CN115704005A - Method for synthesizing phenylpropanol derivative - Google Patents

Abstract

The invention discloses a method for synthesizing phenylpropanol derivatives, which relates to the field of biochemistry and comprises the following steps: the method comprises the following steps: extracting genes of carboxylic acid reductase, alcohol dehydrogenase and olefinic bond reductase to construct engineering bacteria; step two, culturing engineering bacteria, screening strains with effective expression, and storing the strains: step three, mixing

Dissolving adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate and flavin mononucleotide to form solution, and addingCarrying out biotransformation to finally obtain a crude product; purified by adjusting pH value, stirring, suction filtering, washing and drying

The phenylpropanol derivative synthesized by the method has high purity, simple operation and low energy consumption.

Description

Method for synthesizing phenylpropanol derivative

Technical Field

The invention relates to the field of bioengineering, in particular to a method for synthesizing phenylpropanol derivatives.

Background

Phenylpropanol and its derivatives are important raw materials for products in various fields such as medicine, food, feed, cosmetics, etc., and are naturally present in substances such as strawberry, tea, bay leaf, etc., taking phenylpropanol as an example. Phenylpropanol is a food flavor that is approved for use in the food industry; in the cosmetic industry, the compound can be added as a preservative; in the medical field, phenylpropanol is an intermediate of central skeletal muscle relaxant prednisone.

The phenylpropanol and the derivative thereof have a plurality of effects and applications, but the current acquisition mode is still limited to the production of ethyl cinnamate, cinnamyl alcohol and other raw materials through reactions such as hydrogenation, the process is complex in process and high in energy consumption, the current environment-friendly and green production concept is not met, a method in the technical field of biological engineering is needed in the market, the phenylpropanol and the derivative thereof are obtained through a simple process and low energy consumption, and the problem is solved.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a method for synthesizing phenylpropanol derivatives, the synthesis technology is simple, the energy consumption is low, and the purity of the obtained phenylpropanol derivatives is high.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a method of synthesizing a phenylpropanol derivative, comprising: the method comprises the following steps:

step one, extracting enzyme genes, and constructing engineering bacteria:

extracting genes of carboxylic acid reductase, alcohol dehydrogenase and olefinic bond reductase, preparing recombinant expression plasmids after polymerase chain reaction amplification, and introducing the recombinant expression plasmids into escherichia coli for culture to obtain recombinant expressed engineering bacteria;

step two, culturing engineering bacteria, screening strains with effective expression, and storing the strains:

adding the recombinant escherichia coli into a culture medium for culturing to obtain a seed solution of the recombinant escherichia coli; carrying out amplification culture and fermentation on the seed liquid of the recombinant escherichia coli to obtain a converted solution; detecting the content of target enzymes in the strain, and screening the strain with higher enzyme expression content; preparing the screened strains into high-concentration bacterial suspension by using sterile water, taking a strain preservation protective agent, putting the strain preservation protective agent into the bacterial suspension, fully and uniformly mixing, and freezing to obtain a spare seed solution;

step three, converting 3,4-dihydroxycinnamic acid into 3,4-dihydroxyphenylpropanol by using recombinant escherichia coli:

will be provided with

Adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate and flavin mononucleotide in a mass ratio of 1: (1-2): (1-12): (1-3) dissolving to form a solution, and adding the solution into the standby seed solution obtained in the step two for biotransformation to finally obtain a crude product; adjusting the pH value of the crude product to 5-8, stirring, filtering, washing, and drying to obtain purified product

R ₁ The method comprises the following steps: -CH ₃ -OH, -H or-OCH ₃ ；

R ₂ The method comprises the following steps: -CH ₃ -OH, -H or-OCH ₃ ；

R ₃ The method comprises the following steps: -CH ₃ -OH, -H or-OCH ₃ 。

In the method for synthesizing phenylpropanol derivatives, the source of the gene for extracting carboxylate reductase includes: HXW97-RS16665 of Mycobacterium marinaum having the sequence of SEQ01 or OCQ-RS16610 of Mycobacterium parintracellular having the sequence of SEQ02; the sources of the genes for extracting the alcohol dehydrogenase comprise: ADH1 of Saccharomyces cerevisiae, with sequence of SEQ03, or FSUBG-7421 of Fusarium subglutinans, with sequence of SEQ04; the sources of the gene for extracting the ethylenic double bond reductase comprise: LOC104820880 of Tarenaya hasslerana with sequence SEQ05, or LOC1047699854 of Camelina sativa with sequence SEQ06.

In the method for synthesizing phenylpropanol derivatives, a PCR reaction system for PCR amplification comprises: 10 XPCR buffer 20ul, dNTP 1698, forward primer 6ul, reverse primer 6ul, taq DNA polymerase 10ul, dd H ₂ O140 ul, 0.1-2ug of template DNA; amplification procedure and conditions: pre-denaturation at 94 ℃ for 3min; then 35 cycles of denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; finally, extension is carried out for 5min at 72 ℃.

In the method for synthesizing phenylpropanol derivatives, the forward primer comprises: the sequence of the forward primer of HXW97-RS16665 is SEQ07, the sequence of the forward primer of OCQ-RS16610 is SEQ08, the sequence of the forward primer of ADH1 is SEQ09, the sequence of the forward primer of FSUBG-7421 is SEQ10, the sequence of the forward primer of LOC104820880 is SEQ11, and the sequence of the forward primer of LOC1047699854 is SEQ12;

the reverse primer comprises: the sequence of the reverse primer of HXW97-RS16665 is SEQ13, the sequence of the reverse primer of OCQ-RS16610 is SEQ14, the sequence of the reverse primer of ADH1 is SEQ15, the sequence of the reverse primer of FSUBG-7421 is SEQ16, the sequence of the reverse primer of LOC104820880 is SEQ17, and the sequence of the reverse primer of LOC1047699854 is SEQ18.

In the method for synthesizing phenylpropanol derivatives by using immobilized enzyme, the specific method for preparing recombinant expression plasmids in the first step is as follows: the restriction enzyme Nde I/Xho I double enzyme digestion system is adopted to carry out enzyme digestion treatment on the enzyme digestion sites of the gene and the plasmid vector pET-28a (+), and T4 ligase is used for connection overnight at 18 ℃, so as to obtain the recombinant expression plasmid.

In the method for synthesizing phenylpropanol derivatives by using immobilized enzyme, the specific method for obtaining the recombinant expressed engineering bacteria in the first step is as follows: is prepared from CaCl ₂ Adding the recombinant plasmid solution into the treated competent escherichia coli bacterial suspension, and then adding a culture medium to restore the bacteria to a normal growth state; coating the cultured bacterial liquid on a screening flat plate, inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and continuously culturing; selecting newly activated single colony from the screening plate, inoculating the single colony in a culture medium for culture,until late logarithmic growth; the bacterial suspension was inoculated in a medium and cultured to OD600=0.5.

In the method for synthesizing phenylpropanol derivatives using immobilized enzyme, the method for detecting the content of the target enzyme in step two is: the detection method of the carboxylic acid reductase is to detect the absorbance of the converted solution at 340nm by using an ultraviolet spectrophotometer; the detection method of the alcohol dehydrogenase is to detect the absorbance of the converted solution at 340nm by using an ultraviolet spectrophotometer; the detection method of the ethylenic bond reductase is to detect the absorbance of the converted solution at 340nm by using an ultraviolet spectrophotometer.

In the method for synthesizing phenylpropanol derivatives by using immobilized enzyme,

the method comprises the following steps: 3,4-dihydroxycinnamic acid, 2-hydroxy-4-methoxycinnamic acid, or 2-hydroxy-3,4-dimethoxycinnamic acid.

In the method for synthesizing phenylpropanol derivatives by using immobilized enzyme,

the method comprises the following steps: 3,4-dihydroxyphenylpropanol, 2-hydroxy-4-methoxypropiophenol, 2-hydroxy-3,4-dimethoxyphenylpropanol.

The invention has the advantages that:

compared with the conventional synthesis technology, the method has the advantages of simple operation and low energy consumption;

the phenylpropanol derivative obtained by synthesizing the phenylpropanol derivative by preparing the genetic engineering bacteria has high purity.

Drawings

FIG. 1 is a UV absorption spectrum of NADPH in the carboxylic acid reductase assay of the present invention;

FIG. 2 is a graph showing the ultraviolet absorption spectrum of NADH in the alcohol dehydrogenase assay of the present invention;

FIG. 3 is a graph showing the ultraviolet absorption spectrum of NADH in the measurement of the ethylenic reductase of the present invention;

FIG. 4 is a 3,4-dihydroxyphenylpropanol standard curve of example 1 of the present invention;

FIG. 5 is a standard curve of 2-hydroxy-4-methoxyphenylpropanol from example 2 of the present invention;

FIG. 6 is a standard curve of 2-hydroxy-3,4-dimethoxyphenylpropanol in example 3 of the present invention.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

The synthesis principle of the invention is shown as the following formula:

R ₁ comprising-CH ₃ -OH, -H or-OCH ₃ ；

R ₂ comprising-CH ₃ -OH, -H or-OCH ₃ ；

R ₃ comprising-CH ₃ -OH, -H or-OCH ₃ 。

Example 1:

the synthetic scheme is shown as the following formula:

the method specifically comprises the following steps:

1. extracting enzyme gene and constructing engineering bacteria

Extracting carboxyreductase gene HXW97-RS16665 and its sequence SEQ01 from Mycobacterium marinum, extracting alcohol dehydrogenase gene ADH1 and its sequence SEQ03 from Saccharomyces cerevisiae, extracting olefinic bond reductase gene LOC104820880 and its sequence SEQ05 from Tarnanya hassleiana; performing polymerase chain reaction amplification on the extracted gene; the PCR reaction system for PCR amplification is shown in Table 1; procedure and conditions set for PCR instrument: pre-denaturation at 94 ℃ for 3min; then 35 cycles, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; finally, extending for 5min at 72 ℃; primer design is shown in table 2. Adopting a restriction enzyme Nde I/Xho I double-enzyme digestion system to aim at the enzyme digestion site of a gene and plasmid vector pET-28a (+)Performing enzyme digestion treatment, and connecting overnight at 18 ℃ by using T4 ligase to obtain a recombinant expression plasmid, and obtaining a recombinant expression plasmid with three target genes; introducing the recombinant expression plasmid into Escherichia coli, culturing recombinant strain, collecting CaCl ₂ 200ul of the treated competent escherichia coli bacterial suspension is added with 10ul of recombinant plasmid solution, the recombinant plasmid solution is gently shaken up, the mixture is placed on ice for 30 minutes, then the mixture is subjected to heat shock for 45 to 90 seconds in a water bath at 37 ℃, and then the mixture is rapidly placed on ice for cooling for 3 to 5 minutes after the heat shock. Adding 1ml LB liquid culture medium into the tube, mixing uniformly, and culturing for 1 hour at 37 deg.C under shaking to restore the normal growth state of bacteria. Shaking the cultured bacterial liquid evenly, coating 100 μ l on a screening plate, standing for half an hour with the front side upward, inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and culturing at 37 deg.C for 16-24 hours. Newly activated single colonies were picked from the plate, inoculated in 3-5ml of LB liquid medium, and cultured with shaking at 37 ℃ for about 12 hours until late logarithmic growth. The bacterial suspension was mixed in a 1:100 in 100ml LB liquid medium, 37 ℃ shaking culture 2-3 hours to OD600=0.5. Obtaining the recombinant expression engineering bacteria.

TABLE 1

TABLE 2

2. Culturing engineering bacteria, screening effective expressed strain

Adding the recombinant escherichia coli into an LB culture medium, and culturing for 24 hours under the conditions that the pH is 6-8, the temperature is 20-37 ℃ and the rotating speed is 100-200rpm to obtain a seed solution of the recombinant escherichia coli; inoculating the seed liquid of the recombinant escherichia coli into an M9 culture medium for amplification culture and fermentation; the volume ratio of the seed solution to the M9 medium was 1 ₆₀₀ A value of 0.6-0.8. Under the condition of the enlarged culture of the recombinant escherichia coli, the content of target enzymes in the recombinant escherichia coli is detected, and strains with higher enzyme expression content are screened.

And (3) detecting carboxylic acid reductase:

while the carboxyreductase catalyzes the conversion of a corresponding structure, the reduced coenzyme II (NADPH) is oxidized into oxidized coenzyme II (NADP +), NADP + has no absorption peak, and NADPH has an absorption peak at 340nm, so that the carboxyreductase activity can be detected by detecting the absorbance of the converted solution by using an ultraviolet spectrophotometer, and the obtained NADPH ultraviolet absorption spectrum is shown in FIG. 1.

1.5mL of a sodium pyrophosphate buffer solution having a pH of 7.0, 1.0mL of 27mmol/L reduced coenzyme II (NADPH), and 0.5mL of an 11.5% acetic acid solution were added to a measuring tube, and after mixing, the mixture was incubated in a water bath at 25 ℃ for 5min, 0.1mL of a suspension obtained by expanding the cultured strain was added thereto, and after sufficient conversion, the absorbance was measured (in the control group, acetic acid was replaced with distilled water).

And (3) detecting alcohol dehydrogenase:

when the alcohol dehydrogenase catalyzes the conversion of a corresponding structure, oxidized coenzyme I (NAD +) is reduced into reduced coenzyme I (NADH), NAD + has no absorption peak, and NADH has an absorption peak at 340nm, so that the alcohol dehydrogenase activity can be detected by detecting the absorbance of the converted solution by using an ultraviolet spectrophotometer, and the ultraviolet absorption spectrum of NADH is shown in figure 2.

1.5mL of a sodium pyrophosphate buffer solution having a pH of 8.8, 1.0mL of 27mmol/L oxidized coenzyme I (NAD +) and 0.5mL of an 11.5% ethanol solution were added to a measuring tube, and after mixing, the mixture was incubated in a water bath at 25 ℃ for 5min, 0.1mL of a bacterial suspension obtained by expanding the cultured strain was added thereto, and after sufficient conversion, the absorbance was measured (in the control group, distilled water was used instead of ethanol).

And (3) detecting an ethylenic bond reductase:

the enzyme activity of the ethylenic reductase was measured by comparing the absorbance at 340nm of a reaction system of NADH final concentration of 0.3mM, cyclohexenone at 3.5mM, tris-HCl (pH 7.5) at 50mM, and a proper amount of bacterial suspension, and the ultraviolet absorption spectrum of NADH is shown in FIG. 3.

3. Strain preservation

Preparing the strains screened in the step 2 into high-concentration bacterial suspension by using sterile water, taking 1-3ml of sterilized glycerol, fully and uniformly mixing the sterilized glycerol with the bacterial suspension to ensure that the concentration of the glycerol is about 10% -30%, and freezing and storing the mixture at the temperature of-70 ℃.

4. Converting 3,4-dihydroxycinnamic acid into 3,4-dihydroxyphenylpropanol by using recombinant Escherichia coli

3,4-dihydroxycinnamic acid, adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate and flavin mononucleotide are mixed according to the mass ratio of 1: (1-2): (1-12): and (1-3) dissolving to form a solution, and adding the solution into the seed solution obtained in the step (2) for biotransformation to finally obtain a 3,4-dihydroxyphenylpropanol crude product. Centrifuging the crude product, collecting supernatant, and respectively filtering with plate-frame filter, microporous filter, and ultrafilter; the mesh number of the plate frame filter is 400 meshes, the pore diameter of the microporous filter is 0.5um, and the molecular weight cut-off of the ultrafilter is 10 ten thousand. And adding the filtered product into a chromatographic column filled with D101 macroporous resin for adsorption, washing with distilled water to remove impurities, and sucking out surface water. Eluting the product with ethanol water solution as mobile phase, placing the eluate obtained by chromatography in a rotary evaporator, and removing ethanol to obtain purified 2-hydroxy-3,4-dimethoxy phenylpropanol.

Macroporous resin pretreatment: activating the D101 macroporous resin, soaking in absolute ethyl alcohol, stirring to remove bubbles, standing for 24h, and washing with distilled water until the resin has no ethyl alcohol smell. The resin was soaked in 5% NaOH solution for 4 hours, washed with distilled water to neutrality, then soaked in 5% HCl solution for 4 hours, washed with distilled water to neutrality, and then dried by blotting for further use.

5. Detecting and calculating the product yield and purity

Precisely weighing the purified and dried product 3,4-dihydroxy phenylpropanol, calculating the corresponding mole number according to the molar mass of the product, and according to a product yield formula: the product yield = ((mass of product) × relative molecular mass of product)/(initial amount of key components before reaction) × relative molecular mass of reactant) was calculated to give a product yield of about 71.55%.

0.5ml of 3,4-dihydroxyphenylpropanol standard solution is measured and diluted to 25ml by absolute ethyl alcohol. And scanning the ultraviolet spectrum of the sample by using an ultraviolet spectrophotometer, and selecting the ultraviolet absorption detection wavelength at the position of 252 nm. Accurately weighing a proper amount of 3,4-dihydroxyphenylpropanol standard solution, adding ethanol for dissolving to prepare 1.0mg/ml stock solution, respectively absorbing 1ml, 1.5ml, 2.0 ml, 2.5 ml and 3.0ml of the stock solution into a 100ml measuring flask, adding ethanol for diluting to a scale, measuring an absorption value at a wavelength of 252nm, wherein the detection data of the concentration and the absorbance of the 3,4-dihydroxyphenylpropanol standard solution are shown in Table 3.

TABLE 3

The absorbance (A) is linearly regressed by concentration (C) with the regression equation: c (ug/ml) =0.0223+40.8903A, r =0.9997 (n = 5), as shown in FIG. 4.

Weighing 5mg of the purified 3,4-dihydroxy phenylpropanol, diluting to 100mL with absolute ethanol, mixing uniformly, filtering with a microporous membrane, taking the filtrate, dividing the filtrate into 5 parts, and performing absorbance determination, wherein the absorbance determination and content calculation data of 3,4-dihydroxy phenylpropanol are shown in Table 4.

TABLE 4

As a result of the measurement, the content of 3,4-dihydroxyphenylpropanol after the purification is about 69.18%.

Example 2:

the synthetic scheme is shown as the following formula:

1. extracting enzyme gene and constructing engineering bacteria

Extracting carboxyreductase gene OCQ-RS16610 and its sequence SEQ02 and Fusarium subglutarans from Mycobacterium paragenic, extracting ethanol dehydrogenase gene FSUBG-7421 and its sequence SEQ04, and extracting olefinic bond reductase gene LOC104769985 and its sequence SEQ06 from Camelina sativa; performing polymerase chain reaction amplification on the extracted gene; carrying out enzyme digestion treatment on enzyme digestion sites of a gene and a plasmid vector pET-28a (+) by adopting a restriction enzyme Nde I/Xho I double enzyme digestion system, and connecting overnight at 18 ℃ by using T4 ligase to obtain a recombinant expression plasmid, thereby obtaining a recombinant expression plasmid with three target genes; and introducing the recombinant expression plasmid into escherichia coli to obtain the recombinant expression engineering bacteria.

The PCR reaction system for PCR amplification is shown in Table 1; procedure and conditions set for PCR instrument: pre-denaturation at 94 ℃ for 3min; then 35 cycles, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; finally extending for 5min at 72 ℃; primer design is shown in table 2.

And (3) culturing the competent escherichia coli and recombinant strain introduced with the plasmid:

is prepared from CaCl ₂ 200ul of treated competent escherichia coli bacterial suspension is added with 10ul of recombinant plasmid solution, the mixture is shaken gently, and after the mixture is placed on ice for 30 minutes, the mixture is subjected to heat shock for 45 seconds to 90 seconds in a water bath at 37 ℃, and after the heat shock, the mixture is quickly placed on ice for cooling for 3 minutes to 5 minutes. Adding 1ml LB liquid culture medium into the tube, mixing uniformly, and culturing for 1 hour at 37 deg.C under shaking to restore the normal growth state of bacteria. And shaking the cultured bacterial liquid uniformly, coating 100 mu l of the cultured bacterial liquid on a screening plate, standing for half an hour with the front side upward, inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and culturing for 16-24 hours at 37 ℃.

Newly activated single colonies were picked from the above plates, inoculated in 3-5ml of LB liquid medium, and shake-cultured at 37 ℃ for about 12 hours until late logarithmic growth. The bacterial suspension was mixed in a 1:100 in 100ml LB liquid medium, 37 ℃ shaking culture 2-3 hours to OD600=0.5.

2. Culturing engineering bacteria, screening effective expressed strain

Adding the recombinant escherichia coli into an LB culture medium, and culturing for 24 hours under the conditions that the pH is 6-8, the temperature is 20-37 ℃ and the rotating speed is 100-200rpm to obtain a seed solution of the recombinant escherichia coli; inoculating the seed liquid of the recombinant escherichia coli into an M9 culture medium for amplification culture and fermentation; the volume ratio of the seed solution to the M9 medium was 1 ₆₀₀ The value reaches 0.6-0.8. Under the condition of the enlarged culture of the recombinant escherichia coli, the content of target enzymes in the recombinant escherichia coli is detected, and strains with higher enzyme expression content are screened.

And (3) detecting carboxylic acid reductase:

the carboxyreductase catalyzes the conversion of a corresponding structure, simultaneously oxidizes reduced coenzyme II (NADPH) into oxidized coenzyme II (NADP +), NADP + has no absorption peak, and NADPH has an absorption peak at 340nm, so that the carboxyreductase activity can be detected by detecting the absorbance of a converted solution by using an ultraviolet spectrophotometer.

1.5mL of a sodium pyrophosphate buffer solution having a pH of 7.0, 1.0mL of 27mmol/L reduced coenzyme II (NADPH), and 0.5mL of an 11.5% acetic acid solution were added to a measuring tube, and after mixing, the mixture was incubated in a water bath at 25 ℃ for 5min, 0.1mL of a suspension obtained by expanding the cultured strain was added thereto, and after sufficient conversion, the absorbance was measured (in the control group, acetic acid was replaced with distilled water).

And (3) detecting alcohol dehydrogenase:

when the alcohol dehydrogenase catalyzes the conversion of a corresponding structure, oxidized coenzyme I (NAD +) is reduced into reduced coenzyme I (NADH), NAD + has no absorption peak, and NADH has an absorption peak at 340nm, so that the alcohol dehydrogenase activity can be detected by detecting the absorbance of the converted solution by using an ultraviolet spectrophotometer.

1.5mL of a sodium pyrophosphate buffer solution having a pH of 8.8, 1.0mL of 27mmol/L oxidized coenzyme I (NAD +) and 0.5mL of an 11.5% ethanol solution were added to a measuring tube, and after mixing, the mixture was incubated in a water bath at 25 ℃ for 5min, 0.1mL of a bacterial suspension obtained by expanding the cultured strain was added thereto, and after sufficient conversion, the absorbance was measured (in the control group, distilled water was used instead of ethanol).

And (3) detecting an ethylenic bond reductase:

ethylenic reductase

The enzyme activity of the enzyme ethylenic reductase was measured by comparing the absorbance at 340nm with the reaction system of NADH of final concentration of 0.3mM, cyclohexenone of final concentration of 3.5mM, tris-HCl of final concentration of 50mM (pH 7.5) as a control group and the reaction system of NADH of final concentration of 0.3mM, cyclohexenone of final concentration of 3.5mM, tris-HCl of final concentration of 50mM (pH 7.5) and a proper amount of bacterial suspension as an experimental group.

3. Strain preservation

Preparing the strains screened in the step 2 into high-concentration bacterial suspension by using sterile water, taking 1-3ml of sterilized glycerol, putting the sterilized glycerol into the bacterial suspension, fully and uniformly mixing the sterilized glycerol and the bacterial suspension to ensure that the concentration of the glycerol is about 10-30%, and freezing and storing the mixture at the temperature of-70 ℃.

4. 2-hydroxy-4-methoxy cinnamic acid is converted into 2-hydroxy-4-methoxy phenylpropanol by recombinant escherichia coli

2-hydroxy-4-methoxy cinnamic acid, adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate and flavin mononucleotide are mixed according to the mass ratio of 1: (1-2): (1-12): and (1) dissolving the raw materials in the proportion of (1-3) to form a solution, adding the solution into the seed solution obtained in the step (2) for biotransformation, and finally obtaining a crude product of 2-hydroxy-4-methoxyphenylpropanol. -centrifuging the crude product, taking the supernatant, and respectively filtering through a plate-and-frame filter, a microporous filter and an ultrafilter; the mesh number of the plate frame filter is 400 meshes, the pore diameter of the microporous filter is 0.5um, and the molecular weight cut-off of the ultrafilter is 10 ten thousand. And adding the filtered product into a chromatographic column filled with D101 macroporous resin for adsorption, washing with distilled water to remove impurities, and sucking out surface water. Eluting the product with ethanol water solution as mobile phase, placing the eluate obtained by chromatography in a rotary evaporator, and removing ethanol to obtain purified 2-hydroxy-3,4-dimethoxy phenylpropanol.

Macroporous resin pretreatment: activating the D101 macroporous resin, soaking in absolute ethyl alcohol, stirring to remove bubbles, standing for 24h, and washing with distilled water until the resin has no ethyl alcohol smell. Soaking the resin in 5% NaOH solution for 4h, washing with distilled water to neutrality, soaking in 5% HCl solution for 4h, washing with distilled water to neutrality, and sucking off water for use.

5. Detecting and calculating the product yield and purity

Precisely weighing the purified and dried product 2-hydroxy-4-methoxyphenylpropanol, calculating the corresponding mole number according to the mole mass of the product, and according to a product yield formula: the product yield = ((mass of product content) · relative molecular mass of product)/(initial amount of critical components before reaction) · relative molecular mass of reactant) was calculated to yield about 69.59%.

0.5ml of a weight standard solution of 2-hydroxy-4-methoxyphenylpropanol was measured and diluted to 25ml with absolute ethanol. And scanning the ultraviolet spectrum of the sample by using an ultraviolet spectrophotometer, and selecting the ultraviolet absorption detection wavelength at the position of 252 nm. Accurately weighing a proper amount of 2-hydroxy-4-methoxypropiophenol standard solution, adding ethanol to dissolve the solution to prepare 1.0mg/ml stock solution, respectively absorbing 1ml, 1.5ml, 2.0 ml, 2.5 ml and 3.0ml of the stock solution in a 100ml measuring flask, adding ethanol to dilute the solution to a scale, and measuring an absorption value at a wavelength of 252nm, wherein the detection data of the concentration and the absorbance of the 2-hydroxy-4-methoxypropiophenol standard solution are shown in Table 5.

TABLE 5

The absorbance (A) is linearly regressed by concentration (C) with the regression equation: c (ug/ml) =0.0217+41.3089A, r =0.9999 (n = 5), 2-hydroxy-4-methoxyphenylpropanol standard curve is shown in the following FIG. 5.

Measuring the purified 2-hydroxy-4-methoxy phenylpropanol 5mg, diluting to 100mL with anhydrous ethanol, mixing well, filtering with microporous membrane, collecting filtrate, dividing into 5 parts, and measuring absorbance, wherein the absorbance measurement and content calculation data of 2-hydroxy-4-methoxy phenylpropanol are shown in Table 6.

TABLE 6

As a result of the measurement, the content of 3,4-dihydroxyphenylpropanol after the purification is about 71.44%.

Example 3:

1. extracting enzyme gene and constructing engineering bacteria

Extracting carboxyreductase gene HXW97-RS16665 and its sequence SEQ01 and ethanol dehydrogenase gene FSUBG-7421 and its sequence SEQ04 from Mycobacterium marinum and extracting olefinic bond reductase gene LOC104820880 and its sequence SEQ05 from Tarnanya hassleiana; performing polymerase chain reaction amplification on the extracted gene; carrying out enzyme digestion treatment on enzyme digestion sites of a gene and a plasmid vector pET-28a (+) by adopting a restriction enzyme Nde I/Xho I double enzyme digestion system, and connecting overnight at 18 ℃ by using T4 ligase to obtain a recombinant expression plasmid, thereby obtaining a recombinant expression plasmid with three target genes; and introducing the recombinant expression plasmid into escherichia coli to obtain the recombinant expression engineering bacteria. The PCR reaction system for PCR amplification is shown in Table 1; procedure and conditions set for PCR instrument: pre-denaturation at 94 ℃ for 3min; then 35 cycles of denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; finally extending for 5min at 72 ℃; primer design is shown in table 2. And (3) culturing the competent escherichia coli and recombinant strain introduced with the plasmid:

200ul of the competent Escherichia coli bacterial suspension treated by CaCl2 is taken, 10ul of the recombinant plasmid solution is added, the mixture is gently shaken up, placed on ice for 30 minutes, then thermally shocked in a water bath at 37 ℃ for 45-90 seconds, and rapidly placed on ice for cooling for 3-5 minutes. Adding 1ml LB liquid culture medium into the tube, mixing uniformly, and culturing for 1 hour at 37 deg.C under shaking to restore the normal growth state of bacteria. And shaking the cultured bacterial liquid uniformly, coating 100 mu l of the cultured bacterial liquid on a screening plate, standing for half an hour with the front side upward, inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and culturing for 16-24 hours at 37 ℃.

Newly activated single colonies were picked from the above plates, inoculated in 3-5ml of LB liquid medium, and shake-cultured at 37 ℃ for about 12 hours until late logarithmic growth. The bacterial suspension is mixed with a mixture of 1:100 in 100ml LB liquid medium, 37 ℃ shaking culture 2-3 hours to OD600=0.5.

2. Culturing engineering bacteria, screening effective expressed strain

Adding the recombinant escherichia coli into an LB culture medium, and culturing for 24 hours under the conditions that the pH is 6-8, the temperature is 20-37 ℃ and the rotating speed is 100-200rpm to obtain a seed solution of the recombinant escherichia coli; inoculating the seed liquid of the recombinant escherichia coli into an M9 culture medium for amplification culture and fermentation; the volume ratio of the seed solution to the M9 medium was 1 ₆₀₀ The value reaches 0.6-0.8. Under the condition of the enlarged culture of the recombinant escherichia coli, the content of target enzymes in the recombinant escherichia coli is detected, and strains with higher enzyme expression content are screened.

And (3) detecting carboxylic acid reductase:

the carboxyreductase catalyzes the conversion of a corresponding structure, simultaneously oxidizes reduced coenzyme II (NADPH) into oxidized coenzyme II (NADP +), NADP + has no absorption peak, and NADPH has an absorption peak at 340nm, so that the carboxyreductase activity can be detected by detecting the absorbance of a converted solution by using an ultraviolet spectrophotometer.

1.5mL of a sodium pyrophosphate buffer solution having a pH of 7.0, 1.0mL of 27mmol/L reduced coenzyme II (NADPH), and 0.5mL of an acetic acid solution having a concentration of 11.5% were added to the measurement tube, and after mixing, the tube was placed in a water bath at 25 ℃ and incubated for 5min, 0.1mL of a suspension obtained by expanding the culture strain was added thereto and sufficiently transformed, and then the absorbance was measured (in the control group, acetic acid was replaced with distilled water).

And (3) detecting alcohol dehydrogenase:

when the alcohol dehydrogenase catalyzes the conversion of a corresponding structure, oxidized coenzyme I (NAD +) is reduced into reduced coenzyme I (NADH), NAD + has no absorption peak, and NADH has an absorption peak at 340nm, so that the alcohol dehydrogenase activity can be detected by detecting the absorbance of the converted solution by using an ultraviolet spectrophotometer.

1.5mL of a sodium pyrophosphate buffer solution having a pH of 8.8, 1.0mL of 27mmol/L oxidized coenzyme I (NAD +) and 0.5mL of an 11.5% ethanol solution were added to a measuring tube, and after mixing, the mixture was incubated in a water bath at 25 ℃ for 5min, 0.1mL of a bacterial suspension obtained by expanding the cultured strain was added thereto, and after sufficient conversion, the absorbance was measured (in the control group, distilled water was used instead of ethanol).

And (3) detecting an ethylenic bond reductase:

ethylenic reductase

The enzyme activity of the enzyme ethylenic reductase was measured by comparing the absorbance at 340nm with the reaction system of NADH of final concentration of 0.3mM, cyclohexenone of final concentration of 3.5mM, tris-HCl of final concentration of 50mM (pH 7.5) as a control group and the reaction system of NADH of final concentration of 0.3mM, cyclohexenone of final concentration of 3.5mM, tris-HCl of final concentration of 50mM (pH 7.5) and a proper amount of bacterial suspension as an experimental group.

3. Strain preservation

Preparing the strains screened in the step 2 into high-concentration bacterial suspension by using sterile water, taking 1-3ml of sterilized glycerol, fully and uniformly mixing the sterilized glycerol with the bacterial suspension to ensure that the concentration of the glycerol is about 10% -30%, and freezing and storing the mixture at the temperature of-70 ℃.

4. 2-hydroxy-3,4-dimethoxy cinnamic acid is converted into 2-hydroxy-3,4-dimethoxy phenylpropanol by utilizing recombinant escherichia coli

2-hydroxy-3,4-dimethoxy cinnamic acid, adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate and flavin mononucleotide are mixed according to the mass ratio of 1: (1-2): (1-12): and (1) dissolving the raw materials in the proportion of (1-3) to form a solution, adding the solution into the seed solution obtained in the step (2) for biotransformation, and finally obtaining a crude product of 2-hydroxy-3,4-dimethoxyphenylpropanol. Centrifuging the crude product, taking the supernatant, and respectively filtering the supernatant through a plate-and-frame filter, a microporous filter and an ultrafilter; the mesh number of the plate frame filter is 400 meshes, the pore diameter of the microporous filter is 0.5um, and the molecular weight cut-off of the ultrafilter is 10 ten thousand. And adding the filtered product into a chromatographic column filled with D101 macroporous resin for adsorption, washing with distilled water to remove impurities, and sucking out surface water. Eluting the product with ethanol water solution as mobile phase, placing the eluate in a rotary evaporator, and removing ethanol to obtain purified 2-hydroxy-3,4-dimethoxyphenylpropanol.

Macroporous resin pretreatment: activating the D101 macroporous resin, soaking in absolute ethyl alcohol, stirring to remove bubbles, standing for 24h, and washing with distilled water until the resin has no ethyl alcohol smell. The resin was soaked in 5% NaOH solution for 4 hours, washed with distilled water to neutrality, then soaked in 5% HCl solution for 4 hours, washed with distilled water to neutrality, and then dried by blotting for further use.

5. Detecting and calculating product yield and purity

Precisely weighing the purified and dried product 2-hydroxy-3,4-dimethoxy phenylpropanol, calculating the corresponding mole number according to the molar mass of the product, and according to a product yield formula: the product yield = ((mass of product) × relative molecular mass of product)/(initial amount of key components before reaction) × relative molecular mass of reactant) was calculated to give a product yield of about 80.56%.

0.5ml of 2-hydroxy-3,4-dimethoxyphenylpropanol standard solution is weighed out and diluted to 25ml with absolute ethyl alcohol. And scanning the ultraviolet spectrum of the sample by using an ultraviolet spectrophotometer, and selecting the ultraviolet absorption detection wavelength at the position of 252 nm. Accurately weighing a proper amount of 2-hydroxy-3,4-dimethoxy phenylpropanol standard solution, adding ethanol for dissolving to prepare 1.0mg/ml stock solution, respectively sucking 1, 1.5, 2.0, 2.5 and 3.0ml of the stock solution into a 100ml measuring flask, adding ethanol for diluting to scale, and measuring an absorption value at a wavelength of 252nm, wherein the concentration and absorbance detection data of the 2-hydroxy-3,4-dimethoxy phenylpropanol standard solution are shown in Table 7.

TABLE 7

The absorbance (A) is linearly regressed by concentration (C) with the regression equation: c (ug/ml) =0.0235+42.1023A, r =0.9998 (n = 5), 2-hydroxy-3,4-dimethoxyphenylpropanol standard curve is shown in FIG. 6.

Measuring 5mg of the purified 2-hydroxy-3,4-dimethoxy phenylpropanol, diluting to 100mL with absolute ethanol, mixing uniformly, filtering with a microporous membrane, dividing the filtrate into 5 parts, and measuring the absorbance, wherein the absorbance measurement and content calculation data of the 2-hydroxy-3,4-dimethoxy phenylpropanol are shown in Table 8.

TABLE 8

As a result of the measurement, the content of 3,4-dihydroxyphenylpropanol after the purification was about 91.98%.

Experiment two:

comparing the product contents and yields of example 1 and example 2 with those of example 3, it was found that the product contents and yields of example 3 were much higher than those of the other 2 examples.

The experiments of 3 examples all belong to the same experimental conditions except for the difference of the gene combinations and reaction products of the corresponding enzymes, so that the experiments are supplemented to judge the preference of different groups in the recombinant strains for converting the phenylpropanol derivatives.

The gene combinations of the recombinant strains in the preferred experiment of example 3 were preferably selected for the experiments of examples 1 and 2.

Preferably experiment 1 is modified only for the enzyme combination of example 1, using a combination of enzymes of carboxylate reductase (OCQ-RS 16610), alcohol dehydrogenase (ADH 1), and ethylenic reductase (LOC 104820880) to convert 3,4-dihydroxycinnamic acid to 3,4-dihydroxyphenylpropanol, with the remaining experimental conditions, procedure flow referring to example 1, and the experimental data obtained are shown in tables 9-10 below.

TABLE 9 preferred data for experiment 1

Table 10 preferred experiment 1 comparative example 1

Index (I)	Original experiment	Preferred experiments
			Carboxylic acid reductase gene	HXW97-RS16665	OCQ-RS16610
Alcohol dehydrogenase gene	FSUBG-7421	ADH1
			Ethylenic bond reductase gene	LOC104769985	LOC104820880
Purity/content of the product	69.18％	79.68％
			Yield of the product	71.55％	82.78％

Experiment 2 is preferably modified only for the enzyme combination of example 2, using a combination of enzymes carboxylate reductase (OCQ-RS 16610), alcohol dehydrogenase (ADH 1), and ethylenic reductase (LOC 104820880) to convert 2-hydroxy-4-methoxycinnamic acid to 2-hydroxy-4-methoxyphenylpropanol, with the remaining experimental conditions, procedure flow referring to example 1, and the experimental data obtained are shown in tables 11-12 below.

TABLE 11 preferred Experimental 2 data

Table 12 preferred experiment 2 comparative example 2

Index (I)	Original experiment	Preferred experiments
			Carboxylic acid reductase gene	OCQ-RS16610	OCQ-RS16610
Alcohol dehydrogenase gene	FSUBG-7421	ADH1
			Ethylenic bond reductase gene	LOC104769985	LOC104820880
Purity/content of the product	71.44％	81.69％
			Yield of the product	69.59％	80.55％

The comparison of the preferable experiments shows that when the OCQ-RS16610, ADH1 and LOC104820880 genes are combined to construct the strain, the synergistic effect is achieved, and the good promotion effect is achieved on the biological fermentation and transformation of the recombinant strain.

It should be noted that: the invention discloses all preparation processes, conditions and reagent contents of the genetic engineering bacteria, and the genetic engineering bacteria can be fully and repeatedly realized by the technical personnel in the field according to the specification of the invention, so that the strain does not need to be preserved.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.

Claims (9)

1. A method for synthesizing a phenylpropanol derivative, comprising: the method comprises the following steps:

step one, extracting enzyme genes, and constructing engineering bacteria:

extracting genes of carboxylic acid reductase, alcohol dehydrogenase and olefinic bond reductase, preparing recombinant expression plasmids after polymerase chain reaction amplification, and introducing the recombinant expression plasmids into escherichia coli for culture to obtain recombinant expressed engineering bacteria;

step two, culturing engineering bacteria, screening strains with effective expression, and storing the strains:

adding the recombinant escherichia coli into a culture medium for culturing to obtain a seed solution of the recombinant escherichia coli; carrying out amplification culture and fermentation on the seed liquid of the recombinant escherichia coli to obtain a converted solution; detecting the content of target enzymes in the strain, and screening the strain with higher enzyme expression content; preparing the screened strains into high-concentration bacterial suspension by using sterile water, taking a strain preservation protective agent, putting the strain preservation protective agent into the bacterial suspension, fully and uniformly mixing, and freezing to obtain a spare seed solution;

step three, converting 3,4-dihydroxycinnamic acid into 3,4-dihydroxyphenylpropanol by using recombinant escherichia coli:

will be provided with

Adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate and flavin mononucleotide in a mass ratio of 1: (1-2): (1-12): (1-3) dissolving to form a solution, and adding the solution into the standby seed solution obtained in the step two for biotransformation to finally obtain a crude product; adjusting the pH value of the crude product to 5-8, stirring, filtering, washing, and drying to obtain purified product

R ₁ The method comprises the following steps: -CH ₃ -OH, -H or-OCH ₃ ；

R ₂ The method comprises the following steps: -CH ₃ -OH, -H or-OCH ₃ ；

R ₃ The method comprises the following steps: -CH ₃ -OH, -H or-OCH ₃ 。

2. The method of claim 1, wherein the source of the extracted carboxylate reductase gene comprises: HXW97-RS16665 of Mycobacterium marinaum having the sequence of SEQ01 or OCQ-RS16610 of Mycobacterium parintracellular having the sequence of SEQ02; the sources of the genes for extracting the alcohol dehydrogenase comprise: ADH1 of Saccharomyces cerevisiae, with sequence of SEQ03, or FSUBG-7421 of Fusarium subglutinans, with sequence of SEQ04; the sources of the gene for extracting the ethylenic double bond reductase comprise: LOC104820880 of Tarenaya hasslerana with sequence SEQ05, or LOC1047699854 of Camelina sativa with sequence SEQ06.

3. The method of claim 1, wherein the step is a polymerase chain reaction amplificationThe PCR reaction system comprises: 10 XPCR buffer 20ul, dNTP 1698, forward primer 6ul, reverse primer 6ul, taq DNA polymerase 10ul, dd H ₂ O140 ul, template DNA0.1-2ug; amplification procedure and conditions: pre-denaturation at 94 ℃ for 3min; then 35 cycles of denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; finally, extension is carried out for 5min at 72 ℃.

4. The method of claim 3, wherein the forward primer comprises: the sequence of the forward primer of HXW97-RS16665 is SEQ07, the sequence of the forward primer of OCQ-RS16610 is SEQ08, the sequence of the forward primer of ADH1 is SEQ09, the sequence of the forward primer of FSUBG-7421 is SEQ10, the sequence of the forward primer of LOC104820880 is SEQ11, and the sequence of the forward primer of LOC1047699854 is SEQ12; the reverse primer comprises: the sequence of the reverse primer of HXW97-RS16665 is SEQ13, the sequence of the reverse primer of OCQ-RS16610 is SEQ14, the sequence of the reverse primer of ADH1 is SEQ15, the sequence of the reverse primer of FSUBG-7421 is SEQ16, the sequence of the reverse primer of LOC104820880 is SEQ17, and the sequence of the reverse primer of LOC1047699854 is SEQ18.

5. The method for synthesizing phenylpropanol derivatives using immobilized enzyme as claimed in claim 1, wherein the specific method for preparing recombinant expression plasmid in the first step is: the restriction enzyme Nde I/Xho I double enzyme digestion system is adopted to carry out enzyme digestion treatment on the enzyme digestion sites of the gene and the plasmid vector pET-28a (+), and T4 ligase is used for connection overnight at the temperature of 18 ℃, so as to obtain the recombinant expression plasmid.

6. The method for synthesizing phenylpropanol derivatives by using immobilized enzyme as claimed in claim 1, wherein the specific method for obtaining the recombinant expressed engineering bacteria in the first step is: is prepared from CaCl ₂ Adding the recombinant plasmid solution into the treated competent escherichia coli bacterial suspension, and then adding a culture medium to restore the bacteria to a normal growth state; coating the cultured bacterial liquid on a screening plate, and inverting the culture dish after the bacterial liquid is completely absorbed by the culture mediumContinuing to culture; selecting a newly activated single colony from the screening plate, and inoculating the newly activated single colony in a culture medium for culture until the late logarithmic growth phase; the bacterial suspension was inoculated in a medium and cultured to OD600=0.5.

7. The method for synthesizing phenylpropanol derivatives using immobilized enzyme as claimed in claim 1, wherein the step two of detecting the content of the objective enzyme is: the detection method of the carboxylic acid reductase is to detect the absorbance of the converted solution at 340nm by using an ultraviolet spectrophotometer; the detection method of the alcohol dehydrogenase is to detect the absorbance of the converted solution at 340nm by using an ultraviolet spectrophotometer; the detection method of the ethylenic bond reductase is to detect the absorbance of the converted solution at 340nm by using an ultraviolet spectrophotometer.

8. The method for synthesizing phenylpropanol derivatives using immobilized enzyme as claimed in claim 1, wherein the method comprises immobilizing the immobilized enzyme on the phenylpropanol derivative

The method comprises the following steps: 3,4-dihydroxycinnamic acid, 2-hydroxy-4-methoxycinnamic acid, or 2-hydroxy-3,4-dimethoxycinnamic acid.

9. The method for synthesizing phenylpropanol derivatives using immobilized enzyme as claimed in claim 1, wherein the method comprises immobilizing the immobilized enzyme on the phenylpropanol derivative

The method comprises the following steps: 3,4-dihydroxyphenylpropanol, 2-hydroxy-4-methoxyphenylpropanol, and 2-hydroxy-3,4-dimethoxyphenylpropanol.

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