CN115521891A - Recombinant bacterium for producing acetaminophen and application thereof - Google Patents

Recombinant bacterium for producing acetaminophen and application thereof Download PDF

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CN115521891A
CN115521891A CN202110707846.9A CN202110707846A CN115521891A CN 115521891 A CN115521891 A CN 115521891A CN 202110707846 A CN202110707846 A CN 202110707846A CN 115521891 A CN115521891 A CN 115521891A
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gene
recombinant bacterium
aminobenzoic acid
recombinant
acetaminophen
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咸漠
黄伟
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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    • C12N9/88—Lyases (4.)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
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    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13027—4-Aminobenzoate 1-monooxygenase (1.14.13.27)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01005—Arylamine N-acetyltransferase (2.3.1.5)
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    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/03—Oxo-acid-lyases (4.1.3)
    • C12Y401/03027—Anthranilate synthase (4.1.3.27)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/03—Oxo-acid-lyases (4.1.3)
    • C12Y401/03038—Aminodeoxychorismate lyase (4.1.3.38)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A recombinant bacterium for producing acetaminophen and application thereof, belonging to the technical field of genetic engineering. In order to solve the problems of flammability and explosiveness, high toxicity, serious environmental pollution caused by waste liquid, high price of a catalyst required by reaction, multiple reaction process steps, fussy subsequent treatment and the like in the production of p-acetaminophenol by a chemical synthesis method, the invention provides a recombinant bacterium for producing p-acetaminophenol, wherein the recombinant bacterium is escherichia coli over-expressing p-aminobenzoic acid synthase genes, 4-amino-4-deoxychorismate lyase genes, 4-aminobenzoic acid hydroxylase genes and arylamine-N-acetyltransferase genes. The recombinant bacterium is utilized to realize the process of directly fermenting and producing the acetaminophen by taking the glucose as a substrate, the generation of precursors or intermediates such as nitrobenzene and the like in chemical reaction and the use of metal catalytic substances are avoided, the whole process is safe, efficient, green and easy to operate, and a new technical method is provided for the production of the acetaminophen.

Description

Recombinant bacterium for producing acetaminophen and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant bacterium for producing acetaminophen and application thereof.
Background
Paracetamol is a common antipyretic analgesic, is one of the most widely applied medicaments all over the world, is one of the varieties with the largest yield in raw material medicaments in China, and is listed in the basic medicament list of the world health organization in the 20 th century. In recent years, demand for acetaminophen is increasing at home and abroad, and the original production process is synthesized by a chemical method, so that the following problems exist: on one hand, the precursors or intermediates in the synthesis process all relate to benzene derivatives, such as nitrobenzene and the like, and are flammable and explosive, the toxicity is high, and the waste liquid has serious pollution to the environment; on the other hand, the catalyst required by the reaction is expensive, the reaction process has many steps, and the post-treatment is complicated. Therefore, the development of the acetaminophen synthesis process with less environmental pollution and production cost saving has important significance. The biological synthesis method has the characteristics of environmental protection, high efficiency, safety and the like, can convert simple and easily obtained precursors into target products through organisms, and solves the problems of the chemical synthesis method.
Disclosure of Invention
In order to solve the problems of producing p-acetaminophenol by using a chemical synthesis method, the invention provides a recombinant bacterium for producing p-acetaminophenol and application thereof, and the specific technical scheme is as follows:
the first purpose of the invention is to provide a recombinant bacterium for producing p-acetamidophenol, which is characterized in that the recombinant bacterium is escherichia coli over-expressing a p-aminobenzoic acid synthase gene, a 4-amino-4-deoxychorismate lyase gene, a 4-aminobenzoic acid hydroxylase gene and an arylamine-N-acetyltransferase gene.
In one embodiment of the present invention, the p-aminobenzoic acid synthase gene is derived from Corynebacterium (YS-314) and has a nucleotide sequence shown in SEQ ID NO. 1; the 4-amino-4-deoxy-chorismate lyase gene is derived from escherichia coli (E.coli BW 25113), and the nucleotide sequence is shown as SEQ ID NO. 2; the nucleotide sequence of the 4-aminobenzoate hydroxylase gene is shown as SEQ ID NO. 3; the nucleotide sequence of the arylamine-N-acetyltransferase gene is shown in SEQ ID NO. 4.
In one embodiment of the invention, the p-aminobenzoic acid synthase gene and the 4-amino-4-deoxychorismate lyase gene are expressed from the pACYCDuet-1 plasmid; the pETDuet-1 plasmid was used to express the 4-aminobenzoate hydroxylase gene and the arylamine-N-acetyltransferase gene.
The second object of the present invention is to provide a method for constructing the above recombinant bacterium, comprising the steps of:
(1) Optimizing and synthesizing a p-aminobenzoic acid synthetase gene, a 4-amino-4-deoxy chorismate lyase gene, a 4-aminobenzoic acid hydroxylase gene and an arylamine-N-acetyltransferase gene;
(2) Connecting the p-aminobenzoic acid synthase gene and the 4-amino-4-deoxychorismate lyase gene obtained in the step (1) to the multiple cloning site of pACYCDuet-1 to obtain a recombinant plasmid pACYCDuet-ABC;
(3) Connecting the 4-aminobenzoic acid hydroxylase gene and the arylamine-N-acetyltransferase gene obtained in the step (1) to a multiple cloning site of pETDuet-1 to obtain a recombinant plasmid pETDuet-1-HP;
(4) The plasmids obtained in the step (1) and the step (2) are introduced into Escherichia coli BL21 (DE 3) to obtain a recombinant bacterium Escherichia coli BL21 (DE 3) -ABCHP.
The third purpose of the invention is to provide a method for producing p-acetaminophenol, which takes glucose as a substrate and utilizes the recombinant bacteria to ferment and produce p-acetaminophenol.
In one embodiment of the invention, the method for producing acetaminophen is to inoculate the recombinant bacteria in an M9 modified liquid medium and shake-flask culture to OD 600 When the fermentation time reaches 0.6, adding isopropyl thiogalactoside into the fermentation liquor, and stopping fermentation after induction for 42-52 h.
In one embodiment of the present invention, the M9 modified liquid medium groupTo be 10g/L glucose, na 2 HPO 4 6g/L，KH 2 PO 4 3g/L，NaCl 0.5g/L，NH 2 Cl 2g/L，MgSO 4 ·7H 2 O0.2465 g/L, the balance being water.
In one embodiment of the present invention, the M9 modified liquid medium further comprises 100mg/L ampicillin and 50mg/L chloramphenicol.
In one embodiment of the present invention, the amount of the recombinant bacterium inoculated is 1% to 2%.
In one embodiment of the present invention, the shake flask culture is performed at 37 ℃ and 180 to 200rpm.
The invention has the beneficial effects that:
the invention realizes the purpose of obtaining the acetaminophen by microbial fermentation for the first time, completes the production of the acetaminophen by fermentation by taking escherichia coli as a chassis cell and taking common carbohydrate glucose and the like as substrates, avoids the generation of toxic and explosive precursor intermediates and the use of a metal catalyst in the chemical synthesis process, is safe, green, simple, convenient and efficient because the whole synthesis process is carried out at normal temperature and normal pressure, and provides a new technical method for the production of the acetaminophen.
Drawings
FIG. 1 is a schematic diagram of the metabolic pathway for the synthesis of acetaminophen using glucose;
FIG. 2 shows LC-MS detection of recombinant E.coli fermentation product acetaminophen.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are all conventional in the art and are commercially available.
The enzyme reagents used in the following examples were purchased from NEW ENGLAND BioLabs; the kit for extracting the plasmid and the kit for recovering the DNA fragment are purchased from American OMEGA company, and the corresponding operation steps are carried out according to the product instruction; all media were prepared with deionized water unless otherwise specified.
The formula of the culture medium is as follows:
LB medium: 5g/L yeast powder, 10g/L NaCl,10g/L peptone and the balance of water, and sterilizing at 121 ℃ for 20min.
M9 modified liquid medium: glucose 10g/L, na 2 HPO 4 6g/L，KH 2 PO 4 3g/L，NaCl 0.5g/L，NH 2 Cl 2g/L，MgSO 4 ·7H 2 O0.2465 g/L, the balance being water, sterilizing at 115 ℃ for 30min.
During the actual culture process, antibiotics with a certain concentration, such as 100mg/L ampicillin and 50mg/L chloramphenicol, can be added to the above culture medium to maintain the stability of plasmids.
Example 1: construction of recombinant bacterium
1. Obtaining of genes
Optimizing and synthesizing p-aminobenzoate synthase gene, 4-amino-4-deoxychorismate lyase gene, 4-aminobenzoate hydroxylase gene and arylamine-N-acetyltransferase gene, wherein the p-aminobenzoate synthase gene is derived from Corynebacterium (Corynebacterium efficiens YS-314), and the nucleotide sequence is shown as SEQ ID NO. 1; 4-amino-4-deoxy-chorismate lyase gene, which is derived from Escherichia coli (E.coli BW 25113) and has a nucleotide sequence shown in SEQ ID NO. 2; the nucleotide sequence of the 4-aminobenzoate hydroxylase gene is shown as SEQ ID NO. 3; the nucleotide sequence of the arylamine-N-acetyltransferase gene is shown in SEQ ID NO. 4.
2. Construction of recombinant plasmid pACYCDuet-ABC
1) Carrying out enzyme digestion on the plasmid pACYCDuet-1 and a p-aminobenzoic acid synthase gene fragment synthesized by the whole gene by using EcoR I and Hind III, recovering the enzyme digestion product, connecting, and carrying out the following steps of mixing the vector and the p-aminobenzoic acid synthase gene fragment according to the molar ratio of 1:3, ligation at 25 ℃ for 5min, transformation of the ligation product to E.coli DH 5. Alpha. And application to a plate containing 50. Mu.g.mL -1 PCR screening positive clone (the sequence of the forward primer is shown as SEQ ID NO. 5; the sequence of the reverse primer is shown as SEQ ID NO. 5) on LB solid plate of chloramphenicolID No. 6), recombinant plasmid pACYCDuet-AB was extracted from the positive clone and verified.
2) Carrying out enzyme digestion on a 4-amino-4-deoxy-chorismate lyase gene fragment synthesized by plasmid pACYCDuet-AB and a whole gene by Nde I and Xho I, recovering enzyme digestion products, then carrying out connection, transforming E.coli DH5 alpha by the connection product, and carrying out PCR screening on positive clones (the sequence of the used forward primer is shown as SEQ ID NO. 7; the sequence of the reverse primer is shown as SEQ ID NO. 8) to obtain the recombinant plasmid pACYCDuet-ABC.
3. Construction of recombinant plasmid pETDuet-1-HP
1) Digesting the plasmid pETDuet-1 and the 4-aminobenzoic acid hydroxylase gene fragment synthesized by the whole gene by using EcoR I and Hind III, recovering the digested product, connecting, and mixing the vector and the 4-aminobenzoic acid hydroxylase gene fragment according to the molar ratio of 1:3, ligation at 25 ℃ for 5min, transformation of the ligation product to E.coli DH 5. Alpha. And application to a plate containing 100. Mu.g.mL -1 Ampicillin LB solid plate, PCR screening positive clone (the sequence of the forward primer is shown as SEQ ID NO. 9; the sequence of the reverse primer is shown as SEQ ID NO. 10), extracting the recombinant plasmid pETDuet-1-H from the positive clone and verifying.
2) Carrying out enzyme digestion on plasmid pETDuet-1-H and arylamine-N-acetyltransferase gene fragments synthesized by the whole gene by Nde I and Xho I, recovering enzyme digestion products, then carrying out connection, transforming E.coli DH5 alpha by the connection products, and screening positive clones (the sequence of the used forward primer is shown as SEQ ID NO. 11; the sequence of the reverse primer is shown in SEQ ID NO. 12), and the recombinant plasmid pETDuet-1-HP is obtained.
4. Construction of recombinant bacterium
The recombinant plasmids pETDuet-1-HP and pACYCDuet-ABC are used for transforming BL21 (DE 3) competent cells and are coated on an LB solid plate added with ampicillin and chloramphenicol to obtain recombinant bacteria BL21 (DE 3) -ABCHP. A schematic diagram of the metabolic pathway for the synthesis of acetaminophen using glucose is shown in FIG. 1.
Example 2: production of p-acetaminophenol by recombinant bacteria
The activated recombinant strain was inoculated at a ratio of 1 (v/v) to 100 (v/v) to a strain containing 50mL of the modified M9 strainIn a 250mL baffled shake flask of liquid medium (containing 100. Mu.g mL of -1 Ampicillin of (1); 50 μ g mL -1 Chloramphenicol) was cultured at 37 ℃ under shaking at 180 rpm. OD 600 When the concentration reached about 0.6, isopropyl thiogalactoside (final concentration 0.1 mM) was added for induction for 48h, and the fermentation was terminated.
Centrifuging the fermentation liquor to obtain fermentation liquor supernatant, extracting with equal volume of ethyl acetate, concentrating, dissolving with 1mL of methanol, centrifuging at 13000rpm for 10min, filtering with a membrane, and detecting the fermentation product by comparing LC-MS with a standard substance (figure 2) to confirm that the product paracetamol is obtained. The yield of paracetamol obtained by the method is 11.43mg/L fermentation liquor.
It will be understood by those skilled in the art that the above experiments for the insertion of the E.coli (E.coli) gene were carried out according to standard molecular cloning techniques; the four genes overexpressed above were co-cloned into E.coli (E.coli), and each step was performed according to standard molecular cloning techniques.
The present invention is described in detail in order to make those skilled in the art understand the content and practice the invention, and the invention is not limited to the above embodiments, and all equivalent changes or modifications made according to the spirit of the invention should be covered by the scope of the invention.
SEQUENCE LISTING
<110> institute for bioenergy and Process in Qingdao of Chinese academy of sciences
<120> recombinant bacterium for producing acetaminophen and application thereof
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 1866
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gaagtgaccc aggaagaatt ttttcacacc tatcagggct ttgacgagga ggtgctggcg 900
ctgattcatt gcattaaaaa accgaccaaa tgggcgctgc atgtgctgga tcatctggat 960
atttttagca aacagcaggt gtttctgatg ggcgatgcgg cgcatgcgat gctgccacat 1020
ttaggtgcgg gtgcgaccgt tggcattgaa gatgcgtata ttctggcgag catgctgacc 1080
catcagagca ccagccgtcc attgaatagc gaaaaaatta aactgatcag caccatctac 1140
aacaccgtgc tggtgccgca tgcgacccgt atgagcaaat tgaccaatga taccggccat 1200
ctgctggatc tgaccgcgcc aggttttgat ctggaacgct ataccctggg cgatcgcatt 1260
ccgctggaaa ccttaattaa tgcgtttcgc caggtggaac gcaattggat ttggagcagc 1320
agcgatccgg aagaagatcg ccgcaaagtg gaagatctgc tggaaagctg gagcggcccg 1380
cgtttaagca ttagccatca a 1401
<210> 4
<211> 897
<212> DNA
<213> Artificial Synthesis
<400> 4
atgggcagca gccatcatca tcatcatcat agcagcggct tagttcctcg tggcagccac 60
atgactcctc tgacccctga acagacccat gcgtatctgc atcatattgg cattgatgat 120
cctggtcctc cgagcctggc gaatttagat cgcctgatcg atgcgcattt acgccgcgtt 180
gcgtttgaaa acctggatgt gctgctggat cgcccgattg aaattgatgc ggacaaagtg 240
tttgcgaaag tggtggaagg tagccgtggc ggctattgct ttgaactgaa cagcctgttt 300
gcgcgtctgt tactggcgtt aggctatgaa ctggaactgc tggtggcacg tgttcgttgg 360
ggtttacctg atgatgcgcc gttaacccag cagagccatt taatgctgcg cctgtatctg 420
gcggaaggcg aatttctggt ggatgtgggc tttggttcag cgaacccgcc tcgtgcatta 480
cctttaccgg gcgatgaagc agatgcgggt caggttcatt gtgttcgcct ggtggatcct 540
catgcgggct tatatgaaag cgcggtgcgt ggtcgtagcg gttggttacc tctgtatcgc 600
tttgatctgc gcccgcagct gtggattgat tatattccgc gcaactggta taccagcacc 660
catccgcata gcgtgtttcg ccagggtctg aaagcggcga ttaccgaagg tgatctgcgt 720
ctgaccttag cggatggtct gtttggtcag cgtgcgggta acggtgaaac cctgcagcgt 780
cagttacgcg atgtggaaga actgctggat attctgcaga cccgctttcg tttacgtctg 840
gatccggcgt cagaagttcc tgcactggcg cgtcgtttag cgggcttaat tagcgcg 897
<210> 5
<211> 18
<212> DNA
<213> Artificial Synthesis
<400> 5
atgcgcgtgc tgatcatt 18
<210> 6
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 6
cggaaattcc acacccagc 19
<210> 7
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 7
atgttcctga ttaacggcca taaac 25
<210> 8
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 8
attcggacgt tcgcataacg 20
<210> 9
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 9
atgagtcagc aggaacgtac cc 22
<210> 10
<211> 18
<212> DNA
<213> Artificial Synthesis
<400> 10
cagaacggcg cgcggacc 18
<210> 11
<211> 13
<212> DNA
<213> Artificial Synthesis
<400> 11
atgagtcagc agg 13
<210> 12
<211> 13
<212> DNA
<213> Artificial Synthesis
<400> 12
cagaacggcg cgc 13

Claims (10)

1. A recombinant bacterium for producing acetaminophen, which is Escherichia coli overexpressing a p-aminobenzoic acid synthase gene, a 4-amino-4-deoxychorismate lyase gene, a 4-aminobenzoic acid hydroxylase gene and an arylamine-N-acetyltransferase gene.
2. The recombinant bacterium according to claim 1, wherein the p-aminobenzoic acid synthase gene is derived from Corynebacterium (Corynebacterium efficiens YS-314) and has a nucleotide sequence shown in SEQ ID No. 1; the 4-amino-4-deoxy-chorismate lyase gene is derived from escherichia coli (E.coli BW 25113), and the nucleotide sequence is shown as SEQ ID NO. 2; the nucleotide sequence of the 4-aminobenzoate hydroxylase gene is shown as SEQ ID NO. 3; the nucleotide sequence of the arylamine-N-acetyltransferase gene is shown in SEQ ID NO. 4.
3. The recombinant bacterium according to any one of claims 1 or 2, wherein a p-aminobenzoic acid synthase gene and a 4-amino-4-deoxychorismate lyase gene are expressed in a pacycdue-1 plasmid; the pETDuet-1 plasmid was used to express the 4-aminobenzoate hydroxylase gene and the arylamine-N-acetyltransferase gene.
4. The method for constructing a recombinant bacterium according to any one of claims 1 to 3, wherein the method comprises the steps of:
(1) Optimizing and synthesizing p-aminobenzoate synthase gene, 4-amino-4-deoxychorismate lyase gene, 4-aminobenzoate hydroxylase gene and arylamine-N-acetyltransferase gene;
(2) Connecting the p-aminobenzoic acid synthase gene and the 4-amino-4-deoxychorismate lyase gene obtained in the step (1) to the multiple cloning site of pACYCDuet-1 to obtain a recombinant plasmid pACYCDuet-ABC;
(3) Connecting the 4-aminobenzoic acid hydroxylase gene and the arylamine-N-acetyltransferase gene obtained in the step (1) to a multiple cloning site of pETDuet-1 to obtain a recombinant plasmid pETDuet-1-HP;
(4) And (3) introducing the recombinant plasmid obtained in the step (1) and the step (2) into Escherichia coli BL21 (DE 3) to obtain a recombinant bacterium Escherichia coli BL21 (DE 3) -ABCHP.
5. A method for producing acetaminophen, which comprises producing acetaminophen by fermentation using the recombinant bacterium according to any one of claims 1 to 3, using glucose as a substrate.
6. The method according to claim 5, wherein the recombinant bacterium according to any one of claims 1 to 3 is inoculated into M9 modified liquid medium and shake-flask cultured to OD 600 When the fermentation time reaches 0.6, adding isopropyl thiogalactoside into the fermentation liquor, and stopping fermentation after induction for 42-52 h.
7. The method of claim 6, wherein the M9 modified liquid medium has a composition of glucose 10g/L, na 2 HPO 4 6g/L，KH 2 PO 4 3g/L，NaCl 0.5g/L，NH 2 Cl 2g/L，MgSO 4 ·7H 2 O0.2465 g/L, the balance being water.
8. The method as claimed in claim 7, wherein the M9 modified liquid medium further comprises 100mg/L ampicillin and 50mg/L chloramphenicol.
9. The method according to claim 6, wherein the amount of the recombinant bacterium inoculated is 1% to 2%.
10. The method according to claim 6, wherein the shake flask culture is carried out at 37 ℃ and 180 to 200rpm.
CN202110707846.9A 2021-06-24 2021-06-24 Recombinant bacterium for producing acetaminophen and application thereof Pending CN115521891A (en)

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