CN111647545A - Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde - Google Patents

Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde Download PDF

Info

Publication number
CN111647545A
CN111647545A CN202010640818.5A CN202010640818A CN111647545A CN 111647545 A CN111647545 A CN 111647545A CN 202010640818 A CN202010640818 A CN 202010640818A CN 111647545 A CN111647545 A CN 111647545A
Authority
CN
China
Prior art keywords
escherichia coli
recombinant
hydroxybenzaldehyde
coli
recombinant escherichia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010640818.5A
Other languages
Chinese (zh)
Inventor
郑璞
郑义培
吴丹
陈鹏程
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN202010640818.5A priority Critical patent/CN111647545A/en
Publication of CN111647545A publication Critical patent/CN111647545A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C12N9/0079—Steroid 11 beta monooxygenase (P-450 protein)(1.14.15.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0095—Oxidoreductases (1.) acting on iron-sulfur proteins as donor (1.18)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
    • C12Y114/15004—Steroid 11-beta-monooxygenase (1.14.15.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y118/00—Oxidoreductases acting on iron-sulfur proteins as donors (1.18)
    • C12Y118/01—Oxidoreductases acting on iron-sulfur proteins as donors (1.18) with NAD+ or NADP+ as acceptor (1.18.1)
    • C12Y118/01002—Ferredoxin-NADP+ reductase (1.18.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a recombinant escherichia coli and application thereof in synthesizing p-hydroxybenzaldehyde, wherein cytochrome monooxygenase (CYP199A4), iron redox protein (HaPuR) and iron redox protein reductase (HaPux) which are derived from Rhodopseudomonas palustris (Rhodopseudomonas palustris) are used as target genes, pRSFduet-1 and pETduet-1 are used as vectors, escherichia coli E.coli BL21 is used as a host, and genetically engineered bacteria which generate p-hydroxybenzaldehyde by using anisaldehyde as a substrate are successfully constructed. The recombinant Escherichia coli whole cell is used as a catalyst, and 1g/L anisaldehyde can be converted into 250mg/L p-hydroxybenzaldehyde within 12 h. The method for transforming the p-hydroxybenzaldehyde into the microorganism whole cells has the advantages of mild reaction conditions, simple process, easy control and the like, is beneficial to environmental protection and is easy to popularize and apply.

Description

Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde
Technical Field
The invention relates to recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde, belonging to the technical field of genetic engineering.
Background
P-hydroxybenzaldehyde is an important organic intermediate for synthesizing spices, medicines and the like, and has wide application in the fields of chemical industry, agriculture, liquid crystal, electroplating, food additives and the like. In the field of pesticides, p-hydroxybenzaldehyde is mainly used for synthesizing herbicides bromoxynil and hydroxydiuril; in the field of medicine, p-hydroxybenzaldehyde can be used for synthesizing amoxicillin, antibacterial synergist trimethoprim, 3,4, 5-trimethoxybenzaldehyde, p-hydroxy glycine, amoxicillin and cefamycin, artificial rhizoma gastrodiae, rhododendron, eisenlol and the like; in the perfume industry, p-hydroxybenzaldehyde is mainly used for synthesizing vanillin, ethyl vanillin, heliotropin, syringaldehyde, anisaldehyde, raspberry ketone and other perfumes. In addition, p-hydroxybenzaldehyde can also be used for producing bactericides, photographic emulsifiers, nickel-plating gloss agents, liquid crystals and the like.
The production of the p-hydroxybenzaldehyde has a plurality of process routes, and the current industrial production mainly comprises phenol; p-cresol; a raw material route of p-nitrotoluene and the like. (1) The phenol method is also divided into Reimer-Tiemann reaction; gattermann reaction; the phenol-chloral route; the phenol-glyoxylic acid route; phenol-formaldehyde routes and other various synthetic process routes. The phenol method has the technical characteristics of easily obtained raw materials, simpler manufacturing process, lower yield and higher cost. (2) A p-nitrotoluene method, wherein the technical process for producing the p-hydroxybenzaldehyde by the p-nitrotoluene method comprises oxidation reduction; diazotization and hydrolysis. (3) The catalytic oxidation process of p-cresol is to oxidize p-cresol directly with air or oxygen to synthesize p-hydroxybenzaldehyde under the action of catalyst.
However, the above methods are all chemical synthesis methods, and have problems of environmental pollution, safety, and the like. At present, the strain capable of directly fermenting to generate the parahydroxybenzaldehyde is adopted to ferment to produce the parahydroxybenzaldehyde, but the fermentation product obtained by directly fermenting to produce the parahydroxybenzaldehyde by adopting the microorganism has complex components and higher purification difficulty. Therefore, the method for producing the p-hydroxybenzaldehyde by using the biological catalysis method meets the requirement of green chemistry, has bright application prospect in industrial production, but has few and immature related technologies at present.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of natural equivalent P-hydroxybenzaldehyde by constructing recombinant escherichia coli and catalyzing natural anisic aldehyde to synthesize P-hydroxybenzaldehyde through whole cells by utilizing the demethylation characteristic of P450 enzyme CYP199A 4.
The first purpose of the invention is to provide a recombinant Escherichia coli, wherein the recombinant Escherichia coli is obtained by co-expressing cytochrome monooxygenase (CYP199A4) and ferredoxin reductase (HaPux) in an Escherichia coli host by a pRSFduet-1 vector and co-expressing ferredoxin (HaPuR) and ferredoxin reductase (HaPux) in a pETduet-1 vector.
Further, the nucleotide sequence of the gene for coding the cytochrome monooxygenase is shown as SEQ ID NO. 1.
Further, the nucleotide sequence of the gene for coding the ferroxidation-reduction protein is shown as SEQ ID NO. 2.
Further, the nucleotide sequence of the gene for coding the ferrodoxin reductase is shown as SEQ ID No. 3.
Further, the escherichia coli host is e.coli BL 21.
The second purpose of the invention is to provide the construction method of the recombinant Escherichia coli, which comprises the following steps:
(1) constructing a recombinant plasmid: the cytochrome monooxygenase (CYP199A4) and the iron redox protein reductase (HaPux) genes are respectively inserted into the enzyme cutting sites BamHI/SacI and NdeI/XhoI on the pRSFduet-1 plasmid to obtain a recombinant plasmid pRSFduet-1-CYP199A 4-HaPux; respectively inserting genes of ferroxidation-reduction protein (HaPuR) and ferroxidation-reduction protein reductase (HaPux) into enzyme cutting sites NdeI/XhoI and XbaI/EcoRI on a pETduet-1 plasmid to obtain a recombinant plasmid pETduet-1-HaPuR-Ha Pux;
(2) and sequentially transferring the recombinant plasmid pRSFduet-1-CYP199A4-HaPux and the recombinant plasmid pETduet-1-Ha PuR-HaPux into an escherichia coli host, and screening positive transformants to obtain the recombinant escherichia coli.
The third purpose of the invention is to provide the application of the recombinant Escherichia coli in synthesizing p-hydroxybenzaldehyde.
Furthermore, the recombinant escherichia coli whole cell is used as a catalyst to catalyze the conversion of anisaldehyde into p-hydroxybenzaldehyde.
Further, the application specifically comprises the steps of re-suspending recombinant escherichia coli by adopting an LB culture medium until the wet weight of the cells is 3-5 g/L, adding a 2-4 mM substrate anisaldehyde for conversion, wherein the conversion temperature is 25-40 ℃, and the conversion pH is 7-8.
Further, the recombinant escherichia coli whole cell is obtained by culturing the recombinant escherichia coli at 35-40 ℃ to the late logarithmic growth stage, and then transferring the recombinant escherichia coli to 20-28 ℃ for induction culture for 16-24 hours.
The invention has the beneficial effects that:
the invention successfully constructs the genetically engineered bacteria which generate the p-hydroxybenzaldehyde by taking anisaldehyde as a substrate by taking cytochrome monooxygenase (CYP199A4), iron redox protein (HaPuR) and iron redox protein reductase (HaPux) which are derived from Rhodopseudomonas palustris (Rhodopseudomonas palustris) as target genes, pRSFduet-1 and pETduet-1 as vectors and Escherichia coli E.coli BL21 as hosts. The recombinant Escherichia coli whole cell is used as a catalyst, and 1g/L anisaldehyde can be converted into 250mg/L p-hydroxybenzaldehyde within 12 h.
The method for transforming the p-hydroxybenzaldehyde into the microorganism whole cells has the advantages of mild reaction conditions, simple process, easy control and the like, is beneficial to environmental protection and is easy to popularize and apply.
Drawings
FIG. 1 shows the synthesis of p-hydroxybenzaldehyde by catalyzing anisic aldehyde with biological method;
FIG. 2 shows HPLC determination of anisic aldehyde and p-hydroxybenzaldehyde in recombinant E.coli whole cell transformation fluid.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
The media and assay methods designed in the following examples were as follows:
10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl in an LB culture medium (2% agar powder is added in a solid culture medium).
HPLC analysis:
the instrument comprises the following steps: waters 2487; the type of the chromatographic column: amethyl C18-H; mobile phase: acetonitrile, water: formic acid 60:40:0.1 (V/V); flow rate: 1.0 mL/min-1(ii) a Column temperature: 30 ℃; sample introduction amount: 10-20 μ L; a detector: an ultraviolet detector; detection wavelength: 300 nm.
Example 1: plasmid construction
And (3) carrying out codon optimization on the gene sequences for encoding the CYP199A4, the HaPuR and the HaPux, and obtaining cytochrome monooxygenase, ferredoxin and ferredoxin reductase genes with the sequences as encoded by SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 after the codon optimization. The recombinant plasmids pRSFduet-1-CYP199A4-HaPux and pETduet-1-HaPuR-HaPux were obtained by one-step cloning. CYP199A4 (primers P1, P2) and HaPux (primers P3, P4) genes were inserted into the cleavage sites BamHI/SacI and NdeI/XhoI on pRSFduet-1 plasmid, respectively, and HaPuR (primers P5, P6) and HaPux (primers P7, P8) genes were inserted into the cleavage sites NdeI/XhoI and XbaI/EcoRI on pETduet-1 plasmid, respectively. Wherein the PCR reaction conditions are as follows: 5min at 95 ℃; 10s at 98 ℃, 5s at 55 ℃, 5s/kb at 72 ℃ (30 cycles); after 10min at 72 ℃, the PCR amplification product was verified and recovered by electrophoresis on a 1% agarose gel. One-step cloning method the fragment (0.06pmol) and the vector (0.03pmol) were mixed in the same PCR tube, and 2-4. mu.l of recombinase buffer and 1-2. mu.l of recombinase were added, followed by completion with ddH2O to 10-20. mu.l, followed by reaction at 37 ℃ for 30 min.
P1(SEQ ID NO.4)：ccacagccaggatccgaattcATGATCAGCAATAGCAGTGCGG
P2(SEQ ID NO.5)：gcattatgcggccgcaagcttTTACGCCGGGGTGAGTTTC
P3(SEQ ID NO.6)：gcgatcgctgacgtcggtaccATGCCGAGCATCACCTTCAT
P4(SEQ ID NO.7)：ggtttctttaccagactcgagTTAGGTCTGGCGTTCTGGCA
P5(SEQ ID NO.8)：taagaaggagatatacatatgATGAATGACACCGTTCTGATTGC
P6(SEQ ID NO.9)：ggtttctttaccagactcgagTTACGCCATCGCCTTCTTCA
P7(SEQ ID NO.10)：aggcgcgccgagctcgaattcATGCCGAGCATCACCTTCAT
P8(SEQ ID NO.11)：ggataacaattcccctctagaTTAGGTCTGGCGTTCTGGCA。
Example 2: construction of recombinant bacterium
And (3) transforming the recombinant plasmids pRSFduet-1-CYP199A4-HaPux and pETduet-1-HaPuR-HaPux into Escherichia coli E.coli BL21 by a heat shock method to obtain the recombinant Escherichia coli E.coli BL21/pRSFduet-1-CYP199A 4-HaPux/pETduet-1-HaPuR-HaPux.
Basic operation of the thermal shock method:
(1) melting competent cells of Escherichia coli preserved at-80 deg.C in ice for 10 min;
(2) adding 0.1 ng-10 ng of corresponding plasmid into the competent cells of the escherichia coli, gently mixing uniformly, and placing in ice for 30 min;
(3) performing water bath heat shock at 42 ℃ for 45-90 s, and immediately placing in ice for 1-2 min;
(4) add 890 ul LB medium;
(5) culturing in a shaking table at 37 deg.C and 180-300 rmp for 1 hr;
(6) taking a proper amount of bacterial liquid to coat a resistant plate containing 50ug/mL kanamycin and 100ug/mL ampicillin, and inverting the plate in an incubator at 37 ℃ overnight;
(7) single colonies were picked for further experiments.
Screening of Positive transformants of coli BL21/pRSFduet-1-CYP199A4-HaPux/pETduet-1-HaPuR-HaPux recombinant Strain
Colonies growing on kanamycin-and ampicillin-resistant LB plates were selected, cultured in a shake flask, and transformed into whole cells at a temperature of 30 ℃ and a pH of 7 in a concentration of 1g/L in a transformant system of 15 mL. The product p-hydroxybenzaldehyde in the conversion solution was detected by HPLC. The product p-hydroxybenzaldehyde is detected, which shows that the recombinant strain for converting the anisic aldehyde into the p-hydroxybenzaldehyde is successfully constructed.
Blank control: and (3) transforming the plasmids pRSFduet-1 and pETduet-1 into Escherichia coli E.coli BL21 by a heat shock method to obtain the recombinant strain E.coli BL 21/pRSFduet-1/pETduet-1. Colonies growing on kanamycin-and ampicillin-resistant LB plates were selected, cultured in a shake flask, and transformed into whole cells at a temperature of 30 ℃ and a pH of 7 in a concentration of 1g/L in a transformant system of 15 mL. The product p-hydroxybenzaldehyde in the conversion solution was detected by HPLC, and as a result, no p-hydroxybenzaldehyde was detected.
Example 3: recombinant Escherichia coli protein expression conditions
(1) Effect of inducer IPTG concentration on conversion Rate
The recombinant E.coli BL21/pRSFduet-1-CYP199A4-Ha Pux/pETduet-1-HaPuR-HaPux constructed in example 2 was inoculated as a single colony in 30mL of LB medium and cultured for 12 hours. Inoculating 10% of the strain into a 250mL container containing 30mL of LB medium, culturing at 37 deg.C and 220r, and culturing at OD600When the concentration reaches 0.6, 0, 0.2, 0.4, 0.6, 0.8 and 1.0mmol/L IPTG are respectively added, the mixture is cultured for 20 hours at 25 ℃, the thalli are collected by centrifugation, and the recombinant escherichia coli is resuspended by 15mL of LB culture medium. The conversion system was 15mL, the temperature was 30 ℃, the pH was 7, and the substrate concentration was 1 g/L. HPLC detection is carried out on anisaldehyde and p-hydroxybenzaldehyde in the recombinant bacteria whole cell transformation liquid after 3h of whole cell transformation, and the result shows that the transformation rate is highest when 0.2mmol/L IPTG is used, and the concentration of the product p-hydroxybenzaldehyde is 77 mg/L.
(2) Effect of Induction temperature on conversion Rate
The recombinant E.coli BL21/pRSFduet-1-CYP199A4-Ha Pux/pETduet-1-HaPuR-HaPux constructed in example 2 was inoculated as a single colony in 30mL of LB medium and cultured for 12 hours. Inoculating 10% of the strain into a 250mL container containing 30mL of LB medium, culturing at 37 deg.C and 220r, and culturing at OD600Adding 0.2mmol/L IPTG when the concentration reaches 0.6, culturing at 16 deg.C, 19 deg.C, 22 deg.C, 25 deg.C, 28 deg.C and 31 deg.C for 20 hr, centrifuging, and collectingThe cells were collected and the recombinant E.coli was resuspended in 15mL of LB medium. The conversion system was 15mL, the temperature was 30 ℃, the pH was 7, and the substrate concentration was 1 g/L. After 3h of whole-cell transformation, HP LC detection is carried out on anisaldehyde and p-hydroxybenzaldehyde in the recombinant bacteria whole-cell transformation liquid, and the result shows that the transformation rate reaches the highest at 16 ℃, and the concentration of the product p-hydroxybenzaldehyde is 78 mg/L.
(3) Effect of duration of Induction on transformation Rate
The recombinant E.coli BL21/pRSFduet-1-CYP199A4-Ha Pux/pETduet-1-HaPuR-HaPux constructed in example 2 was inoculated as a single colony in 30mL of LB medium and cultured for 12 hours. Inoculating 10% of the strain into a 250mL container containing 30mL of LB medium, culturing at 37 deg.C and 220r, and culturing at OD600When the concentration reached 0.6, 0.2mmol/L IPTG was added, and the cells were cultured at 16 ℃ for 16, 18, 20, 22 and 24 hours, respectively, and the cells were collected by centrifugation, and the recombinant E.coli was resuspended in 15mL of LB medium. The conversion system was 15mL, the temperature was 30 ℃, the pH was 7, and the substrate concentration was 1 g/L. After 3h of whole-cell transformation, HPLC detection is carried out on anisaldehyde and p-hydroxybenzaldehyde in the recombinant bacteria whole-cell transformation liquid, and the result shows that the transformation rate reaches the highest at 22h, and the concentration of the product p-hydroxybenzaldehyde is 130 mg/L.
Example 4: detection of recombinant strain whole cell transformation performance
The recombinant E.coli BL21/pRSFduet-1-CYP199A4-Ha Pux/pETduet-1-HaPuR-HaPux constructed in example 2 was inoculated as a single colony in 30mL of LB medium and cultured for 12 hours. Inoculating 10% of the strain into a 250mL container containing 30mL of LB medium, culturing at 37 deg.C and 220r, and culturing at OD600When the concentration reached 0.6, 0.2mmol/L IPTG was added, and the mixture was cultured at 16 ℃ for 22 hours, centrifuged to collect cells, and the recombinant E.coli was resuspended in 15mL of LB medium. The conversion system was 15mL, the temperature was 30 ℃, the pH was 7, and the substrate concentration was 1 g/L. HPLC detection is carried out on anisaldehyde and p-hydroxybenzaldehyde in the recombinant bacteria whole cell transformation liquid, and the transformation rate reaches the highest after 12h whole cell transformation. Standard HPLC detection of anisaldehyde and p-hydroxybenzaldehyde is shown in FIG. 2.
The results showed that 1g/L anisaldehyde was converted to 250mg/L p-hydroxybenzaldehyde after 12h whole cell transformation.
Comparative example 1:
the codon-optimized CYP199A4 (primers P1, P2) gene in example 1 was inserted into the cleavage site BamHI/SacI on pRS Fduet-1 plasmid, and the HaPuR (primers P5, P6) and HaPux (primers P7, P8) genes were inserted into the cleavage site NdeI/XhoI and XbaI/Eco RI on pETduet-1 plasmid, respectively, to obtain recombinant plasmids pRSFduet-1-CYP199A4 and pETduet-1-HaPuR-HaPux. And (3) transforming the recombinant plasmid into escherichia coli E.coli BL21 by a heat shock method to obtain a recombinant strain.
The recombinant bacterium obtained was cultured and transformed into whole cells under the conditions described in example 4, and it was found that anisaldehyde was not consumed in the fermentation broth and that p-hydroxybenzaldehyde was not produced.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of south of the Yangtze river
<120> recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde
<160>11
<170>PatentIn version 3.3
<210>1
<211>1233
<212>DNA
<213> (Artificial sequence)
<400>1
atgatcagca atagcagtgc ggagagcatc agtgccccgc cgaacgatag cacgatcccg 60
catctggcga tcgatccgtt tagcctcgac ttcttcgatg acccgtaccc ggatcagcaa 120
acgctgcgtg acgccggtcc ggtggtgtac ctcgacaagt ggaatgttta cggcgtggcc 180
cgctatgcgg aagtgcatgc ggttctgaac gacccgacga ccttctgcag cagccgcggt 240
gttggtctga gcgattttaa gaaggagaag ccgtggcgtc cgccgagtct gattctcgaa 300
gccgatccac cagcgcatac gcgtccgcgt gccgttctca gcaaagttct gagcccagcc 360
accatgaaga cgatccgcga tggtttcgcc gcggcggccg atgcgaaggt tgatgaactg 420
ctgcagcgcg gctgcatcga cgcgattgcg gatctggccg aagcctaccc actgagcgtg 480
tttccagatg cgatgggcct caaacaagaa ggccgcgaac atctgctgcc gtatgccggt 540
ctggtgttca atgcgttcgg tccgccgaat gaactccgcc agaccgccat tgaacgtagc 600
gccccgcacc aagcctacgt taacgaacag tgccagcgtc cgaatctcgc gccgggcggt 660
tttggtgcgt gcatccatgc gttcaccgat acgggcgaaa ttaccccgga tgaagcccca 720
ctcctcgttc gcgatctgct gagtgccggt ctcgatacca ccgttaatgg tatcggcgcc 780
gcggtttatt gtctggcccg ctttccgggt gaactccagc gtctgcgtag cgatccaacg 840
ctggcccgca atgcgttcga agaagcggtg cgtttcgaga gcccggtgca gaccttcttt 900
cgcacgacga cccgcgaagt tgaactgggt ggcgcggtga tcggcgaagg cgaaaaggtg 960
ctgatgtttc tgggcagcgc gaatcgcgat ccacgtcgct ggagcgatcc agatctgtac 1020
gacatcaccc gcaaaacgag cggccatgtt ggctttggca gcggcgttca tatgtgtgtt 1080
ggccagctcg tggcccgtct ggaaggcgaa gtgatgctga gtgcgctggc ccgtaaggtt 1140
gccgcgatcg atatcgatgg cccggttaaa cgccgcttca acaatacgct gcgcggtctg 1200
gaaagtctgc cggtgaaact caccccggcg taa 1233
<210>2
<211>1218
<212>DNA
<213> (Artificial sequence)
<400>2
atgaatgaca ccgttctgat tgccggtgcc ggtcatgcgg gcttccaagt tgccgttagt 60
ctgcgtcaag ccaaatacac cggccgcatc gccctcatca acgacgaaaa acatctgccg 120
taccagcgcc cgccactgag caaagcgtat ctcaaaagcg gtggtgaccc gaacagtctg 180
atgtttcgcc cggagaagtt cttccaagat cagaccattg agctgattga tggtcgtgcc 240
gtggccattg atcgcgacgc caaaacgctg ctgctggcga gcggcgataa gatcgaatac 300
ggccatctgg ttctggcgac cggcgcgcgt aatcgccaac tggatgtgcc gaacgcgacg 360
ctggatgatg ttctgtatct ccgcacgctg gacgaaagcg aagttgttcg ccagcgcatg 420
ccggagaaga aacacgtggt ggttatcggc gcgggtttca tcggtctgga atttgccgcg 480
accgcgcgtg gtaaaggtat ggaagtggac gtggtggaac tggccccgcg tgttatggcc 540
cgtgccgtta ccccggagat cagcagttac ttccacgacc gtcacaccgc cgcgggtatc 600
cgcattcatt acggcgttcg cgccaccgaa atcgaaggtg aagatggccg tgttaccggt 660
gtggcgctga gtgatggccg tacgctgccg tgcgatctgg tggttgtggg tgtgggcgtt 720
attccgaacg tggaactcgc gagtgcggcc ggtctgccaa cggcggccgg catcatcgtg 780
aacgaacagc tgctcaccga agacccgaat atcagcgcca ttggcgactg cgcgctgttc 840
aatagtgtgc gtttcggcga ggtgatgcgc gtggaaagcg tgcagaatgc caccgatcaa 900
gcgcgttgcg ttgcggcccg cctcaccggt agtccagcca cctacgacgg ctatccgtgg 960
ttctggagcg atcaaggcga cgataaactg cagatcgcgg gcctcacggc gggctttgat 1020
caagttgttc tgcgcggcag cgttgccgaa cgtagcttca gcgccttctg ctacaaagac 1080
ggccagctga tcggcgttga aagcgttaat cgcgcggccg atcatgtgtt cggtcgcaaa 1140
attctgccac tgggcaaaag tgtgacgcca gagcaagccg cggatctgag ctttgatctg 1200
aagaaggcga tggcgtaa 1218
<210>3
<211>324
<212>DNA
<213> (Artificial sequence)
<400>3
atgccgagca tcaccttcat ccatccggat ggccgcagcg aaatcgttga tgccgcgatc 60
ggcgatagcg ccatgttcgc ggcgctgaat catggcatcg atagcatcgt ggccgaatgc 120
ggcggtaatg cggtttgcgc cacgtgccat gtttacgtgg atgatctgtg gctggccaaa 180
ctgccgccgg ttgatgccaa tgaggatgat ctgctggacg gtaccgccag tgatcgtctg 240
ccgaatagcc gtctgagctg ccagatcaaa atcgccccgg aactggatgg tctggttctg 300
cgtctgccag aacgccagac ctaa 324
<210>4
<211>43
<212>DNA
<213> (Artificial sequence)
<400>4
ccacagccag gatccgaatt catgatcagc aatagcagtg cgg 43
<210>5
<211>40
<212>DNA
<213> (Artificial sequence)
<400>5
gcattatgcg gccgcaagct tttacgccgg ggtgagtttc 40
<210>6
<211>41
<212>DNA
<213> (Artificial sequence)
<400>6
gcgatcgctg acgtcggtac catgccgagc atcaccttca t 41
<210>7
<211>41
<212>DNA
<213> (Artificial sequence)
<400>7
ggtttcttta ccagactcga gttaggtctg gcgttctggc a 41
<210>8
<211>44
<212>DNA
<213> (Artificial sequence)
<400>8
taagaaggag atatacatat gatgaatgac accgttctga ttgc 44
<210>9
<211>41
<212>DNA
<213> (Artificial sequence)
<400>9
ggtttcttta ccagactcga gttacgccat cgccttcttc a 41
<210>10
<211>41
<212>DNA
<213> (Artificial sequence)
<400>10
aggcgcgccg agctcgaatt catgccgagc atcaccttca t 41
<210>11
<211>41
<212>DNA
<213> (Artificial sequence)
<400>11
ggataacaat tcccctctag attaggtctg gcgttctggc a 41

Claims (10)

1. The recombinant Escherichia coli is obtained by coexpressing cytochrome monooxygenase and ferredoxin reductase by a pRSFduet-1 vector and coexpressing ferredoxin and ferredoxin reductase by a pETduet-1 vector in an Escherichia coli host.
2. The recombinant Escherichia coli of claim 1, wherein the nucleotide sequence of the gene encoding cytochrome monooxygenase is represented by SEQ ID NO. 1.
3. The recombinant Escherichia coli of claim 1, wherein the nucleotide sequence of the gene encoding said iron redox protein is represented by SEQ ID No. 2.
4. The recombinant Escherichia coli of claim 1, wherein the nucleotide sequence of the gene encoding said ferrodoxin reductase is represented by SEQ ID No. 3.
5. The recombinant E.coli of claim 1, wherein said E.coli host is E.coli BL21.
6. A method for constructing recombinant Escherichia coli according to any one of claims 1 to 5, comprising the steps of:
(1) constructing a recombinant plasmid: the cytochrome monooxygenase and the iron redox protein reductase gene are respectively inserted into the enzyme cutting sites BamHI/SacI and NdeI/XhoI on the pRSFduet-1 plasmid to obtain a recombinant plasmid pRSFduet-1-CYP199A 4-HaPux; respectively inserting ferroxidase-reductase genes and ferroxidase-reductase genes into enzyme cutting sites NdeI/XhoI and XbaI/EcoRI on a pETduet-1 plasmid to obtain a recombinant plasmid pETduet-1-HaPuR-HaPux;
(2) and sequentially transferring the recombinant plasmid pRSFduet-1-CYP199A4-HaPux and the recombinant plasmid pETduet-1-Ha PuR-HaPux into an escherichia coli host, and screening positive transformants to obtain the recombinant escherichia coli.
7. Use of the recombinant Escherichia coli of any one of claims 1 to 5 in the synthesis of p-hydroxybenzaldehyde.
8. The use of claim 7, wherein the recombinant E.coli whole cells are used as a catalyst to catalyze the conversion of anisaldehyde to p-hydroxybenzaldehyde.
9. The application of claim 8, wherein the application is to resuspend recombinant escherichia coli by adopting an LB culture medium until the wet weight of cells is 3-5 g/L, add 2-4 mM substrate anisaldehyde for conversion, the conversion temperature is 25-40 ℃, and the conversion pH is 7-8.
10. The application of claim 8, wherein the recombinant Escherichia coli whole cell is obtained by culturing recombinant Escherichia coli at 35-40 ℃ to late logarithmic growth phase, and then transferring to 20-28 ℃ for induction culture for 16-24 h.
CN202010640818.5A 2020-07-06 2020-07-06 Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde Pending CN111647545A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010640818.5A CN111647545A (en) 2020-07-06 2020-07-06 Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010640818.5A CN111647545A (en) 2020-07-06 2020-07-06 Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde

Publications (1)

Publication Number Publication Date
CN111647545A true CN111647545A (en) 2020-09-11

Family

ID=72344107

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010640818.5A Pending CN111647545A (en) 2020-07-06 2020-07-06 Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde

Country Status (1)

Country Link
CN (1) CN111647545A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112852697A (en) * 2021-01-22 2021-05-28 湖南中医药大学 Recombinant strain and method for preparing gastrodin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1249779A (en) * 1997-03-07 2000-04-05 诺瓦提斯公司 Cytochrome P450 monooxygenases
CN106103724A (en) * 2013-11-04 2016-11-09 Bgn科技有限公司 The method preparing vanillin from eugenol by the fermentable use plant dehydrogenase of ferulic acid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1249779A (en) * 1997-03-07 2000-04-05 诺瓦提斯公司 Cytochrome P450 monooxygenases
CN106103724A (en) * 2013-11-04 2016-11-09 Bgn科技有限公司 The method preparing vanillin from eugenol by the fermentable use plant dehydrogenase of ferulic acid

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AGNIESZKA ZYGADLO NIELSEN等: "Redirecting Photosynthetic Reducing Power toward Bioactive Natural Product Synthesis", 《ACS SYNTHETIC BIOLOGY》 *
REBECCA R. CHAO等: "Modification of an enzyme biocatalyst for efficient and selective oxidative demethylation of para-substituted benzene derivatives", 《CHEMCATCHEM》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112852697A (en) * 2021-01-22 2021-05-28 湖南中医药大学 Recombinant strain and method for preparing gastrodin
CN112852697B (en) * 2021-01-22 2023-02-10 湖南中医药大学 Recombinant strain and method for preparing gastrodin

Similar Documents

Publication Publication Date Title
US10837036B2 (en) Method for preparing L-aspartic acid with maleic acid by whole-cell biocatalysis
CN111100832B (en) Gene engineering bacterium for synthesizing pyruvic acid and D-alanine and construction method and application thereof
CN108048416B (en) Improved ketoreductase mutant and preparation method and application thereof
CN108949652B (en) Engineering bacterium and application thereof in producing caffeic acid
CN111647545A (en) Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde
CN109371070B (en) Method for high yield of alpha-ketoisovaleric acid
CN113817693B (en) Short-chain carbonyl reductase PpYSDR mutant, encoding gene, recombinant expression vector, genetic engineering bacterium and application
CN111826332A (en) Method for producing piperonal by using recombinant engineering bacteria co-expressing trans-anethole monooxygenase and formate dehydrogenase and engineering bacteria thereof
CN109679978B (en) Recombinant co-expression system for preparing L-2-aminobutyric acid and application thereof
CN105567655B (en) Halogen alcohol dehalogenase and application thereof in synthesis of statin drug intermediate
CN115873881A (en) Genetically engineered bacterium for producing 1,3-butanediol and application thereof
CN108949840B (en) Engineering bacterium and application thereof in production of p-hydroxycinnamic acid
CN110791467B (en) Recombinant bacterium for producing acetylacetone and construction method and application thereof
CN113215120A (en) Method for producing trans-4-hydroxy-L-proline by recombinant escherichia coli transformation
CN112011523B (en) Acetylacetone lyase mutant capable of improving acetylacetone synthesis efficiency, gene, expression vector, cell and application thereof
CN114381412B (en) Recombinant bacterium for synthesizing 3-hydroxy propionic acid and construction method and application thereof
CN115747194B (en) L-threonine aldolase mutant, gene and method for preparing L-anti-p-methylsulfonylphenyl serine
CN113388600B (en) Aldoxime dehydratase and application thereof in catalytic synthesis of aromatic nitrile compounds
CN113444699B (en) Acetylacetone lyase mutant capable of improving acetylacetone synthesis efficiency, nucleotide, expression vector, recombinant bacterium and application
CN111575258B (en) Carbonyl reductase EbSDR8 mutant and construction method and application thereof
CN113444700B (en) Acetylacetone lyase mutant capable of improving acetylacetone synthesis efficiency, nucleotide, expression vector, recombinant bacterium and application
CN113755477B (en) Nitrilase mutant and application thereof in preparation of acetophenone acid compounds
CN115247144B (en) Genetically engineered bacterium for producing L-threo-3-hydroxy aspartic acid and application thereof
CN115029329A (en) Carbonyl reductase mutant and application thereof in preparation of R-mandelic acid
CN114891710A (en) Method for strengthening catalytic synthesis of biliverdin by recombinant escherichia coli through reconstructed coenzyme regeneration system

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20200911