CN108753912B - High-throughput screening method for trans-anethole oxygenase mutant, mutant strain and mutant - Google Patents
High-throughput screening method for trans-anethole oxygenase mutant, mutant strain and mutant Download PDFInfo
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Abstract
The invention discloses a high-throughput screening method of trans-anethole oxygenase mutants, a mutant strain and a mutant, which comprises the steps of carrying out error-prone PCR by taking a trans-anethole oxygenase gene as a template to obtain a trans-anethole oxygenase mutant library; adding the trans-anethole oxygenase reaction substrate into a solution containing a trans-anethole oxygenase mutant for catalytic reaction, carrying out color development reaction on a reaction solution and 2, 4-dinitrophenylhydrazine, adding an alkali solution and an organic solvent for reaction, and measuring absorbance. The invention constructs a mutation library through error-prone PCR, establishes 2, 4-dinitrophenylhydrazine as a color developing agent, and determines OD by an enzyme-labeling instrument470The mutant strain for synthesizing the heliotropin recombinant strain is screened by a high-throughput screening mode which is based on the heliotropin recombinant strain, the mode has the advantages of high efficiency, accuracy and rapidness, and the mutant strain LSQ-3 has good application value and is capable of remarkably improving the catalytic efficiency of synthesizing heliotropin, p-anisaldehyde and vanillin.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a high-throughput screening method of trans-anethole oxygenase mutants, a mutant strain and mutants.
Background
The propenyl benzene type essential oil is a certain propenyl benzene compound extracted from plant essential oil by a physical method, and the compounds have the common characteristics of having propenyl side chains and methoxy substituent groups on benzene rings and are often used as starting materials (such as vanillin, heliotropin, anisaldehyde and the like) for synthesizing rare spices and medicaments with benzene ring structures. Due to the existence of chemical synthetic perfumes in perfume production: the residual chemical substances in the spice compounds bring harm to human health, the chemical synthesis brings environmental pollution, and the stereoselectivity of the chemical synthesis reaction is poor to cause the products to be racemes. Currently, techniques for biosynthesizing flavor compounds are receiving increasing attention.
Trans-anethole oxygenase (TAO), which was first discovered by Han et al in Pseudomonas putida JYR-1, oxidizes trans-anethole to synthesize p-anisaldehyde and also oxidizes isoeugenol, isosafrole and p-methoxyisoeugenol, but the catalytic efficiency is not high, E.coli BL21(DE3) (pET-TAO) in a concentration of 0.5 mmol.L-1Glucose and 1 mmol. L-1React for 6 hours in different substrate solutions to respectively synthesize 0.63 mmol.L-1P-anisaldehyde and vanillin, 0.38 mmol.L-1Heliotropin and veratraldehyde.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, in one aspect of the invention, the invention overcomes the defects in the prior art and provides a method for screening trans-anethole oxygenase mutants in high throughput.
In order to solve the technical problems, the invention provides the following technical scheme: a high throughput screening method of trans-anethole oxygenase mutants comprises the following steps,
taking a trans-anethole oxygenase gene as a template, and carrying out error-prone PCR to obtain a trans-anethole oxygenase mutant library;
adding the trans-anethole oxygenase reaction substrate into a solution containing a trans-anethole oxygenase mutant for catalytic reaction, carrying out color development reaction on a reaction solution and 2, 4-dinitrophenylhydrazine, adding an alkali solution and an organic solvent for reaction, and measuring absorbance.
As a preferred scheme of the high-throughput screening method of the trans-anethole oxygenase mutant, the method comprises the following steps:
the error-prone PCR comprises the following steps of designing error-prone PCR primers:
taoF：TTCCAGGGGCCCCTGGGATCCGGATCCATGGAGGACATCATGCtaoD：
GTCACGATGCGGCCGCTCGAGCTCGAGTCAGTTAGTCCTCAAGTCG
as a preferred scheme of the high-throughput screening method of the trans-anethole oxygenase mutant, the method comprises the following steps: the method for obtaining the trans-anethole oxygenase mutant library comprises the steps of using a recombinant plasmid PGEX-6P-1-tao as a template, carrying out error-prone PCR (polymerase chain reaction) by using rTaq polymerase to obtain random mutation, wherein the addition concentration in a PCR system is 7-10 mmol.L-1Mg of (2)2+And 30 to 250. mu. mol. L-1Mn of (2)2+The PCR product was ligated to the linear plasmid PGEX-6P-1, and the ligation product was introduced into competent cells E.coli BL21(DE3) to obtain a trans-anethole oxygenase mutant library.
As a preferred scheme of the high-throughput screening method of the trans-anethole oxygenase mutant, the method comprises the following steps: the color reaction comprises coating a plate with a trans-anethole oxygenase mutant library introduced into competent cells, and selecting a single colony to be inoculated into a culture medium containing 100 mg.L-1The ampicillin-containing LB medium of (1), culturing under induction, centrifuging to obtain cells, adding 100-200. mu.L of a buffer solution, mixing the cells, and adding the buffer solution to a final concentration of 0.1-1.0 g.L-1The trans-anethole oxygenase reaction substrate is subjected to catalytic reaction at 28-37 ℃ for 20-120 min, reaction supernatant is taken to react with 2, 4-dinitrophenylhydrazine color developing agent at 10-40 ℃ for 5-40 min, and then 0.5-3 mol.L is added-1Dissolving the precipitate in 0-80% ethanol solution at 10-40 deg.C, and measuring absorbance OD with enzyme-labeling instrument470。
As a preferred scheme of the high-throughput screening method of the trans-anethole oxygenase mutant, the method comprises the following steps: the trans-anethole oxygenase reaction substrate comprises 4-propenyl-2-methoxyphenol, trans-1-methoxy-4-propenyl benzene and 1, 2-dimethoxy-4-propenyl benzene.
As a preferred scheme of the high-throughput screening method of the trans-anethole oxygenase mutant, the method comprises the following steps: the recombinant plasmid PGEX-6P-1-tao is prepared by connecting trans-anethole oxidase gene derived from Pseudomonas putida JYR-1 to plasmid PGEX-6P-1.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides a trans-anethole oxygenase mutant obtained by a high-throughput screening method, which is characterized in that: the trans-anethole oxygenase mutant is E.coli BL21(DE3) -3G2, and the amino acid characteristic sequence of the mutant is shown as SEQ ID NO 1.
As a preferred embodiment of the trans-anethole oxygenase mutant, the invention is characterized in that: the trans-anethole oxygenase mutant is E.coli BL21(DE3) -3G2, and has three base mutations of 211G to T, 330A to G and 746G to T; compared with the amino acid sequence of the trans-anethole oxygenase wild type, two amino acid changes are carried out, namely 71Val is changed into Leu, and 249Gly is changed into Cys.
As another aspect of the present invention, the present invention overcomes the disadvantages of the prior art by providing a trans-anethole oxygenase mutant strain in which: the trans-anethole oxygenase mutant strain comprises the trans-anethole oxygenase mutant, the trans-anethole oxygenase mutant strain is LSQ-3, and the catalysis efficiency of the LSQ-3 whole cell catalysis of 4-allyl-1, 2-methylenedioxybenzene and trans-1-methoxy-4-propenyl benzene synthesis heliotropin and p-anisaldehyde is obviously improved.
The invention has the beneficial effects that: the invention uses the recombinant plasmid PGEX-6P-1-tao constructed in the laboratory as a template, constructs a mutation library in an error-prone PCR mode, establishes 2, 4-dinitrophenylhydrazine as a color developing agent, and measures OD by an enzyme labeling instrument470Based high-throughput screening method for screening mutant strains for synthesizing heliotropin recombinant strains, and the method has high efficiencyThe advantages of accuracy and rapidness. The obtained mutant LSQ-3 has improved catalytic efficiency for synthesizing heliotropin, anisic aldehyde and vanillin, and embodies good application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a flow chart of the directed evolution high throughput screening method of the present invention.
FIG. 2 is a GC-MS diagram of the synthesis of aromatic aldehyde product by LSQ-3 whole cell catalysis propenyl benzene type essential oil single separation.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
establishment of high throughput screening method:
(1) effect of different alkali solutions on color development
Respectively determine 1 mol. L-1The effect of NaOH-50% ethanol solution and KOH-50% ethanol solution on color development, OD was recorded470And stability (OD within 1 h)470The absolute value of the maximum difference to the value of the current use value) as shown in table 1.
TABLE 1 Effect of different alkali solutions on color development
| Alkali liquor | NaOH | KOH |
| OD470 | 2.86 | 1.814 |
| Stability of | 0.016 | 0.055 |
The results show the OD using NaOH-50% ethanol solution470The response is high and the stability is good.
(2) Effect of different alkali concentrations on color development
Respectively measuring 0.5 to 3.0 mol.L-1The effect of NaOH on color development, OD was recorded470And stability, as shown in table 2.
TABLE 2 Effect of different alkali concentrations on color development
The results show that the NaOH concentration is 1.5-2.0 mol.L-1Then, OD470And (4) stabilizing.
(3) Influence of different organic solvents on color development
The influence of ethanol and methanol as organic solvents on color development was measured separately and OD was recorded470And stability, as shown in table 3.
TABLE 3 Effect of different organic solvents on color development
| Organic solvent | Ethanol | Methanol |
| OD470 | 2.463 | 0.33 |
| Stability of | 0.013 | 0.048 |
The results show that ethanol, OD, is selected470The response is high and the stability is good.
(4) Effect of ethanol at different concentrations on color development
Respectively measuring the influence of 0-80% ethanol dissolved NaOH on site, and recording OD470And stability, as shown in table 4.
TABLE 4 Effect of different concentrations of ethanol on color development
The results show that the OD is higher than 50% when the ethanol concentration is higher than470The response value and the stability are better.
After the reaction is finished, scanning is carried out by using an enzyme-labeling instrument under the wavelength ranging from 300 nm to 700nm (the increment is 10nm), and the maximum absorption wavelength of the reaction liquid is 470 nm. This format can be used as a high throughput screening format.
Example 2:
taking the synthesized heliotropin as an example, 50 mu L of heliotropin standard solution (the concentration is 0-1000 mg.L)-1) Adding 50-300 mu L of 2, 4-dinitrophenylhydrazine color developing agent, reacting for 5-40 min at 10-40 ℃, and adding 0.5-3 mol.L-1Reacting NaOH- (0-80%) ethanol solution at 10-40 ℃ for 5-60 min, taking out, and measuring the light absorption value OD of the solution by using an enzyme-labeling instrument470Taking the heliotropin concentration as an abscissa and the OD470 value as an ordinate, drawing a standard curve, and obtaining a calculation formula that y is 0.00496x +0.09445, and R is20.9995, and the detection concentration range of the heliotropin is 0-700 mg.L-1. This format can be used as a high throughput screening format.
Example 3:
tao creation of mutation library:
(1) error-prone PCR establishment of tao mutation library
Tao is synthesized according to trans-anethole oxygenase gene in Pseudomonas putida JYR-1 and connected to PGEX-6P-1 to obtain recombinant plasmid PGEX-6P-1-tao, error-prone PCR primers taoF and taoD are designed by using the recombinant plasmid as a template, and Mg is set2+Concentration, Mn2+The concentration is 7 to 10 mmol. L respectively-1And 30 to 250. mu. mol. L-1Error-prone PCR was performed using common rTaq polymerase. The primers are as follows:
taoF：ttccaggggcccctgggatccGGATCCATGGAGGACATCATGC
taoD：gtcacgatgcggccgctcgagCTCGAGTCAGTTAGTCCTCAAGTCG
the PCR program was set up as follows:
and detecting the PCR product by using nucleic acid gel electrophoresis, purifying and recovering the PCR product, connecting the PCR product to a linear vector PGEX-6P-1 by using a one-step cloning kit, converting the PCR product into E.coli BL21(DE3) super competence, performing coating culture, selecting a single colony for colony PCR verification, and sequencing to obtain 1-6 average amino acid mutation amounts in the mutation library.
Example 4:
screening of mutation libraries:
(1) primary screen of mutation storehouse
The single colonies obtained were streaked on a 48-well plate, which was placed in a 37 ℃ incubator overnight for culture and stored at 4 ℃.
The thallus picked and preserved by adopting toothpick is respectively inoculated in a 96 deep-well plate with 100-1000 mu L, after overnight culture at 37 ℃, the thallus is transferred to a 96 deep-well plate with LB with 100-1000 mu L by the inoculum size of 1-10 percent, the thallus is cultured for 2-5 h at 37 ℃ and 110rpm, and the final concentration is added to be 0.05-1.0 mmol.L-1The IPTG inducer is induced for 6-12 h at the temperature of 20-25 ℃. After the culture, the cells were collected by centrifugation using a well plate centrifuge. Adding 100-200 μ L buffer (pH 7-8.5) into the cells, resuspending and mixing the cells with a shaker, and adding the mixture to a final concentration of 0.1-1.0 g.L-1The total reaction system of the 4-allyl-1, 2-methylenedioxybenzene is 300 mu L, the catalytic reaction is carried out at 28-37 ℃ for 10-120 min, the centrifugation is carried out after the reaction is finished, 50 mu L of reaction supernatant is taken, and the OD after the color development is measured according to the method in the embodiment 1470Namely, 50 mu L of supernatant is reacted with 50-300 mu L of 2, 4-dinitrophenylhydrazine color developing agent at 10-40 ℃ for 5-40 min, and 800 mu L of 0.5-3 mol.L is added-1Reacting NaOH- (0-80%) ethanol solution at 10-40 ℃ for 5-60 min, and determining OD by using an enzyme-labeling instrument470And comparing with a control group, selecting 53 strains with high light absorption values, and rescreening.
The preparation method of the 2, 4-dinitrophenylhydrazine color developing agent comprises the following steps: dissolving 0.5g of 2, 4-dinitrophenylhydrazine powder in 0.03L of concentrated sulfuric acid completely, slowly adding 0.3L of 95% ethanol solution, mixing, diluting to 1.0L by using a volumetric flask, storing in a brown reagent bottle, sealing, and storing in a cool place.
(2) Rescreening of mutation storehouse
The rescreened strain was induced at 20 ℃ for 6 hours in 30mL/250mL LB medium with IPTG (0.4 mmol. multidot.L-1) added to the strain, and the cells were collected. Adding a substrate of 4-allyl-1, 2-methylenedioxybenzene with the concentration of 0.1-3 g.L < -1 > into PBS buffer solution, carrying out catalytic reaction for 10-120 min at 28-37 ℃ by using wet thalli with the wet weight of 1-3%, terminating the reaction by using methanol with the volume twice, measuring the concentration of heliotropin by using high performance liquid chromatography after treating reaction liquid, and calculating the conversion rate to obtain a mutant strain E.Coli BL21(DE3) -3G2 (named as LSQ-3 for short), wherein valine at the 71 th position of trans-anethole oxygenase is replaced by leucine (V71L), and glycine at the 249 th position is replaced by cysteine (G249C). The amino acid sequence of the mutant is shown in SEQ ID NO 1. The mutant LSQ-3 can catalyze the heliotropin and p-anisaldehyde synthesized by 4-allyl-1, 2-methylenedioxybenzene and trans-1-methoxyl-4-propenyl benzene in the whole-cell catalysis process, so that the catalysis efficiency is improved.
Example 5:
the mutant strain LSQ-3 catalyzes four propenyl benzene type essential oils to be singly separated and polymerized into aromatic aldehyde:
after overnight culture at 37 ℃ in an LB medium, LSQ-3 was transferred to a fresh LB medium and cultured, and then the final concentration was 0.05 to 1.0 mmol.L-1The IPTG is induced for 8-12 h at the temperature of 20-25 ℃, and thalli are collected by centrifugation. Taking a proper amount of thallus LSQ-3 in a buffer solution reaction system, respectively using four propenylbenzenes, namely 4-allyl-1, 2-methylenedioxybenzene, 4-propenyl-2-methoxyphenol, 1, 2-dimethoxy-4-propenylbenzene and trans-1-methoxy-4-propenylbenzene, as substrates, converting for 15min at the temperature of 28-37 ℃ and the speed of 90-180 rpm, terminating the reaction by using twice the volume of methanol, and measuring the product concentration by using high performance liquid chromatography, wherein the results are shown in Table 5. The conversion capacity per cell unit time for aromatic aldehyde synthesis was calculated and compared to that reported in the literature for the wild-type trans-anethole oxygenase recombinant strain e.coli BL21(DE3) (pET-TAO) (R) as shown in table 5.
TABLE 5 comparison of the catalytic results of LSQ-3 with E.coli BL21(DE3) (pET-TAO)
A: prior art data, reaction conditions 1mM substrate, OD600The reaction time is 6h with the bacterium amount of 2;
b: the reaction conditions of the invention are as follows: 8mM substrate, OD600The bacterial quantity is 1, and the reaction time is 15 min;
from the table, it was found that mutant LSQ-3 had significantly higher total cell synthesis capacities for heliotropin and anisaldehyde than the wild type, and had catalytic efficiencies that were increased by 79 and 42 times, respectively, as compared to the wild type, R ═ B × 6 × 60 × (2/(a × 15 × (8) (i.e., conversion of substrate per unit time unit cell B/conversion of substrate per unit time unit cell a).
The invention uses the recombinant plasmid PGEX-6P-1-tao constructed in the laboratory as a template, constructs a mutation library in an error-prone PCR mode, establishes 2, 4-dinitrophenylhydrazine as a color developing agent, and measures OD by an enzyme labeling instrument470The high-throughput screening method based on the heliotropin can screen mutant strains for synthesizing heliotropin recombinant strains, and has the advantages of high efficiency, accuracy and rapidness. The obtained mutant LSQ-3 has improved catalytic efficiency for synthesizing heliotropin, anisic aldehyde and vanillin, and embodies good application value.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (3)
1. A trans-anethole oxygenase mutant characterized in that: the amino acid characteristic sequence is shown as SEQ ID NO 1.
2. The trans anethole oxygenase mutant according to claim 1, characterized in that: the trans-anethole oxygenase mutant has three base mutations, namely 211G to T, 330A to G and 746G to T; compared with the amino acid sequence of the trans-anethole oxygenase wild type, two amino acid changes are carried out, namely 71Val is changed into Leu, and 249Gly is changed into Cys.
3. A recombinant strain characterized by: the recombinant strain comprising the trans-anethole oxygenase mutant of claim 1 or 2.
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| WO2009155527A2 (en) * | 2008-06-19 | 2009-12-23 | Progenics Pharmaceuticals, Inc. | Phosphatidylinositol 3 kinase inhibitors |
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| WO2009155527A2 (en) * | 2008-06-19 | 2009-12-23 | Progenics Pharmaceuticals, Inc. | Phosphatidylinositol 3 kinase inhibitors |
| CN102206659A (en) * | 2011-04-11 | 2011-10-05 | 四川农业大学 | Method for enhancing activity of endoglucanase on basis of error-prone PCR (Sequential Error-Prone) technology |
| CN105802983A (en) * | 2016-03-28 | 2016-07-27 | 中国科学院青岛生物能源与过程研究所 | High-flux screening method of aliphatic hydrocarbon generation gene, obtained mutant and application |
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