CN116622688A - Phenylpyruvic acid decarboxylase mutant for converting and synthesizing tyrosol and application thereof - Google Patents
Phenylpyruvic acid decarboxylase mutant for converting and synthesizing tyrosol and application thereof Download PDFInfo
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- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-hydroxyphenyl)ethanol Chemical compound OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 title claims abstract description 85
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- 235000004330 tyrosol Nutrition 0.000 title claims abstract description 42
- 230000002194 synthesizing effect Effects 0.000 title abstract description 7
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- 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
- C12N9/88—Lyases (4.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
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- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01043—Phenylpyruvate decarboxylase (4.1.1.43)
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Abstract
The invention discloses a phenylpyruvate decarboxylase mutant for synthesizing tyrosol by conversion and application thereof, belonging to the technical field of biology. The invention provides phenylpyruvate decarboxylase mutant ARO10 D331S 、ARO10 D331V And ARO10 D331C And E.coli YMGR is used as host strain, pKK223-3 plasmid is used as carrier to express phenylpyruvate decarboxylase mutant ARO10 in Saccharomyces cerevisiae D331S 、ARO10 D331V And ARO10 D331C . The strain is fermented in a 250mL shake flask with 50mL M9Y culture medium, and the tyrosol yield is 1.91g/L, 2.02g/L and 2.04g/L respectively, which are higher than the current results of most research teams.
Description
Technical Field
The invention relates to a phenylpyruvate decarboxylase mutant for synthesizing tyrosol by conversion and application thereof, belonging to the technical field of biology.
Background
Tyrosol (tyrosol) is a natural monophenol compound and is widely used in the food, cosmetic, nutritional supplement and pharmaceutical industries. It is one of the strongest natural antioxidants known, e.g. antioxidant and anticancer, and has the potential to act as a cardiac and neuroprotective agent. Thus, tyrosol has great commercial application value, and mass production of tyrosol is a necessary trend. At present, the production of tyrosol in China is still in the natural extraction and chemical synthesis stages. The natural extraction is mainly carried out from salidroside, olive fruits and leaves and residues and waste water generated in the olive oil preparation process, and has the advantages of cheap and abundant raw materials, but has the disadvantages of using strong acid steam, low recovery rate and easy secondary pollution. The substrates used in chemical synthesis are relatively expensive, and these processes typically require protection and deprotection steps, reducing overall yields; high cost, serious pollution and low safety, and can not be applied to the addition of foods and medicines.
There are a number of known tyrosol effects: (1) Synthesis of cardiovascular drugs such as metoprolol and betaxolol: preventing and treating arteriosclerosis, hypertension, heart disease, cerebral hemorrhage, etc.; (2) safe and efficient antioxidant: the product is applied to a beauty product health care product; (3) high potency stable anti-inflammatory: inhibiting allergic inflammation and other diseases; (4) anti-aging, beneficial to skeletal system: is helpful for the absorption of minerals by human body and reduces the osteoporosis; (5) Improving endocrine system function, promoting metabolism, promoting wound healing, scavenging free radicals, recovering health state of viscera and organs, and delaying aging; (6) anticancer and cancer preventing: promoting cancer later recovery and improving chemotherapy effect. Thus, mass production of tyrosol is a necessary trend.
In the prior report, although safe and efficient biological methods for synthesizing tyrosol exist, for example: tyrosol was produced by expressing phenylpyruvate decarboxylase ARO10 from Saccharomyces cerevisiae in E.coli (article "High-level production of tyrosol with noninduced recombinant escherichia coli by metabolic engineering"), the yield was to be further improved.
Disclosure of Invention
The invention aims to provide a high-activity phenylpyruvate decarboxylase mutant and a safe, efficient and low-cost biological method for synthesizing tyrosol.
The invention provides a phenylpyruvate decarboxylase ARO10 mutant, which is characterized in that on the basis of a parent enzyme ARO10 with an amino acid sequence shown as SEQ ID NO.5, any one of the following mutations is carried out:
(1) Mutation of aspartic acid at position 331 to serine, designated ARO10 D331S The method comprises the steps of carrying out a first treatment on the surface of the The amino acid sequence is shown as SEQ ID NO. 1;
(2) Mutation of aspartic acid at position 331 to valine, designated ARO10 D331V The method comprises the steps of carrying out a first treatment on the surface of the The amino acid sequence is shown as SEQ ID NO. 2;
(3) Mutation of aspartic acid at position 331 to cysteine, designated ARO10 D331C The method comprises the steps of carrying out a first treatment on the surface of the The amino acid sequence is shown as SEQ ID NO. 3;
in one embodiment, the parent enzyme phenylpyruvate decarboxylase ARO10 is derived from saccharomyces cerevisiae.
In one embodiment, the nucleotide sequence of the gene encoding the phenylpyruvate decarboxylase ARO10 is shown in SEQ ID No. 4.
The invention also provides a mutant ARO10 encoding the phenylpyruvate decarboxylase D331S 、ARO10 D331V Or ARO10 D331C Is a gene of (a).
In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO. 6-8.
The invention also provides a recombinant vector carrying the gene.
The invention also provides a method for expressing the phenylpyruvate decarboxylaseVariant ARO10 D331S 、ARO10 D331V Or ARO10 D331C Or a recombinant microbial cell containing said gene or said recombinant vector.
In one embodiment of the invention, the recombinant microbial cell is a bacterial or fungal host cell.
The invention also provides a recombinant escherichia coli which expresses the phenylpyruvate decarboxylase ARO10 D331S 、ARO10 D331V Or ARO10 D331C 。
In one embodiment, the recombinant E.coli is a host cell of E.coli BL21 (DE 3) or E.coli MG 1655.
In one embodiment, the recombinant E.coli is an expression vector of pEtac or pkk 223-3.
The invention also provides a method for constructing the recombinant escherichia coli, which comprises the following steps:
(1) The feaB, pheA, tyrB, tyrR gene in E.coli MG1655 was knocked out to construct strain E.coli YMGR.
(2) Chemically synthesizing phenylpyruvate decarboxylase ARO10 and mutant genes.
(3) Preparing recombinant plasmid pKK223-ARO10 by double-enzyme cutting the gene of phenylpyruvate decarboxylase ARO10 and plasmid pKK-223-3 and connecting to obtain recombinant plasmid pKK223-ARO10.
(4) Synthesis of phenylpyruvate decarboxylase ARO10 mutant by full plasmid PCR to obtain recombinant plasmid
pKK223-ARO10 D331S 、pKK223-ARO10 D331V 、pKK223-ARO10 D331C 。
(5) The recombinant plasmid is introduced into a strain E.coli YMGR to obtain a recombinant tyrosol production strain E.coli YMGR/pKK223-ARO10 or a recombinant tyrosol high-yield strain E.coli YMGR/pKK223-ARO10 mutant.
The invention also provides a method for producing tyrosol by adopting the recombinant E.coli YMGR/pKK223-ARO10 mutant.
In one embodiment, the method comprises inoculating the recombinant E.coli YMGR/pKK223-ARO10 mutant into a seed culture medium for culture to prepare a seed solution; inoculating the seed liquid into a reaction system containing glucose, and fermenting to prepare tyrosol.
In one embodiment, the glucose-containing reaction system is a fermentation medium.
In one embodiment, the fermentation medium is an M9Y medium comprising, in g/L: na (Na) 2 HPO 4 ·12H 2 O17.1,KH 2 PO 4 3,NaCl 0.5,NH 4 Cl 1, glucose 20, yeast powder 0.25, mgSO 4 5mmol·L -1 。
In one embodiment, the seed solution is prepared by the following steps: inoculating the recombinant E.coli YMGR/pKK223-ARO10 mutant to an LB solid medium for culture to obtain single bacterial colonies, picking the single bacterial colonies, inoculating the single bacterial colonies to an LB liquid medium, and culturing for 10-14 h at the temperature of 35-39 ℃ under the condition of 180-220 r/min to obtain seed liquid.
In one embodiment, the prepared seed liquid is inoculated into LB liquid culture medium, cultured for 10-14 h under the conditions of 35-39 ℃ and 180-220 r/min, and then the thalli are collected; inoculating the collected thalli into an M9Y liquid culture medium for fermentation culture, and preparing the tyrosol.
In one embodiment, the seed solution is inoculated into LB liquid medium in a ratio of 1% (v/v) of the inoculum size.
In one embodiment, the fermentation is at 28-32 ℃ for at least 48 hours.
In one embodiment, the ARO10 mutant is ARO10 D331S 、ARO10 D331V Or ARO10 D331C 。
The invention also provides application of the mutant or the recombinant escherichia coli in preparation of tyrosol and tyrosol-containing products.
Advantageous effects
(1) The phenylpyruvate decarboxylase mutant obtained by the invention has higher synthesis efficiency, and the tyrosol is synthesized by using glucose.
(2) On the basis of the prior art, the invention utilizes the technology of modern biological genetic engineering to construct recombinant E.coli YMGR/pKK223-ARO10 expressing the high-yield tyrosol of the phenylpyruvate decarboxylase mutant on the basis of the existing tyrosol high-yield strain, and can realize the conversion of glucose into tyrosol. Recombinant strain E.coli YMGR/pKK223-ARO10 D331S 、E.coli YMGR/pKK223-ARO10 D331V 、E.coli YMGR/pKK223-ARO10 D331C The yield of the product can reach 1.91 g.L respectively -1 ,2.02g·L -1 ,2.04g·L -1 。
Drawings
FIG. 1 shows the tyrosol production during fermentation of the strains E.coli YMGR, E.coli YMGR/pKK223-ARO10 constructed according to the invention.
FIG. 2 shows the strains E.coli YMGR/pKK223-ARO10, E.coli YMGR/pKK223-ARO10 constructed in this example D331S 、E.coli YMGR/pKK223-ARO10 D331V 、E.coli YMGR/pKK223-ARO10 D331C Tyrosol yield during fermentation.
Detailed Description
The following examples relate to the following media:
LB medium formulation (g/L): yeast powder 5, peptone 10, naCl 10, and 1.5% -2.0% agar powder.
M9Y Medium formulation (g/L): na (Na) 2 HPO 4 ·12H 2 O 17.1,KH 2 PO 4 3,NaCl 0.5,NH 4 Cl 1, glucose 20, yeast powder 0.25, mgSO 4 5mmol·L -1 。
The detection method involved in the following examples is as follows:
the tyrosol detection method comprises the following steps: high Performance Liquid Chromatography (HPLC) is used for detection. The chromatographic detection conditions are specifically as follows: agela Innoval C18 column (4.6X1250 mm, pore size 5 μm); a mobile phase of 80% aqueous solution of 0.1% formic acid, 20% methanol; the flow rate is 1mL/min; the sample injection amount is 10 mu L; an ultraviolet detector for detecting a wavelength of 280nm; the column temperature was 28 ℃.
Table 1: primer sequences
Table 2: preparation of PCR System
Example 1: construction of recombinant plasmid pKK223-ARO10
The ARO10 gene with the nucleotide sequence shown in SEQ ID NO.4 is chemically synthesized, ecoR I and Hind III cleavage sites are designed in primers p-ARO10-L and p-ARO10-R (shown in table 1), target fragments and pKK223-3 plasmids are subjected to cleavage purification at two sites of EcoR I and Hind III, then are connected by using Solution I ligase, are transferred into E.coli JM109 by a chemical conversion method, are coated on LB solid medium plates containing kanamycin resistance, are cultured for 10-12 hours in a 37 ℃ incubator, and the single colonies which are grown are subjected to cleavage verification, and the recombinant plasmid pKK223-ARO10 is extracted after the correct strains are cultured.
Example 2: construction of recombinant plasmid containing phenylpyruvate decarboxylase mutant
Site-directed mutagenesis primers (shown in Table 1) were designed, and single-point mutagenesis was performed using the recombinant plasmid pKK223-ARO10 constructed in example 1 as a template, and the PCR reaction system was as shown in Table 2. Obtaining ARO10 containing mutants respectively D331S 、ARO10 D331V 、ARO10 D331C Recombinant plasmid pKK223-ARO10 of (E) D331S 、pKK223-ARO10 D331V 、pKK223-ARO10 D331C 。
Example 3: construction of recombinant strains containing phenylpyruvate decarboxylase mutants
Construction of E.coli YMGR: construction of the feaB gene knockout cassette: primers NfeaBU and NfeaBD with homology arms of the feaB gene were designed based on the gene sequence of feaB (GenBank ID: 945933), and the size of the knockout cassette containing the Kana-resistant gene feaB was 1.3kb by PCR amplification using plasmid pKD13 as a template and NfeaBU and NfeaBD as primers. Primers were designed in a similar manner, npheAU, npheAD; ntyrBU, ntyrBD; ntyrRU, ntyrRD and amplified with pKD13 as template to obtain the gene pheA, tyrB, tyrR knockout cassette of 1.3kb. Four pairs of verification primers, yfeaBU, yfeaBD respectively, were designed for the feaB, pheA, tyrB, tyrR four genes; ypheAU, ypheAD; ytyrBU, ytyrBD; ytyrRU, ytyrRD (shown in Table 3).
After constructing the deletion cassette, sequencing and verifying correct, and then converting the linearized deletion cassette into E.coli MG1655 competence, and obtaining the recombinant strain E.coli YMGR with the gene feaB, pheA, tyrB and tyrR knocked out by a Red homologous recombination method.
TABLE 3 primers
The recombinant plasmids with correct sequencing verification, which are prepared in the example 2, are respectively transferred into E.coli JM109 by a chemical transformation method, coated on an LB solid culture medium plate containing ampicillin resistance, cultured for 10-12 hours in a 37 ℃ incubator, and recombinant plasmids pKK223-ARO10 and pKK223-ARO10 are extracted after the correct strains are cultured D331S 、pKK223-ARO10 D331V 、pKK223-ARO10 D331C The method comprises the steps of carrying out a first treatment on the surface of the Transferring the recombinant plasmids into E.coli YMGR respectively by an electrotransformation method, coating the E.coli YMGR on an LB solid culture medium plate containing ampicillin resistance, culturing for 10-12 hours in a 37 ℃ incubator, picking single colony for culturing to obtain recombinant escherichia coli E.coli YMGR/pKK223-ARO10 and E.coli YMGR/pKK223-ARO10 respectively D331S 、E.coli YMGR/pKK223-ARO10 D331V 、E.coli YMGR5/pKK223-ARO10 D331C 。
Example 4: construction of tyrosol production metabolic pathway by shake flask fermentation verification
(1) The recombinant E.coli YMGR/pKK223-ARO10 and E.coli YMGR strains obtained in example 3 were streaked on LB solid medium to obtain single colonies, and the single colonies were picked up and inoculated in 100mL conical flasks containing 20mL LB liquid medium and cultured at 37℃for 12h at 200 r/min; seed solutions are prepared respectively.
(2) Respectively inoculating the seed solution prepared in the step (1) into a 250mL conical flask filled with 50mL LB liquid medium according to the inoculum size of 1% (v/v), culturing at 37 ℃ for 10 hours at 200r/min, and collecting thalli;
(3) And (3) washing the thalli obtained in the step (2) once by using normal saline, transferring the thalli into a 250mL conical flask filled with 50mL M9Y liquid culture medium, culturing for 72 hours at 30 ℃ and 200r/min to prepare a fermentation broth, and detecting the tyrosol yield in the fermentation broth. Tyrosol production of each recombinant strain is shown in table 4 and figure 1.
TABLE 4 tyrosol production by different recombinant E.coli
Time h | E.coli YMGR(g/L) | E.coli YMGR/Pkk223-ARO10(g/L) |
0 | 0.00 | 0.00 |
12 | 0.00 | 0.32 |
24 | 0.00 | 0.63 |
36 | 0.00 | 0.97 |
48 | 0.00 | 1.29 |
60 | 0.00 | 1.31 |
72 | 0.00 | 1.34 |
Example 5: shake flask fermentation to verify mutant strain high-yield tyrosol
(1) Recombinant E.coli YMGR/pKK223-ARO10 and E.coli YMGR/pKK223-ARO10 constructed in example 3 were used respectively D331S 、E.coli YMGR/pKK223-ARO10 D331V 、E.coli YMGR5/pKK223-ARO10 D331C Streaking culture on LB solid medium to obtain single colony, selecting single colony, inoculating into 100mL conical flask containing 20mL LB liquid medium, and culturing at 37deg.C and 200r/min for 12 hr; seed solutions are prepared respectively.
(2) Respectively inoculating the seed solution prepared in the step (1) into a 250mL conical flask filled with 50mL LB liquid medium according to the inoculum size of 1% (v/v), culturing at 37 ℃ for 10 hours at 200r/min, and collecting thalli;
(3) After the thalli obtained in the step (2) are washed once by normal saline, the thalli are transferred into a 250mL conical flask filled with 50mL M9Y liquid culture medium, cultured for 84 hours at 30 ℃ and 200r/min, sampling is carried out every 12 hours, and the content of tyrosol in the fermentation broth is detected, and the results of tyrosol yield are shown in Table 5 and figure 2.
TABLE 5 tyrosol production by different recombinant E.coli
The results show that the recombinant escherichia coli E.coli YMGR/pKK223-ARO10 and E.coli YMGR/pKK223-ARO10 provided by the invention D331S 、E.coli YMGR/pKK223-ARO10 D331V 、E.coli YMGR5/pKK223-ARO10 D331C Can convert glucose into tyrosol, and the yield of the tyrosol produced by fermentation in a glucose culture medium can respectively reach 1.34g/L, 1.91g/L, 2.02g/L and 2.04g/L, and the mutant yield is respectively improved by 42.5 percent, 50.7 percent and 52.2 percent compared with the wild type.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A high-activity phenylpyruvate decarboxylase mutant, which is characterized by carrying out any one of the following mutations on the basis of a parent enzyme ARO10 with an amino acid sequence shown as SEQ ID NO. 5:
(1) Mutating aspartic acid at position 331 to serine;
(2) Mutating aspartic acid at position 331 to valine;
(3) Aspartic acid at position 331 was mutated to cysteine.
2. A gene encoding the phenylpyruvate decarboxylase mutant of claim 1.
3. A recombinant vector carrying the gene of claim 2.
4. A recombinant microbial cell expressing the phenylpyruvate decarboxylase mutant of claim 1, or comprising the gene of claim 2, or comprising the recombinant vector of claim 3.
5. The recombinant microbial cell of claim 4, wherein the microbial cell is a bacterial cell or a fungal cell.
6. A recombinant E.coli, wherein the phenylpyruvate decarboxylase mutant of claim 1 is expressed.
7. The recombinant E.coli according to claim 6, wherein E.coli BL21 (DE 3), E.coli MG1655 or E.coli CCTCC NO: m2019390 is a host cell; pEtac or pkk223-3 is used as an expression vector.
8. A process for producing tyrosol, which comprises fermenting the recombinant E.coli of claim 6 in a medium containing glucose.
9. The method of claim 7, wherein the fermentation is in M9Y liquid medium at 28-32 ℃ for at least 48 hours.
10. Use of the phenylpyruvate decarboxylase mutant of claim 1, or the recombinant escherichia coli of claim 6, or the method of any one of claims 8-9 for the preparation of tyrosol and tyrosol containing products.
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