTACTGCCTGGCCTTTAATCGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGACATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTAACATGAGAATTACAACTTATATCGTATGGGGCTGACTTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAAGCATATTAAACCTTTACCCAACGTTAGGTAAATAATGAACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTCAACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGTTGGCCCTTGAGCATGAGTCTTACGAGATGGGTGAAGCACGCTTCCGCAAGATGTTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACGCTGCCGCCAAGCCTCTCATCACTACCCTACTCCCTAAGATGATTGCACGCATCAACGACTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTCCAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATTAAGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAGCGCAATCGGTCGGGCCATTGAGGACGAGGCTCGCTTCGGTCGTATCCGTGACCTTGAAGCTAAGCACTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTACAAGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTGGCGAGGCGTGGTCTTCGTGGCATAAGGAAGACTCTATTCATGTAGGAGTACGCTGCATCGAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGCTGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATACGCTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGATGTTCCAACCTTGCGTAGTTCCTCCTAAGCCGTGGACTGGCATTACTGGTGGTGGCTATTGGGCTAACGGTCGTCGTCCTCTGGCGCTGGTGCGTACTCACAGTAAGAAAGCACTGATGCGCTACGAAGACGTTTACATGCCTGAGGTGTACAAAGCGATTAACATTGCGCAAAACACCGCATGGAAAATCAACAAGAAAGTCCTAGCGGTCGCCAACGTAATCACCAAGTGGAAGCATTGTCCGGTCGAGGACATCCCTGCGATTGAGCGTGAAGAACTCCCGATGAAACCGGAAGACATCGACATGAATCCTGAGGCTCTCACCGCGTGGAAACGTGCTGCCGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTCTCGCCGTATCAGCCTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCATAAGGCCATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGTGTCAATGTTCAACCCGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGGCGAAAGGTAAACCAATCGGTAAGGAAGGTTACTACTGGCTGAAAATCCACGGTGCAAACTGTGCGGGTGTCGATAAGGTTCCGTTCCCTGAGCGCATCAAGTTCATTGAGGAAAACCACGAGAACATCATGGCTTGCGCTAAGTCTCCACTGGAGAACACTTGGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCTTGCGTTCTGCTTTGAGTACGCTGGGGTACAGCACCACGGCCTGAGCTATAACTGCTCCCTTCCGCTGGCGTTTGACGGGTCTTGCTCTGGCATCCAGCACTTCTCCGCGATGCTCCGAGATGAGGTAGGTGGTCGCGCGGTTAACTTGCTTCCTAGTGAAACCGTTCAGGACATCTACGGGATTGTTGCTAAGAAAGTCAACGAGATTCTACAAGCAGACGCAATCAATGGGACCGATAACGAAGTAGTTACCGTGACCGATGAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAGAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACCATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCAATCCGTGGCATTGTTGAGCGCTATTTTACGTGTTTTTTCGCGGCGGCGGTTTGTTCAGACGCAAATTAACGCCGCCAGATAATTTTACTGACTTTCCAGTTTTTCAGGGTATCGTCCGAAGGCATTAACCCTTCAGGACCACGCCATTCCCCGCTGAACTGGTAGCTAATATGTTGACTGCCTTCGGCTTTACATTCGACGGCACGATTATCATCACCATCAGCTTTTTGGCAATTGCCAAATGCTTTGCTGTAAAGGTCGCTAAACGGTGTACCGATTTTAACACCGGTGTCAGCAGGAATATCGCTATCCAGCACGTCAATGCGGCTGATCGTGCCTTGCTCGCCGTTAATCACCATCGCCACATTGTCGTCTTTCATCACTTCAAAAAAGCGCACCACGTTGCCGTTCGCGGTTTTCATTCCGCTGCGCAGGCGATAATCGCCATCAAGCGCATCAGCAATGGCTTGTTCTTGCAGTGGTGTGGACGCCGTTAATTCACATCTTTCTAGAAGATCTCCTACAATATTCTCAGCTGCCATGGAAAATCGATGTTCTTCTTTTATTCTCTCAAGATTTTCAGGCTGTATATTAAAACTTATATTAAGAACTATGCTAACCACCTCATCAGGAACCGTTGTAGGTGGCGTGGGTTTTCTTGGCAATCGACTCTCATGAAAACTACGAGCTAAATATTCAATATGTTCCTCTTGACCAACTTTATTCTGCATTTTTTTTGAACGAGGTTTAGAGCAAGCTTCAGGAAACTGAGACAGGAATTTTATTAAAAATTTAAATTTTGAAGAAAGTTCAGGGTTAATAGCATCCATTTTTTGCTTTGCAAGTTCCTCAGCATTCTTAACAAAAGACGTCTCTTTTGACATGTTTAAAGTTTAAACCTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCAATTGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCC
SEQ ID No：4：GCCCCTGCAGCCGAATTATATTATTTTTGCCAAATAATTTTTAACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATGATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATCACGCTGTGAGTAAGTTCTAAACCATTTTTTTATTGTTGTATTATCTCTAATCTTACTACTCGATGAGTTTTCGGTATTATCTCTATTTTTAACTTGGAGCAGGTTCCATTCATTGTTTTTTTCATCATAGTGAATAAAATCAACTGCTTTAACACTTGTGCCTGAACACCATATCCATCCGGCGTAATACGACTCACTATAGGGAGAGCGGCCGCCAGATCTTCCGGATGGCTCGAGTTTTTCAGCAAGATGCACAGTGTATTACGCAGTCAGGTACGCGGGGCGATTATTCCCGGCCTGCTGGGCGTTGGCGAACCGTTGATTTATGGCGTCACCCTGCCCCGCATGAAACCGTTTATTACCGCCTGTTTAGGCGGCGCGGCGGGCGGTTTGTTTATCGGATTGATAGCCTGGTGGGGTCTGCCGATGGGCTTAAACAGCGCCTTTGGGCCGTCTGGTCTGGTGGCGCTGCCGCTGATGACTTCCGCACAAGGCATCCTTCCGGCAATGGCGGTTTATGCTGGCGGGATTCTGGTGGCATGGGTTAGTGGGTTTATTTTCACCACGCTCTTTGGCTGCCGTAACGTCAATCTGGACTGATATCATGAAACGGACAATGCTCTATCTTTCTCTGCTGGCTGTTAGTTGTAGCGTCAGCGCCGCAAAATACCCTGTTCTGATAGAAGGTAGTTGATCAAGTATATGCGGCGTTAAAGCAGTAATAAAAAGGCCAGTCAGTAATGTTACTGCCTGGCCTTTAATCGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGACATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTAACATGAGAATTACAACTTATATCGTATGGGGCTGACTTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAACACCCCCCCTCTCCCACTATTAAGGACATCAACAATGAACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTCAACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGTTGGCCCTTGAGCATGAGTCTTACGAGATGGGTGAAGCACGCTTCCGCAAGATGTTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACGCTGCCGCCAAGCCTCTCATCACTACCCTACTCCCTAAGATGATTGCACGCATCAACGACTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTCCAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATTAAGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAGCGCAATCGGTCGGGCCATTGAGGACGAGGCTCGCTTCGGTCGTATCCGTGACCTTGAAGCTAAGCACTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTACAAGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTGGCGAGGCGTGGTCTTCGTGGCATAAGGAAGACTCTATTCATGTAGGAGTACGCTGCATCGAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGCTGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATACGCTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGATGTTCCAACCTTGCGTAGTTCCTCCTAAGCCGTGGACTGGCATTACTGGTGGTGGCTATTGGGCTAACGGTCGTCGTCCTCTGGCGCTGGTGCGTACTCACAGTAAGAAAGCACTGATGCGCTACGAAGACGTTTACATGCCTGAGGTGTACAAAGCGATTAACATTGCGCAAAACACCGCATGGAAAATCAACAAGAAAGTCCTAGCGGTCGCCAACGTAATCACCAAGTGGAAGCATTGTCCGGTCGAGGACATCCCTGCGATTGAGCGTGAAGAACTCCCGATGAAACCGGAAGACATCGACATGAATCCTGAGGCTCTCACCGCGTGGAAACGTGCTGCCGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTCTCGCCGTATCAGCCTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCATAAGGCCATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGTGTCAATGTTCAACCCGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGGCGAAAGGTAAACCAATCGGTAAGGAAGGTTACTACTGGCTGAAAATCCACGGTGCAAACTGTGCGGGTGTCGATAAGGTTCCGTTCCCTGAGCGCATCAAGTTCATTGAGGAAAACCACGAGAACATCATGGCTTGCGCTAAGTCTCCACTGGAGAACACTTGGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCTTGCGTTCTGCTTTGAGTACGCTGGGGTACAGCACCACGGCCTGAGCTATAACTGCTCCCTTCCGCTGGCGTTTGACGGGTCTTGCTCTGGCATCCAGCACTTCTCCGCGATGCTCCGAGATGAGGTAGGTGGTCGCGCGGTTAACTTGCTTCCTAGTGAAACCGTTCAGGACATCTACGGGATTGTTGCTAAGAAAGTCAACGAGATTCTACAAGCAGACGCAATCAATGGGACCGATAACGAAGTAGTTACCGTGACCGATGAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAGAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACCATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCAATCCGTGGCATTGTTGAGCGCTATTTTACGTGTTTTTTCGCGGCGGCGGTTTGTTCAGACGCAAATTAACGCCGCCAGATAATTTTACTGACTTTCCAGTTTTTCAGGGTATCGTCCGAAGGCATTAACCCTTCAGGACCACGCCATTCCCCGCTGAACTGGTAGCTAATATGTTGACTGCCTTCGGCTTTACATTCGACGGCACGATTATCATCACCATCAGCTTTTTGGCAATTGCCAAATGCTTTGCTGTAAAGGTCGCTAAACGGTGTACCGATTTTAACACCGGTGTCAGCAGGAATATCGCTATCCAGCACGTCAATGCGGCTGATCGTGCCTTGCTCGCCGTTAATCACCATCGCCACATTGTCGTCTTTCATCACTTCAAAAAAGCGCACCACGTTGCCGTTCGCGGTTTTCATTCCGCTGCGCAGGCGATAATCGCCATCAAGCGCATCAGCAATGGCTTGTTCTTGCAGTGGTGTGGACGCCGTTAATTCACATCTTTCTAGAAGATCTCCTACAATATTCTCAGCTGCCATGGAAAATCGATGTTCTTCTTTTATTCTCTCAAGATTTTCAGGCTGTATATTAAAACTTATATTAAGAACTATGCTAACCACCTCATCAGGAACCGTTGTAGGTGGCGTGGGTTTTCTTGGCAATCGACTCTCATGAAAACTACGAGCTAAATATTCAATATGTTCCTCTTGACCAACTTTATTCTGCATTTTTTTTGAACGAGGTTTAGAGCAAGCTTCAGGAAACTGAGACAGGAATTTTATTAAAAATTTAAATTTTGAAGAAAGTTCAGGGTTAATAGCATCCATTTTTTGCTTTGCAAGTTCCTCAGCATTCTTAACAAAAGACGTCTCTTTTGACATGTTTAAAGTTTAAACCTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCAATTGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCC
In the step 2) of constructing a recombinant strain expressing T7RNAP activity according to the present invention, preferably, a DNA sequence expressing T7RNAP for insertion is amplified while deleting the yfeX gene, a plasmid vector for deleting a target yfeX gene is constructed, an electrotransformation competent cell is prepared, a target fragment of the DNA sequence expressing T7RNAP is inserted and the target yfeX gene is deleted, to obtain the recombinant strain, and plasmids in the recombinant strain are deleted.
The method comprises the steps of constructing a plasmid vector for strengthening a heme synthesis path, wherein the heme synthesis path can be constructed by dividing into three plasmids:
a first plasmid, from alpha-ketoglutarate, into which aminolevulinic acid is synthesized, and in which this pathway is constructed;
a second plasmid, from 5-aminolevulinic acid, forming uroporphyrinogen III, in which the pathway is constructed;
a third plasmid for converting uroporphyrinogen III into protoporphyrin IX via successive decarboxylation and oxidation reactions via coproporphyrinogen III and protoporphyrinogen IX, allowing the ferrochelatase to catalyze Fe 2+ Chelate protoporphyrin IX to form heme, and this pathway is constructed in the third plasmid.
In the above method, the 5-aminolevulinic acid is synthesized from the α -ketoglutarate by catalysis of a glutamyl-tRNA synthetase, a glutamyl-tRNA reductase, and a glutamate semialdehyde aminotransferase. Starting from the 5-aminolevulinic acid, 5-aminolevulinic acid dehydrogenase condenses 2 molecules of 5-aminolevulinic acid to form porphobilinogen, porphobilinogen deaminase condenses four molecules of porphobilinogen to hydroxymethyl bilin, and uroporphyrinogen synthase cyclizes the hydroxymethyl bilin to form uroporphyrinogen III. Enzymes involved in the construction of the third plasmid are uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and protoporphyrinogen oxidase.
The present invention can be carried out by the following experimental materials, reagents, detection methods, etc., but the present invention is not limited thereto.
< Experimental Material >
Plasmid vector: pJet1.2/blunt (Thermofisher), pETDuet-1 (Novagen), pEcCas9 (Addgene), pEcgRNA (Addgene)
Strains: e.coli DH 5. Alpha. And E.coli BL21
DNA sequence: the complete gene sequence of E.coli BL21 (No.: CP 110816) can be obtained from Genebank
Culture medium: LB medium (NaCl 1%, tryptone 1%, yeast extract 0.5%)
< reagent >
FeSO 4 .7H 2 O, glucose, fructose, all purchased from Shanghai Michael chemical technologies Co. IPTG (Isopropyl beta-D-thiogalactopyranoside), kanamycin, ampicillin and spectinomycin were all purchased from Beijing Soy Corp technology Co. DNA ligase, DNA polymerase for PCR was purchased from Nanjinouzan Biotechnology Co., ltd. PCR primer synthesis and DNA sequence determination were performed by Jin Weizhi Biotechnology Inc., and gene sequence synthesis was performed by nine days Gene technology (Tianjin) Inc. Other restriction enzymes and molecular biology enzyme preparations were purchased from New England Biolabs.
The conventional methods of constructing plasmids and vectors, expressing proteins, transforming cells, and the like, according to the present invention, may be referred to methods of molecular biology and genetics known in the art, and for example, the corresponding methods described in publications such as "the latest molecular biology experimental methods assembly (Current Protocols in Molecular Biology, wiley publication)", "the molecular cloning experimental guidelines (Molecular Cloning: A Laboratory Manual, cold spring harbor laboratory publication)", and the like, may be referred to.
< detection of heme content by HPLC >
Mobile phase: a:0.1% (v/v) trifluoroacetic acid, B: methanol/acetonitrile=1/9 (v/v)
C18 reverse column:C18(2)5μ，4.6mm×250mm
program elution: the flow rate of the mobile phase at 40 ℃ and 400nm is 1ml/min, and the mobile phase B is 5% and the mobile phase is 0-5min;5% -100% of mobile phase B,5-35min;100% mobile phase B,35-40min; and 5% of mobile phase B, and carrying out mixed elution of mobile phases A and B in the sequence of 40-50 min.
And finally, manufacturing a standard curve by using a heme standard sample, and quantitatively detecting the heme content through the standard curve.
The present invention is further explained below with reference to examples, which are given for the purpose of illustration only, and the present invention is not limited thereto.
Example 1: construction of BL21 strains with different T7RNAP Activity
1.1 construction of integration vectors expressing different T7RNAP Activity
The yfeX gene codes heme degrading enzyme in BL21 strain, so that DNA sequences expressing different T7 RNAPs are inserted into the position of the yfeX gene, and simultaneously the yfeX gene is deleted, and the sequences of the yfeX gene are shown as SEQ ID No: 1. The constructed vector LT7P, MT7P, HT7P is shown in FIGS. 1-3, wherein the DNA sequence for insertion of T7RNAP expression is shown in SEQ ID No:2 to 4. The constructed vector mainly comprises the following steps:
(1) A 3' -end genome homologous region of the yfeX gene;
(2) lacI gene, which contains lacI gene promoter, lacI gene coding region and other transcription and translation essential sequences;
(3) Lactose operon promoter (Plac), lactose operon repressor protein (LacI) binding domain (O), ribosome Binding Site (RBS), where the amount of protein translation synthesis of T7RNAP is regulated by altering the DNA sequence of the RBS domain, thereby altering the intracellular T7RNAP activity of the host;
(4) A T7RNAP protein coding region;
(5) Terminator (rrnB);
(6) The 3' -end genome homologous region of yfeX gene.
The Ribosome Binding Site (RBS) sequences of different translational strengths are shown in Table 1.
TABLE 1
1.2 construction of BL21 recombinant strains expressing different T7RNAP Activity
The DNA sequence expressing T7RNAP for insertion described in 1.1 was amplified using the nuuzan (Vazyme) Phanta high fidelity enzyme, and pEcCas9 and pEcgRNA-yfeX were used for the insertion of the target fragment and deletion of the target yfeX gene, see in particular the following steps:
1.2.1 amplification of target fragments using the following primers, respectively, with plasmid LT7P, MT7P, HT P as template:
primer 1:5'GCACAGTGTATTACGCAGTCAGGTAC 3' (SEQ ID NO: 8), and
primer 2:5'GTGAATTAACGGCGTCCACAC 3' (SEQ ID NO: 9)
And purifying and recovering the target fragment by using the gel recovery kit.
1.2.2 construction of pEcgRNA-yfeX plasmid, see FIG. 4, for the preparation of the gRNA of the target yfeX gene
Cas9 protein binding cleavage site of yfeX gene was analyzed using CRISPOR (crispor.tefor.net) software, and finally 5'CAGACCGTGAGTGCCACTGG 3' sequence was selected as gRNA sequence, and pEcgRNA-yfeX plasmid vector was constructed with complete DNA sequence as shown in SEQ ID NO: shown at 10.
SEQ ID No：10：
TTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGCTGGATCCTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTCAGACCGTGAGTGCCACTGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAATTCTCTAGAGTCGACCTGCAGAAGCTTAGATCTATTACCCTGTTATCCCTACTCGAGTTCATGTGCAGCTCCATAAGCAAAAGGGGATGATAAGTTTATCACCACCGACTATTTGCAACAGTGCCGTTGATCGTGCTATGATCGACTGATGTCATCAGCGGTGGAGTGCAATGTCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAAGATGCCGCTCGCCAGTCGATTGGCTGAGCTCATAAGTTCCTATTCCGAAGTTCCGCGAACGCGTAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCT
1.2.3 preparation of BL21 electrotransformation competent cells containing pEcCas9 plasmid
The pEcCas9 plasmid is transferred into BL21 strain, BL21 single colony containing pEcCas9 plasmid is picked from solid plate, added into liquid LB culture medium containing kanamycin resistance, and cultured overnight. The next day, according to 1:100 adding the cultured bacterial liquid overnight into fresh LB liquid medium containing kanamycin, when the OD of the bacterial liquid 600 When the concentration is=0.2, the arabinose solution is added to make the final concentration be 10mM, and the bacterial cells are cultured until the OD 600 =0.6 to 0.8. Centrifuging at 4 ℃, collecting the thalli, and washing the thalli for 3-4 times by 10% glycerol. Finally, 10% glycerol is used for suspending thalli, 100 mu L of competent cells per tube are respectively packaged into a precooled 1.5mL centrifuge tube, the temperature is minus 80 ℃, and the obtained glycerol tube cells are the competent cells of the subsequent electrotransformation experiment.
1.2.4 insertion of target fragment and deletion of target Gene
mu.L of pEcgRNA-yfeX plasmid and 10. Mu.L of 1.2.1 gel recovered linear fragment (10. Mu.g or more) were mixed with competent cells prepared in 1.2.3, and added to a 2mm cuvette under electric shock conditions: 2.5kV, 25. Mu.F, 200Ω,2mm. After electric shock, 900mL of LB culture medium is added into the electric rotating cup rapidly, and after the LB culture medium and electric shock cells are mixed uniformly, the culture is carried out for more than 2 hours at 37 ℃ and 200 rpm. The cells were then collected by centrifugation and plated on LB solid medium containing calicheamicin (50. Mu.g/ml) and spectinomycin (50. Mu.g/ml), and cultured overnight at 37℃in a constant temperature incubator.
Colony PCR was performed on single colonies grown on LB solid medium containing kanamycin and spectinomycin using the following primers:
primer 1:5'GCACAGTGTATTACGCAGTCAGGTAC 3' (SEQ ID NO: 8), and
primer 2:5'GTGAATTAACGGCGTCCACAC 3' (SEQ ID NO: 9)
BL21 bacteria are used as starting bacteria, a target sequence is inserted into a genome, namely a DNA sequence for expressing T7RNAP is constructed, and a recombinant strain of yfeX genes is deleted, and the target recombinant strain is further confirmed by sequencing PCR products.
1.2.5 elimination of pEcgRNA-yfeX and pEcCas9 plasmids in the recombinant strains of interest
1.2.4 recombinant strains obtained by the screening were streaked on LB solid medium containing calicheamicin (50. Mu.g/ml) and spectinomycin (50. Mu.g/ml), and cultured overnight. Then, single colonies were picked, inoculated into fresh liquid LB medium (containing 10mM rhamnose and 50. Mu.g/ml of kanamycin), and cultured overnight at 37 ℃. Then, the bacterial liquid was streaked on a solid LB medium containing kanamycin, and single colonies on a plate were picked up and cultured on a solid LB medium containing spectinomycin or kanamycin. Finally, there was no growth in the spectinomycin plates, while the strain grown in the kanamycin plates was the successful elimination of the pEcgRNA-yfeX plasmid.
The recombinant strain from which the pEcgRNA-yfeX plasmid was eliminated was cultured overnight in LB medium containing 5g/L glucose, then plated on LB solid plates containing both glucose (5 g/L) and sucrose (10 g/L), and cultured overnight. Subsequently, single colonies were picked on solid LB medium containing kanamycin or no resistance, and kanamycin resistance verification was performed, with no growth in kanamycin, i.e., successful elimination of the pEcCas9 plasmid.
1.3 determination of T7RNAP Activity in different BL21 recombinant strains
1.3.1 preparation of cell disruption solution of recombinant strains of different BL21
BL21 recombinant strains were streaked on antibiotic-free LB solid medium and cultured overnight at 37 ℃. The next day, single colonies were picked from the plates, inoculated into LB liquid medium, and cultured overnight at 37 ℃. Overnight culture broth was used at 1:100, inoculating into fresh LB liquid culture medium for culturing until the bacterial liquid OD 600 When=0.6 to 0.8, IPTG was added at a final concentration of 1mM, and the culture was continued for 12 hours. The cells were collected by centrifugation at 4℃and PBS (137mM NaCl,2.7mM KCl,10mM Na) was added 2 HPO 4 ,2mM KH 2 PO 4 pH 7.4) suspending the thalli in a buffer solution, ultrasonically crushing the obtained thalli at 4 ℃, centrifuging and taking the supernatant for detecting the activity of T7RNAP in the recombinant strain.
1.3.2T7RNAP Activity assay
T7RNAP activity in the cell disruption solution collected in 1.3.1 was detected by the method described in J.biochem,2023,173 (5): 343-352. The total reaction volume in the detection method was 20. Mu.L, including 7. Mu.L of the cell-free line reaction solution, 6. Mu.L of BL21 cell extract, 2. Mu.L of the template DNA and 5. Mu.L of the cell disruption solution collected in 1.3.1.
The T7RNAP activity assay system was reacted in a 37℃water bath with shaking at 100rpm for 1 hour, and then quenched by the addition of 980. Mu.L of 4℃cold water. 200 mu L of reaction solution is taken and added into a 96-well plate, and the fluorescence intensity of the reaction solution is measured in a multifunctional enzyme-labeled instrument. The multifunctional enzyme-labeled instrument is set to have an excitation wavelength of 460nm and an emission wavelength of 510nm, and the measured fluorescence intensity value is calculated by referring to a standard curve of the T7RNAP activity to obtain the T7RNAP activity in the final cell disruption solution.
FIG. 5 shows the T7RNAP activity of different strains, H, M and L representing host bacteria with high, medium and low translational strength RBS sequences T7RNAP, respectively. As can be seen from FIG. 5, the enzyme activity of H strain is highest after induction of IPTG by the host strain having T7RNAP activity, and when it is set to 1, the enzyme activities of both M and L strains relative to H strain are less than 1, and the enzyme activity of L strain is less than that of M strain.
Example 2: construction of plasmid vector for enhancing heme Synthesis pathway
Because the heme metabolic synthesis pathway is long, the heme C5 synthesis pathway is divided into three plasmids for construction: ALA was synthesized from alpha-ketoglutarate by catalysis of glutaryl-tRNA synthetase (GluRS), glutaryl-tRNA reductase (GluTR) and glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde aminotransferase, GSAM), and this pathway was constructed in a plasmid.
Starting from 5-aminolevulinic acid (ALA), the pathway was constructed in a plasmid by condensing two molecules of ALA with ALA dehydrogenase (hemB) to form Porphobilinogen (PBG), condensing four molecules of PBG to Hydroxymethylcholine (HMB) with PBG deaminase (hemC), and then cyclizing HMB with uroporphyrinogen synthase (hemD) to form uroporphyrinogen III.
Uroporphyrinogen III is converted to protoporphyrin IX by successive decarboxylation and oxidation reactions, with uroporphyrinogen III and protoporphyrinogen IX being the enzymes involved in the process, uroporphyrinogen decarboxylase (hemE), coproporphyrinogen oxidase (hemF) and protoporphyrinogen oxidase (hemG). Finally, ferrochelatase (hemH) catalyzes Fe 2+ Chelate protoporphyrin IX to form heme, and construct this pathway in a plasmid.
Construction of 1p1 plasmid vector
The structure of the p1 plasmid vector is shown in FIG. 6, and the DNA sequence is shown in SEQ ID NO: 11. Among them, hemA (fbr), hemL genes and their related DNA sequences related to heme synthesis pathway are distributed in the following order:
(1) A T7 promoter, a lactose operon repressor (LacI) binding region (lac operator) and a Ribosome Binding Site (RBS);
(2)hemA(fbr)；
(3) Ribosome Binding Site (RBS), henl gene;
(4) T7 terminator.
SEQ ID No：11：
GGGGAATTGTGAGCGGATAACAATTCCATAAAAGGAGGAAAATATATGACCAAGAAACTTTTAGCACTCGGTATCAACCATAAAACGGCACCTGTATCGCTGCGAGAACGTGTATCGTTTTCGCCGGATAAGCTCGATCAGGCGCTTGACAGCCTGCTTGCGCAGCCGATGGTGCAGGGCGGCGTGGTGCTGTCGACGTGCAACCGCACGGAACTTTATCTTAGCGTTGAAGAGCAGGATAACCTGCAAGAGGCGTTAATCCGCTGGCTTTGCGATTATCACAATCTTAATGAAGAAGATCTGCGTAAAAGCCTCTACTGGCATCAGGATAACGACGCGGTTAGCCATTTAATGCGTGTTGCCAGCGGCCTGGATTCATTGGTTCTTGGGGAGCCGCAGATCCTCGGTCAGGTTAAAAAAGCGTTTGCCGATTCGCAAAAAGGCCATATGAAGGCCAGCGAACTGGAACGCATGTTCCAGAAATCTTTCTCTGTAGCGAAACGCGTTCGCACTGAAACAGATATCGGTGCCAGCGCTGTGTCTGTCGCTTTTGCGGCTTGTACGCTGGCGCGGCAGATCTTTGAATCGCTCTCTACGGTCACAGTGTTGCTGGTAGGCGCGGGCGAAACCATCGAGCTGGTAGCGCGTCATCTGCGCGAACATAAAGTACAGAAGATGATTATCGCCAACCGCACTCGCGAACGTGCCCAAATACTGGCAGATGAAGTTGGCGCGGAAGTGATTGCCCTGAGTGAGATCGACGAACGTCTGCGCGAAGCCGATATCATCATCAGTTCCACCGCCAGCCCGTTACCGATTATCGGGAAAGGCATGGTGGAGCGCGCATTAAAAAGCCGTCGCAACCAACCAATGCTGTTGGTGGATATTGCCGTTCCGCGCGATGTTGAGCCGGAAGTTGGCAAACTGGCGAATGCTTATCTTTATAGCGTGGACGATCTGCAAAGCATCATTTCGCACAACCTGGCGCAGCGTAAAGCCGCAGCGGTTGAGGCGGAAACTATTGTCGCTCAGGAAACCAGCGAATTTATGGCGTGGCTGCGAGCACAAAGCGCCAGCGAAACCATTCGCGAGTATCGCAGCCAGGCAGAGCAAGTTCGCGATGAGTTAACCGCCAAAGCGTTAGCGGCCCTTGAGCAGGGCGGCGACGCGCAAGCCATTATGCAGGATCTGGCATGGAAACTGACTAACCGCTTGATCCATGCGCCAACGAAATCACTTCAACAGGCCGCCCGTGACGGGGATAACGAACGCCTGAATATTCTGCGCGACAGCCTCGGGCTGGAGTAGTTTGTTTAACTTTAAGAAGGAGATATACCATGAGTAAGTCTGAAAATCTTTACAGCGCAGCGCGCGAGCTGATCCCTGGCGGTGTGAACTCCCCTGTTCGCGCCTTTACTGGCGTGGGCGGCACTCCACTGTTTATCGAAAAAGCGGACGGCGCTTATCTGTACGATGTTGATGGCAAAGCCTATATCGATTATGTCGGTTCCTGGGGGCCGATGGTGCTGGGCCATAACCATCCGGCAATCCGCAATGCCGTGATTGAAGCCGCCGAGCGTGGTTTAAGCTTTGGTGCACCAACCGAAATGGAAGTGAAAATGGCGCAACTGGTGACTGAACTGGTCCCGACCATGGATATGGTGCGCATGGTGAACTCCGGCACCGAGGCGACGATGAGCGCCATCCGCCTGGCCCGTGGTTTTACCGGTCGCGACAAAATTATTAAATTTGAAGGTTGTTACCACGGTCACGCTGACTGCCTGCTGGTGAAAGCCGGTTCTGGCGCACTCACGTTAGGCCAGCCAAACTCGCCGGGCGTTCCGGCAGATTTCGCCAAACATACCTTAACCTGTACTTATAACGATCTGGCTTCTGTACGCGCCGCGTTTGAGCAATACCCGCAAGAGATTGCCTGTATTATCGTCGAGCCGGTGGCAGGCAATATGAACTGCGTTCCACCGCTGCCAGAGTTCCTGCCAGGTCTGCGTGCGCTGTGCGACGAATTTGGCGCATTGCTGATCATCGATGAAGTAATGACCGGCTTCCGCGTGGCACTGGCTGGCGCACAGGATTATTACGGTGTGGAACCGGATCTCACCTGCCTGGGCAAAATCATCGGCGGTGGAATGCCGGTAGGCGCATTCGGTGGTCGTCGTGATGTAATGGATGCGCTGGCCCCGACGGGTCCGGTCTATCAGGCGGGTACGCTTTCCGGTAACCCAATTGCGATGGCAGCGGGTTTCGCCTGTCTGAATGAAGTCGCGCAGCCGGGCGTTCACGAAACGTTGGATGAGCTGACATCACGTCTGGCAGAAGGTCTGCTGGAAGCGGCAGAAGAAGCCGGAATTCCGCTGGTCGTTAACCACGTTGGCGGCATGTTCGGTATTTTCTTTACCGACGCCGAGTCCGTGACGTGCTATCAGGATGTGATGGCCTGTGACGTGGAACGCTTTAAGCGTTTCTTCCATATGATGCTGGACGAAGGTGTTTACCTGGCACCGTCAGCGTTTGAAGCGGGCTTTATGTCCGTGGCGCACAGCATGGAAGATATCAATAACACCATCGATGCTGCACGTCGGGTGTTTGCGAAGTTGTGATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAACCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCATCGTGGCCGGATCTTGCGGCCCCTCGGCTTGAACGAATTGTTAGACATTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACGTAGTGGACAAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTCCAAGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTCGGCAGCGACATCCTTCGGCGCGATTTTGCCGGTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCAGCCCAGTCGGGCGGCGAGTTCCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAAAGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTTTGTCAGCAAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGATAACGCCACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAGCCGAAGTTTCCAAAAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCAAATCAATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGTCGGTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGGCGATCACCGCTTCCCTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGCTAGCTCACTCGGTCGCTACGCTCCGGGCGTGAGACTGCGGCGGGCGCTGCGGACACATACAAAGTTACCCACAGATTCCGTGGATAAGCAGGGGACTAACATGTGAGGCAAAACAGCAGGGCCGCGCCGGTGGCGTTTTTCCATAGGCTCCGCCCTCCTGCCAGAGTTCACATAAACAGACGCTTTTCCGGTGCATCTGTGGGAGCCGTGAGGCTCAACCATGAATCTGACAGTACGGGCGAAACCCGACAGGACTTAAAGATCCCCACCGTTTCCGGCGGGTCGCTCCCTCTTGCGCTCTCCTGTTCCGACCCTGCCGTTTACCGGATACCTGTTCCGCCTTTCTCCCTTACGGGAAGTGTGGCGCTTTCTCATAGCTCACACACTGGTATCTCGGCTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTAAGCAAGAACTCCCCGTTCAGCCCGACTGCTGCGCCTTATCCGGTAACTGTTCACTTGAGTCCAACCCGGAAAAGCACGGTAAAACGCCACTGGCAGCAGCCATTGGTAACTGGGAGTTCGCAGAGGATTTGTTTAGCTAAACACGCGGTTGCTCTTGAAGTGTGCGCCAAAGTCCGGCTACACTGGAAGGACAGATTTGGTTGCTGTGCTCTGCGAAAGCCAGTTACCACGGTTAAGCAGTTCCCCAACTGACTTAACCTTCGATCAAACCACCTCCCCAGGTGGTTTTTTCGTTTACAGGGCAAAAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACTGAACCGCTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCACTATA
Construction of 2.2p2 plasmid vector
The structure of the p2 plasmid vector is shown in FIG. 7, and the DNA sequence is shown in SEQ ID NO: shown at 12. Wherein hemB, hemC and hemD genes and their associated DNA sequences involved in the heme synthesis pathway are distributed in the following order:
(1) A T7 promoter, a lactose operon repressor (LacI) binding region (lac operator) and a Ribosome Binding Site (RBS);
(2) hemB gene;
(3) A T7 terminator;
(4) A T7 promoter, a lactose operon repressor (LacI) binding region (lac operator) and a Ribosome Binding Site (RBS);
(5) hemC gene, ribosome Binding Site (RBS), hemD gene;
(6) T7 terminator.
SEQ ID NO：12：
GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGATATACCATGACAGACTTAATCCAACGCCCTCGTCGCCTGCGCAAATCTCCTGCGCTGCGCGCTATGTTTGAAGAGACAACACTTAGCCTTAACGACCTGGTGTTGCCGATCTTTGTTGAAGAAGAAATTGACGACTACAAAGCCGTTGAAGCCATGCCAGGTGTGATGCGCATTCCAGAGAAACATCTGGCACGCGAAATTGAACGCATCGCCAACGCCGGTATTCGTTCCGTGATGACTTTCGGCATCTCTCACCATACCGATGAAACCGGCAGCGATGCCTGGCGGGAAGATGGACTGGTGGCGCGAATGTCGCGCATCTGCAAGCAGACCGTGCCAGAAATGATCGTCATGTCAGACACCTGCTTCTGCGAATACACATCTCACGGTCACTGCGGTGTGCTGTGCGAGCATGGCGTCGACAACGACGCGACTCTGGAAAATTTAGGCAAGCAAGCCGTGGTTGCAGCTGCTGCAGGCGCAGACTTCATCGCCCCTTCTGCCGCGATGGACGGCCAGGTACAGGCGATTCGCCAGGCGCTGGACGCTGCGGGCTTTAAAGATACGGCGATTATGTCGTATTCGACCAAGTTCGCCTCTTCCTTTTATGGTCCGTTCCGTGAAGCTGCCGGAAGCGCATTAAAAGGCGACCGCAAAAGCTATCAGATGAACCCAATGAACCGTCGTGAGGCGATTCGTGAGTCACTGCTGGATGAAGCCCAGGGCGCAGACTGTCTGATGGTTAAACCTGCCGGAGCGTACCTCGACATCGTGCGTGAGCTGCGTGAACGTACTGAATTGCCGATTGGCGCGTATCAGGTGAGCGGTGAGTACGCGATGATTAAGTTCGCCGCGCTGGCGGGTGCTATAGATGAAGAGAAAGTCGTGCTCGAAAGCTTAGGTTCAATTAAGCGTGCGGGTGCGGATCTGATTTTCAGCTACTTTGCGATGGATTTGGCTGAGAAGAAGATTCTGCGTTAATGCTTAAGTCGAACAGAAAGTAATCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGTATTGTACACGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGTTAGACAATGTTTTAAGAATTGCCACACGCCAAAGCCCACTTGCACTCTGGCAGGCACACTATGTCAAAGACAAGTTGATGGCGAGCCATCCGGGCCTGGTCGTTGAACTGGTACCGATGGTGACGCGCGGCGATGTGATTCTTGATACGCCGCTGGCGAAAGTAGGCGGAAAAGGCTTATTTGTTAAAGAGCTGGAAGTCGCGCTCCTCGAAAATCGCGCCGATATCGCCGTACATTCAATGAAAGATGTGCCGGTTGAATTCCCGCAAGGTCTGGGACTGGTCACTATTTGTGAGCGTGAAGATCCTCGCGATGCCTTTGTGTCCAATAACTATGACAATCTGGATGCGTTACCGGCAGGCAGTATCGTCGGGACGTCCAGTTTACGTCGCCAGTGCCAACTGGCTGAACGCCGCCCGGATCTGATTATCCGCTCCCTGCGAGGCAACGTCGGCACTCGCCTGAGTAAACTGGATAACGGCGAATACGATGCCATCATTCTTGCGGTAGCCGGACTAAAACGTTTAGGTCTGGAGTCCCGCATTCGCGCCGCATTGCCACCCGAGATTTCTCTTCCGGCGGTAGGACAAGGTGCGGTGGGTATTGAATGCCGCCTTGATGATTCTCGCACTCGCGAGCTGCTTGCCGCGCTGAATCACCACGAAACTGCACTGCGCGTTACCGCAGAACGCGCCATGAATACCCGTCTCGAAGGCGGATGTCAGGTGCCAATTGGTAGCTACGCCGAGCTTATTGATGGCGAAATCTGGCTGCGTGCGTTGGTCGGCGCGCCGGACGGTTCGCAGATTATTCGCGGTGAACGCCGCGGTGCGCCGCAAGATGCCGAACAAATGGGGATTTCGCTGGCAGAAGAGCTACTGAATAACGGCGCGCGCGAGATCCTCGCTGAAGTCTATAACGGAGACGCTCCGGCATGAATAAAAGGAGGAAAATATATGAGTATCCTGGTCACCCGCCCGTCTCCCGCTGGAGAAGAGTTAGTGAGCCGTCTGCGCACACTGGGGCAGGTGGCCTGGCATTTTCCACTGATTGAGTTTTCTCCGGGTCGACAATTACCACAACTTGCTGATCAACTGGCGGCGCTGGGGGAGAGCGATCTGTTGTTTGCCCTCTCGCAACACGCGGTTGCTTTTGCCCAATCACAGCTGCATCAGCAAGATCGTAAATGGCCCCGACTACCTGATTATTTCGCCATTGGACGCACCACCGCACTGGCACTACATACCGTAAGCGGACAGAAGATTCTCTACCCGCAGGATCGGGAAATCAGCGAAGTCTTGCTACAATTACCTGAATTACAAAATATTGCGGGCAAACGTGCGCTGATATTACGTGGCAATGGCGGTCGTGAGCTAATTGGGGATACCCTGACGGCGCGCGGTGCTGAGGTCACTTTTTGTGAATGTTATCAACGATGCGCAATCCATTACGATGGTGCAGAAGAAGCGATGCGCTGGCAATCCCGCGAGGTGACGACGGTCGTTGTTACCAGCGGTGAAATGTTGCAGCAACTCTGGTCGCTGATCCCACAATGGTATCGTGAGCACTGGTTACTACACTGTCGACTATTGGTCGTCAGTGAGCGTTTGGCGAAACTCGCCCGGGAACTGGGCTGGCAAGACATTAAGGTCGCCGATAACGCTGACAACGATGCGCTTTTACGGGCATTACAATAATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAACCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACAAGCTGACGACCGGGTCTCCGCAAGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGGTCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGCATGCAGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGTGTCAGCTCACTCAAAAGCGGTAATACGGTTATCCACAGAATCAGGGGATAAAGCCGGAAAGAACATGTGAGCAAAAAGCAAAGCACCGGAAGAAGCCAACGCCGCAGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGCCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTTGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCATTGGTAACTGATTTAGAGGACTTTGTCTTGAAGTTATGCACCTGTTAAGGCTAAACTGAAAGAACAGATTTTGGTGAGTGCGGTCCTCCAACCCACTTACCTTGGTTCAAAGAGTTGGTAGCTCAGCGAACCTTGAGAAAACCACCGTTGGTAGCGGTGGTTTTTCTTTATTTATGAGATGATGAATCAATCGGTCTATCAAGTCAACGAACAGCTATTCCGTTACTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCACTATA
Construction of a 3p3 plasmid vector
The structure of the p3 plasmid vector is shown in FIG. 8, and the DNA sequence of the vector is shown in SEQ ID NO: shown at 13. Wherein the hemE, hemF, hemG and hemH genes and their associated DNA sequences involved in the heme synthesis pathway are distributed in the following order:
(1) A T7 promoter, a lactose operon repressor (LacI) binding region (lac operator) and a Ribosome Binding Site (RBS);
(2) hemE gene;
(3) Ribosome Binding Sites (RBS), hemF genes, ribosome Binding Sites (RBS), hemG genes;
(4) A T7 terminator;
(5) A T7 promoter, a lactose operon repressor (LacI) binding region (lac operator) and a Ribosome Binding Site (RBS);
(6) hemH gene;
(7) T7 terminator.
SEQ ID NO：13：
CCACGATGCGTCCGGCGTAGAGGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGACCGAACTTAAAAACGATCGTTATCTGCGGGCGCTGCTGCGCCAGCCCGTTGATGTCACTCCAGTATGGATGATGCGCCAGGCGGGTCGCTATCTACCGGAATATAAAGCCACGCGCGCCCAGGCGGGCGATTTTATGTCGCTGTGCAAAAACGCCGAGCTGGCGTGCGAAGTGACTTTGCAACCGCTGCGTCGCTACCCGCTGGATGCGGCGATCCTCTTTTCCGATATCCTCACCGTGCCGGACGCGATGGGGTTAGGGCTCTATTTTGAAGCCGGAGAAGGTCCGCGTTTTACCTCGCCAGTCACCTGCAAAGCCGACGTCGATAAACTGCCAATTCCGGACCCGGAAGATGAGCTGGGTTACGTGATGAACGCGGTGCGTACCATTCGTCGCGAACTGAAAGGCGAAGTGCCGCTGATTGGTTTTTCCGGCAGCCCGTGGACGCTGGCGACCTACATGGTGGAAGGCGGCAGCAGCAAAGCGTTCACCGTGATCAAAAAAATGATGTATGCCGATCCGCAGGCGCTGCACGCTCTACTCGATAAACTGGCGAAAAGCGTCACTTTGTATCTGAATGCGCAGATTAAAGCCGGTGCTCAGGCAGTGATGATTTTCGACACCTGGGGCGGTGTGCTTACCGGGCGCGATTATCAACAGTTCTCGCTCTATTACATGCATAAAATTGTTGATGGTTTACTGCGTGAAAACGACGGTCGCCGCGTACCGGTCACGCTGTTTACCAAAGGCGGCGGACAGTGGCTGGAAGCGATGGCAGAAACCGGTTGCGATGCGTTGGGCCTCGACTGGACAACGGATATCGCCGATGCGCGCCGCCGTGTGGGCAATAAAGTCGCGTTGCAGGGTAATATGGATCCGTCGATGCTGTACGCTCCGCCTGCCCGCATTGAAGAAGAAGTAGCGACTATACTTGCAGGTTTCGGTCACGGCGAAGGTCATGTCTTTAACCTTGGTCACGGCATTCATCAGGATGTGCCGCCAGAACATGCTGGCGTATTCGTGGAGGCAGTGCATCGACTGTCTGAACAATATCACCGCTAAATAAAAGGAGGAAAATATATGAAACCCGACGCACACCAGGTTAAACAGTTTCTGCTCAACCTTCAGGATACGATTTGTCAGCAGCTGACCGCCGTCGATGGCGCAGAATTTGTCGAAGATAGTTGGCAGCGCGAAGCTGGCGGCGGCGGGCGTAGTCGGGTGTTGCGTAATGGTGGTGTTTTCGAACAGGCAGGCGTCAACTTTTCGCATGTCCACGGTGAGGCGATGCCTGCTTCCGCCACCGCTCATCGCCCGGAACTTGCCGGGCGCAGTTTCGAGGCGATGGGCGTTTCACTGGTAGTGCATCCGCATAACCCGTATGTTCCCACCAGCCACGCGAATGTGCGGTTTTTTATTGCCGAAAAACCGGGTGCCGATCCCGTCTGGTGGTTTGGCGGCGGCTTCGATTTAACCCCTTTCTATGGTTTTGAAGAAGACGCCATTCACTGGCACCGCACCGCCCGTGACCTGTGCCTGCCATTTGGTGAAGACGTTTATCCCCGTTACAAAAAGTGGTGCGACGATTACTTCTACCTCAAACATCGCAACGAACAGCGCGGTATTGGCGGGCTGTTCTTTGATGATCTGAACACGCCAGATTTCGACCACTGTTTTGCCTTTATGCAGGCGGTAGGCAAAGGCTACACCGACGCTTATTTACCAATTGTAGAGCGACGTAAAGCGATGGCCTACGGCGAGCGCGAGCGCAATTTTCAGCTCTACCGTCGCGGTCGTTATGTCGAGTTCAATCTGGTCTGGGATCGCGGCACGCTGTTTGGCCTGCAAACTGGCGGGCGCACCGAGTCTATCCTGATGTCAATGCCGCCACTGGTACGCTGGGAATATGATTATCAGCCAAAAGATGGCAGCCCAGAAGCGGCGTTAAGTGAGTTTATTAAGGTCAGGGATTGGGTGTAAATAAAAGGAGGAAAATATGTGAAAACATTAATTCTTTTCTCAACAAGGGACGGACAAACGCGCGAGATTGCCTCCTACCTGGCTTCGGAACTGAAAGAACTGGGGATCCAGGCGGATGTCGCCAATGTGCACCGCATTGAAGAACCACAGTGGGAAAACTATGACCGTGTGGTCATTGGTGCTTCTATTCGCTATGGTCACTACCATTCAGCGTTCCAGGAATTTGTCAAAAAACATGCGACGCGGCTGAATTCGATGCCGAGCGCCTTTTACTCCGTGAATCTGGTGGCGCGCAAACCGGAGAAGCGTACTCCACAGACCAACAGCTACGCGCGCAAGTTTCTGATGAACTCGCAATGGCGTCCCGATCGCTGCGCGGTCATTGCCGGGGCGCTGCGTTACCCACGTTATCGCTGGTACGACCGTTTTATGATCAAGCTGATTATGAAGATGTCAGGCGGTGAAACGGATACGCGCAAAGAAGTTGTCTATACCGATTGGGAGCAGGTGGCGAATTTCGCCCGAGAAATCGCCCATTTAACCGACAAACCGACGCTGAAATAATGCTTAAGTCGAACAGAAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGTAATCGTATTGTACACGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGCGTCAGACTAAAACCGGTATCCTGCTGGCAAACCTGGGTACGCCCGATGCCCCCACACCTGAAGCGGTAAAACGCTATCTGAAACAATTTTTAAGCGACAGACGCGTGGTTGATACCTCACGGTTGTTATGGTGGCCATTGCTGCGCGGCGTGATTTTGCCGCTGCGCTCGCCGCGTGTGGCGAAGCTGTATGCCTCTGTCTGGATGGAAGGTGGCTCGCCGCTGATGGTTTACAGCCGTCAGCAACAGCAGGCGCTGGCACAACGTTTACCGGAGACGCCCGTAGCGCTGGGAATGAGCTACGGCTCGCCATCACTGGAAAGCGCCGTAGATGAACTCCTGGCAGAGCATGTAGATCATATTGTGGTGCTGCCGCTTTATCCGCAATACTCCTGTTCAACGGTCGGTGCGGTATGGGATGAACTGGCACGCATTCTGGCGCGCAAACGTAGCATTCCGGGGATATCGTTTATTCGTGATTACGCTGATAACCACGATTACATTAATGCACTGGCGAACAGCGTACGCGCTTCTTTTGCCAAACATGGCGAACCGGATCTGCTGCTGCTCTCTTATCATGGCATTCCCCAGCGTTATGCAGATGAAGGCGATGATTACCCGCAACGTTGCCGCACAACGACTCGCGAACTGGCTTCCGCACTGGGGATGGCACCGGAAAAAGTGATGATGACCTTTCAGTCGCGCTTTGGTCGGGAACCCTGGCTGATGCCTTATACCGACGAAACGCTGAAAATGCTCGGAGAAAAAGGCGTAGGTCATATACAGGTGATGTGCCCGGGCTTTGCTGCGGATTGTCTGGAGACGCTGGAAGAGATTGCCGAGCAAAACCGTGAGGTCTTCCTCGGTGCCGGCGGGAAAAAATATGAATATATTCCAGCGCTTAATGCCACGCCGGAACATATTGAAATGATGGCTAATCTTGTTGCCGCGTATCGCTAAATGGGCAGCAGCCTAGGTTATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCTGGCGGCACGATGGCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATCATGATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGG
Example 3: obtaining recombinant strains for stable synthesis of heme using T7 expression system
3.1 obtaining recombinant strains synthesizing heme Using T7 expression System
And transforming the recombinant plasmid by adopting a heat shock method to obtain the recombinant strain.
First, competent cells of recombinant E.coli were prepared. Then adding the plasmid into competent cells in an ultra clean bench, flicking, mixing uniformly, standing on ice for 30min, heat-shocking at 42 ℃ for 90s, then rapidly returning to ice, standing for 2-3 min, adding 600 mu L of LB culture medium, placing into a shaking table at 37 ℃, and shaking and culturing at 150rpm for 45min. Finally, the colonies were plated on LB solid medium containing resistance, cultured overnight, and the single colonies were the target colonies, see Table 2.
TABLE 2 heme iron synthesis pathway modified E.coli strains
3.2 study of stability and heme yield of recombinant strains synthesizing heme
3.2.1 Synthesis of heme by Induction of recombinant strains
Recombinant strains cultured in the slant culture medium were picked up and inoculated on a liquid LB medium (containing antibiotics corresponding to plasmids), and cultured at 37℃for 12 hours. Then, transferring the strain into fresh LB culture medium with 1% of inoculation amount by volume fraction, adding 0.1mmol/L IPTG to induce gene expression and corresponding antibiotics at 0h, culturing at 37deg.C and 220r/min for 4h, and adding FeSO with final concentration of 100 μmol/L 4 Culturing was continued for 48 hours. Collecting thallus and fermentation liquor, suspending thallus with 1M sodium hydroxide, ultrasonic crushing, collecting thallus crushed liquor and fermentation liquor supernatant, respectivelyHeme production was determined by the HPLC method described above.
See 1.3.2 detection of T7RNAP Activity As shown in FIG. 5, if the T7RNAP Activity of H strain is set to 1, then the T7RNAP Activity of M and L strains relative to H strain is 0.61 and 0.23, respectively. However, after the strain was subjected to IPTG induction to synthesize heme for 48 hours, H was set as shown in Table 3 and FIG. 9 2H In the case where the enzyme activity of the strain is 1, H 2M And H 2L The activity of the strains was 1.15 and 1.19, respectively, H 2H 、H 2M And H 2L The heme contents of the strains are 40.61, 88.35 and 79.28mg/L, H respectively 2H 、H 2M And H 2L Mutation rates of the bacteria were 72.31%, 66.35% and 64.29%, respectively.
TABLE 3T 7RNAP Gene mutation Rate of recombinant strains synthesizing heme Using T7 expression System after IPTG Induction
The mutation rate was measured as follows:
after induction of heme synthesis for 48h, the zymophyte liquid is streaked on LB solid medium containing antibiotics, then 15 single colonies are picked up, and colony PCR is carried out by using the following primers and high-fidelity enzymes:
primer 1:5'GCACAGTGTATTACGCAGTCAGGTAC 3' (SEQ ID NO: 8), and
primer 2:5'GTGAATTAACGGCGTCCACAC 3' (SEQ ID NO: 9)
Amplifying the T7RNAP gene, and carrying out DNA sequencing on the amplified product to determine the mutation condition of the T7RNAP gene.
From the above, H, M and L bacteria express T7RNAP under IPTG induction, and the T7 expression system is started to enhance heme synthesis. Because the high activity of the T7 expression system and toxicity generated by the mass synthesis of heme generate larger stress on host bacteria, the T7RNAP on the genome is mutated, and the activity of the T7RNAP is reduced to reduce the stress pressure on the host bacteria. Wherein H is 2H The mutation rate of the strain is highest and reaches 72.31%, and the activity of the strain is reduced most. T7 of initial M and L strainsRNAP activity was 0.61-fold and 0.23-fold that of H strain (FIG. 5), H after mutation 2M And H 2L The activity of the strain is H 2H 1.15-fold and 1.19-fold of strain activity (FIG. 9). Thus, after mutation of the T7RNAP gene, the T7RNAP enzyme activity is significantly reduced.
Further, as shown in FIG. 9, H 2H 、H 2M And H 2L The heme yields of the strains were 40.61, 88.35 and 79.28mg/L, respectively. It is shown that the initial T7RNAP activity of H strain is highest, and that the high activity of T7RNAP is the most damaging to the synthetic heme of T7 expression system. Comparative H 2M And H 2L Strain discovery, H 2M The strain has higher heme yield than H 2L Strains due to H 2M The T7RNAP activity of the strain is higher than that of H 2L Strains.
The invention improves the yield of heme by utilizing the T7 expression system to regulate the activity of T7RNAP, and obtains the H for stably synthesizing heme by the method 2M Strains.
Those skilled in the art can implement the invention by varying the relevant parameters in light of the present disclosure. The methods described in the embodiments and the results thereof may be implemented by modification, variation and combination without departing from the content, spirit and scope of the invention.
Industrial applicability
The invention provides a method for stably synthesizing a recombinant strain of heme by using a T7 expression system, which can be used for stably synthesizing a large amount of heme by regulating the activity of T7RNAP in a host cell and solving the problem of unstable recombinant strain caused by overhigh activity of the T7 RNAP. The recombinant strain with the T7 expression system constructed by the method can be used for industrialized mass production of heme.

Claims (8)

1. A method for constructing a recombinant strain for stably synthesizing heme using a T7 expression system, comprising the steps of:
1) Constructing an integration vector for expressing T7RNAP,
2) Constructing recombinant strain expressing T7RNAP to synthesize heme,
wherein the recombinant strain of stable synthetic heme is obtained by modulating T7RNAP activity.
2. The method of claim 1, wherein the activity of T7RNAP is modulated by altering the sequence of the ribosome binding site in the DNA sequence of T7 RNAP.
3. The method according to claim 1 or 2, characterized in that the DNA sequence expressing T7RNAP is inserted into the position of the yfeX gene while deleting the yfeX gene.
4. The method according to claim 3, wherein in step 2), the DNA sequence expressing T7RNAP for insertion is amplified, a plasmid vector for deleting the target yfeX gene is constructed, an electrotransformation competent cell is prepared, the DNA sequence expressing T7RNAP is inserted and the target yfeX gene is deleted, the recombinant strain is obtained, and the plasmid in the recombinant strain is eliminated.
5. The method of claim 3 or 4, wherein the DNA sequence for inserting expressed T7RNAP while deleting the yfeX gene comprises a sequence selected from the group consisting of SEQ ID nos: 2. SEQ ID No: 3. SEQ ID No:4, and any one of the following.
6. The method of claim 4, wherein the sequence of the plasmid vector constructed to delete the yfeX gene of interest is SEQ ID NO: 10.
7. The method of any one of claims 1 to 6, comprising constructing a plasmid vector that enhances the heme synthesis pathway.
8. The method of claim 7, wherein the heme synthesis pathway is constructed by dividing into three plasmids:
a first plasmid, from alpha-ketoglutarate, into which aminolevulinic acid is synthesized, and in which this pathway is constructed;
a second plasmid from which uroporphyrinogen III is formed, this pathway being constructed in the second plasmid;
a third plasmid for converting uroporphyrinogen III into protoporphyrin IX via successive decarboxylation and oxidation reactions via coproporphyrinogen III and protoporphyrinogen IX, allowing the ferrochelatase to catalyze Fe 2+ Chelate protoporphyrin IX to form heme, and this pathway is constructed in the third plasmid.
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