CN114350697A - Preparation method and catalytic application of strain for improving caffeic acid yield - Google Patents

Abstract

The invention discloses a preparation method and catalytic application of a strain for improving caffeic acid yield, wherein a starting plasmid is selected, a monooxygenase gene HpaBC is connected to a restriction enzyme site of the plasmid to obtain a circular plasmid pHG04, the obtained circular plasmid pHG04 is used as a template, a dehydrogenase gene FDH is connected to the restriction enzyme site of the circular plasmid pHG04 to obtain a circular recombinant plasmid pHG05, and then escherichia coli metabolic bacteria is used as the starting strain to introduce the circular recombinant plasmid pHG05 by a chemical conversion method to obtain a constructed recombinant strain sHG 05; the constructed recombinant strain sHG05 is used as biological enzyme, and is subjected to cell culture and induction, and then 4-coumaric acid is catalyzed to prepare caffeic acid, and the yield of the caffeic acid is detected. The invention takes escherichia coli metabolic bacteria as an initial strain, introduces recombinant plasmid pHG05 through a chemical conversion method, obtains a new strain for producing caffeic acid, can be used as biological enzyme to catalyze 4-coumaric acid to prepare caffeic acid, can provide sufficient intracellular coenzyme for the reaction process, reduces substrate residue and improves the yield of the caffeic acid.

Description

Preparation method and catalytic application of strain for improving caffeic acid yield

Technical Field

The invention relates to a preparation method and catalytic application of a strain for improving caffeic acid yield, and belongs to the technical field of metabolic engineering.

Background

Caffeic acid is widely distributed in herba Artemisiae Scopariae, herba Cynara scolymus, flos Lonicerae, etc., belongs to phenolic acid compounds, and has pharmacological effects of protecting cardiovascular system, resisting mutagenesis and cancer, resisting bacteria and virus, reducing blood lipid and blood sugar, resisting leukemia, regulating immunity, promoting bile flow, stopping bleeding, and resisting oxidation. Firstly, caffeic acid is extracted from plants, but the content is low, a large number of plants are consumed, the occupied area is large, and the plant culture is greatly influenced by weather; with the development of industry, caffeic acid was synthesized by chemical method, but because the caffeic acid has cis-form and trans-form difference in structure and substrate residue, the yield is low, and the complete separation of single pure caffeic acid is difficult. In view of this, the invention provides a preparation method and a catalytic application of a strain for improving the yield of caffeic acid.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a preparation method of a bacterial strain for improving the yield of caffeic acid, which takes escherichia coli metabolic bacteria as an original bacterial strain, introduces a recombinant plasmid pHG05 through a chemical conversion method to obtain a novel bacterial strain for producing caffeic acid, and takes the bacterial strain as a biological enzyme for catalyzing 4-coumaric acid to prepare caffeic acid, so that sufficient intracellular coenzyme can be provided for the reaction process, the substrate residue is reduced, and the yield of caffeic acid is improved.

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

the invention provides a preparation method of a strain for improving caffeic acid yield, which comprises the following specific preparation steps:

s1, constructing a plasmid pHG 04: using plasmid as a template, carrying out gene amplification by polymerase chain reaction, digesting by DpnI (methylated template digestive enzyme) to obtain a linearized vector, marking the linearized vector as a linearized vector 1, synthesizing a monooxygenase gene HpaBC through a whole gene, marking the monooxygenase gene HpaBC as a gene fragment 1, connecting the gene fragment 1 at the enzyme cutting site of the linearized vector 1 by a connecting kit to obtain a circular plasmid, and marking the circular plasmid as pHG 04;

s2, constructing a recombinant plasmid pHG 05: taking the circular plasmid pHG04 constructed in the step S1 as a template, performing gene amplification through polymerase chain reaction, digesting by DpnI to obtain a linearized vector, marking the linearized vector as a linearized vector 2, synthesizing dehydrogenase gene FDH through a whole gene, marking the dehydrogenase gene FDH as a gene fragment 2, connecting the enzyme cutting sites of the linearized vector 2 with the gene fragment 2 through a connecting kit to obtain a recombinant circular plasmid, and marking the recombinant circular plasmid as pHG 05;

s3, constructing a recombinant strain sHG 05: adding 1 μ l of the circular recombinant plasmid pHG05 constructed in the step S2 into 100 μ l of BL21 (DE 3) competent cells, carrying out ice bath for 30min, placing in a water bath for hot compress for heat shock treatment, immediately carrying out ice bath for 2min, adding 1ml of LB liquid culture medium, placing at 37 ℃ for culture for 1h to recover thalli, finally uniformly coating the cells containing the circular recombinant plasmid pHG05 on an agarose culture dish, and picking a single colony to obtain a recombinant strain, wherein the recombinant strain is marked as sHG 05;

s4, preservation of thallus: and (4) adding glycerol into the recombinant strain sHG05 constructed in the step S3 to enable the final concentration of the recombinant strain sHG05 to be 15-40%, and placing the recombinant strain in a refrigerator at the temperature of-80 ℃ for preservation.

Wherein, the plasmid in the step S1 is one of a plasmid pADuet, a plasmid pCDuet, a plasmid pEDuet or a plasmid pRDuet, the nucleotide sequence of the plasmid pADuet is shown in SEQ ID NO.1, the nucleotide sequence of the plasmid pCDuet is shown in SEQ ID NO.2, the nucleotide sequence of the plasmid pEDuet is shown in SEQ ID NO.3, and the nucleotide sequence of the plasmid pRDuet is shown in SEQ ID NO. 4.

Wherein the nucleotide sequence of the monooxygenase gene HpaBC in the step S1 is any one of the sequences shown in SEQ ID NO. 5-SEQ ID NO. 8.

Wherein the nucleotide sequence of the dehydrogenase gene FDH in the step S2 is any one sequence shown in SEQ ID NO. 9-SEQ ID NO. 11.

Wherein the water bath temperature in step S3 is 42 deg.C, and the hot compress time is 60S.

The invention also provides a catalysis method for preparing caffeic acid by using the prepared bacterial strain, which comprises the following specific catalysis steps:

S.S1, fermentation culture: activating the recombinant strain sHG05 preserved in the glycerol into an LB liquid culture medium according to the inoculum size of 1% in volume ratio, carrying out overnight culture at 37 ℃, and transferring the overnight-cultured recombinant strain sHG05 into 50ml of TB fermentation culture medium according to the inoculum size of 1% in volume ratio;

s.s2, inducible expression: when the OD600 of the cell reaches 0.2-1.2, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.1-0.3 mM, and carrying out induced expression for 12-24 h at 20-40 ℃;

s.s3, biotransformation: after the induction expression is finished, centrifuging for 3-15 min under the conditions that the temperature is 4 ℃ and the rotating speed is 2000-8000 rpm, taking cell sediment, placing the cell sediment into 10ml of phosphate buffer solution, adding 4-coumaric acid and ammonium formate, then carrying out biocatalytic reaction, and then measuring the content of caffeic acid by adopting high performance liquid chromatography.

Wherein the cell biomass in the step S.s3 is 8-18 g/m³The concentration of the 4-coumaric acid is 1-10 g/L, and the concentration of the ammonium formate is 1-5 g/L.

Wherein, in the step S.s3, the biocatalytic reaction temperature is 25-40 ℃, and the reaction time is 2-6 h.

Wherein the formulation of the phosphate buffer in step s.s 3: mixing solution A and solution B according to the volume ratio of 39:61, and adjusting the pH value to 7.0 to obtain a phosphate buffer solution, wherein the solution A is NaH with the concentration of 0.2mM₂PO₄·2H₂O solution, wherein the B solution is Na with the concentration of 0.2mM₂HPO₄·12H₂And (4) O solution.

The invention has the beneficial effects that: the invention uses BL21 (DE 3) competent cells as an original strain, introduces recombinant plasmid pHG05 by a chemical conversion method, and constructs and obtains a recombinant strain sHG 05; the recombinant strain sHG05 constructed by the invention can be used as a biological enzyme for catalyzing 4-coumaric acid to prepare caffeic acid, and during the catalytic reaction process, dehydrogenase oxidizes formate to CO₂Meanwhile, NAD + is reduced into NADH to continuously assist monooxygenase, so that sufficient internal coenzyme is provided for the reaction process, and the yield of caffeic acid is effectively improved; meanwhile, the method for preparing the caffeic acid by catalytic application improves the yield of the caffeic acid, greatly reduces the residual quantity of the substrate, improves the purity of the caffeic acid, and solves the problems that the prior art has more substrate residues, low purity of the caffeic acid and low yield in the biological preparation process of the caffeic acid, and influences the next separation and purification.

Drawings

FIG. 1 is a process for producing caffeic acid according to the present invention; FIG. 2 is a standard graph of caffeic acid according to the present invention; FIG. 3 is a standard curve for 4-coumaric acid of the present invention; FIG. 4 is a plasmid map of recombinant plasmid pHG05 of the present invention; FIG. 5 is a chromatogram of example 1 of the present invention; FIG. 6 is a chromatogram of example 6 of the present invention; FIG. 7 is a chromatogram of example 7 of the present invention.

Detailed Description

In order to more clearly and completely illustrate the present invention, the following examples are given by way of illustration of the present invention, and are not intended to limit the present invention.

The manufacturer of BL21 (DE 3) competent cells used in the present invention is Scintaceae Biotechnology Co., Ltd; na used₂HPO₄·12H₂O、Na₂HPO₄·12H₂The manufacturers of O, 99% grade caffeic acid and 99% grade 4-coumaric acid are the chemical reagents of the national drug group.

The LB liquid culture medium used in the invention has the following formula: in each liter of culture medium, 10g of tryptone, 5g of yeast extract, 10g of NaCl and the balance of water, and the pH is controlled to be 5.5-6.5.

The formula of the TB fermentation medium used in the invention is as follows: adding 12g of tryptone, 24g of yeast extract, 4ml of glycerol and the balance of water into each liter of culture medium, and controlling the pH to be 5.5-6.5.

The phosphate buffer solution used in the invention has the following formula: solution A: taking Na₂HPO₄·12H₂O3.12 g was dissolved in distilled water and dissolved to 100ml to obtain NaH at a concentration of 0.2mM₂PO₄·2H₂O solution; and B, liquid B: taking Na₂HPO₄·12H₂O7.17g was dissolved in distilled water, and the solution was adjusted to 100ml to obtain Na having a concentration of 0.2mM₂HPO₄·12H₂O solution; mixing the solution A and the solution B in a ratio of 39:61, and adjusting the pH value to 7.0 to obtain the phosphate buffer solution.

Standard curves for caffeic acid and 4-coumaric acid

The contents of caffeic acid and 4-coumaric acid are measured by high performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column is characterized in that the temperature of the chromatographic column is 322 ℃, the mobile phase A is 0.1% acetic acid water, the mobile phase D is methanol, the volume ratio of the mobile phase A to the mobile phase D is shown in Table 1, the flow rate is 1ml/min, and the sample injection volume is as follows: 5 mul, retention time of 22 min; by adopting the chromatographic condition, the peak time of caffeic acid is about 9.6min, and the peak time of 4-coumaric acid is about 13.4 min.

TABLE 1

Respectively taking 100mg of 99% grade caffeic acid and 99% grade 4-coumaric acid, respectively dissolving with methanol, transferring into a 50ml volumetric flask, and diluting with methanol to scale; preparing 12 10ml volumetric flasks, transferring 50ml of caffeic acid with constant volume into 1ml, 2ml, 4ml, 6ml, 8ml to 6 l0ml volumetric flasks respectively, and diluting to the scale with methanol; transferring 50ml of constant-volume 4-coumaric acid into 1ml, 2ml, 4ml, 6ml, 8ml to 6 l0ml volumetric flasks respectively, filtering with a 0.22 mu m filter head by using a disposable sterile syringe, sampling according to the chromatographic conditions, drawing a standard curve of the caffeic acid and a standard curve of the 4-coumaric acid respectively by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and obtaining the standard curve of the caffeic acid, wherein the y =3 x 10⁷x - 198377，R² = 1（R²Is a linear fitting constant), the standard curve thereof is shown in fig. 2, and a standard curve y =2 × 10 of 4-coumaric acid is obtained⁷x + 433989，R² = 0.998（R²Is a linear fitting constant) whose standard curve is shown in fig. 3.

Examples

1. Construction of recombinant strain sHG 05:

the method comprises the following steps: using plasmid as a template, carrying out gene amplification through polymerase chain reaction, digesting by DNA digestive enzyme to obtain a linearized vector, marking the linearized vector as a linearized vector 1, synthesizing monooxygenase gene HpaBC through a whole gene, marking the monooxygenase gene HpaBC as a gene fragment 1, connecting the gene fragment 1 at the enzyme cutting site of the linearized vector 1 through a connecting kit to obtain a circular plasmid, and marking the circular plasmid as pHG 04; wherein the plasmid is one of a plasmid pADuet with a nucleotide sequence shown as SEQ ID NO.1, a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.2, a plasmid pEDuet with a nucleotide sequence shown as SEQ ID NO.3 or a pRDuet with a nucleotide sequence shown as SEQ ID NO. 4; the nucleotide sequence of the monooxygenase gene HpaBC is any one sequence shown in SEQ ID NO. 5-SEQ ID NO. 8;

step two: using the circular plasmid pHG04 as a template, performing gene amplification through polymerase chain reaction, digesting by DNA digestive enzyme to obtain a linearized vector, marking the linearized vector as a linearized vector 2, synthesizing dehydrogenase gene FDH through a whole gene, marking the gene fragment 2, connecting the enzyme cutting site of the linearized vector 2 with the gene fragment 2 through a connecting kit to obtain a recombinant circular plasmid, and marking the recombinant circular plasmid as pHG 05; wherein the nucleotide sequence of the dehydrogenase gene FDH is any one sequence shown in SEQ ID NO. 9-SEQ ID NO. 11;

step three: adding 1 μ l of the above circular recombinant plasmid pHG05 into 100 μ l of BL21 (DE 3) competent cells, ice-bathing for 30min, placing in 42 deg.C water bath for hot compress for 60s for heat shock treatment, immediately ice-bathing for 2min, adding 1ml of LB liquid culture medium, placing at 37 deg.C for culture for 1h to recover thallus, uniformly coating the cells containing circular recombinant plasmid pHG05 on agarose culture dish, picking single colony to obtain recombinant strain, and marking as sHG 05; and (4) adding glycerol into the recombinant strain sHG05 constructed in the step S3 to enable the final concentration of the recombinant strain sHG05 to be 15-40%, and placing the recombinant strain in a refrigerator at the temperature of-80 ℃ for preservation.

2. The preparation method of the caffeic acid comprises the following steps:

the method comprises the following steps: activating the recombinant strain sHG05 preserved in the glycerol into an LB liquid culture medium according to the inoculum size of 1% in volume ratio for overnight culture, and transferring the recombinant strain sHG05 subjected to overnight culture into 50ml of TB fermentation medium according to the inoculum size of 1% in volume ratio;

step two: when the OD600 of the cell reaches 0.2-1.2, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.1-0.3 mM, and carrying out induced expression for 12-24 h at 20-40 ℃;

step three: after the induction expression is finished, centrifuging for 3-15 min under the conditions that the temperature is 4 ℃ and the rotating speed is 2000-8000 rpm, taking cell sediment, placing the cell sediment into 10ml of phosphate buffer solution, adding 4-coumaric acid and ammonium formate, placing the cell sediment at a certain temperature for reaction, then sampling, filtering the sample by using a 0.22 mu m filter head through a disposable sterile injector, diluting the sample by 10-20 times according to a caffeic acid standard curve, and measuring the content of caffeic acid by adopting high performance liquid chromatography.

Example 1

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 1: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.2 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.6 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.9 as a gene fragment 2, and ensuring that the cell biomass is 8g/m³The adding amount of 4-coumaric acid is 1g/L, the adding amount of ammonium formate is 1g/L, the reaction temperature is 25 ℃, samples are sampled, filtered and diluted after 1.9h of reaction, the result is shown in Table 2 by high performance liquid chromatography detection, and the chromatogram is shown in figure 5.

Example 2

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 2: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.2 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.6 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.9 as a gene fragment 2, and ensuring that the cell biomass is 8g/m³The addition amount of 4-coumaric acid is 1g/L, the addition amount of ammonium formate is 0.9g/L, the reaction temperature is 30 ℃, samples are sampled, filtered and diluted after 2 hours of reaction, and the results are shown in Table 2 after detection by high performance liquid chromatography.

Example 3

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 3: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.2 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.6 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.9 as a gene fragment 2, and taking the cell biomass as 6g/m³The adding amount of the 4-coumaric acid is 1g/L, the adding amount of the ammonium formate is 1g/L, the reaction temperature is 30 ℃, and the mixture is sampled and filtered after reacting for 2 hoursFiltering, diluting, and detecting by high performance liquid chromatography, the results are shown in table 2.

Example 4

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 4: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.2 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.6 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.9 as a gene fragment 2, and ensuring that the cell biomass is 8g/m³The addition amount of 4-coumaric acid is 1g/L, the addition amount of ammonium formate is 1g/L, the reaction temperature is 30 ℃, samples are sampled, filtered and diluted after 2 hours of reaction, and the results are shown in Table 2 through high performance liquid chromatography detection.

Example 5

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 5: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.2 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.6 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.10 as a gene fragment 2, and ensuring that the cell biomass is 8g/m³The addition amount of 4-coumaric acid is 3g/L, the addition amount of ammonium formate is 1.5g/L, the reaction temperature is 25 ℃, samples are sampled, filtered and diluted after 4 hours of reaction, and the results are shown in Table 2 after detection by high performance liquid chromatography.

Example 6

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 6: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.1 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.8 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.9 as a gene fragment 2, and ensuring that the cell biomass is 12g/m³The addition amount of 4-coumaric acid is 6.5g/L, the addition amount of ammonium formate is 3.2g/L, the reaction temperature is 35 ℃, samples are sampled, filtered and diluted after 4 hours of reaction, and the results are shown in Table 2 through high performance liquid chromatography detection, and the chromatogram is shown in FIG. 6.

Example 7

In this example 7The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid used in the examples: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.4 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.5 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.11 as a gene fragment 2, and taking the cell biomass as 18g/m³The addition amount of 4-coumaric acid is 10g/L, the addition amount of ammonium formate is 5g/L, the reaction temperature is 40 ℃, samples are sampled, filtered and diluted after 6 hours of reaction, and the results are shown in Table 2 and the chromatogram is shown in FIG. 7 through high performance liquid chromatography detection.

Example 8

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 8: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.3 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.7 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.10 as a gene fragment 2, and ensuring that the cell biomass is 12g/m³The addition amount of 4-coumaric acid is 10g/L, the addition amount of ammonium formate is 5g/L, the reaction temperature is 35 ℃, samples are sampled, filtered and diluted after 6 hours of reaction, and the results are shown in Table 2 through high performance liquid chromatography detection.

Example 9

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 9: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.3 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.7 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.11 as a gene fragment 2, and taking the cell biomass as 18g/m³The addition amount of 4-coumaric acid is 10g/L, the addition amount of ammonium formate is 5g/L, the reaction temperature is 42 ℃, samples are sampled, filtered and diluted after 6 hours of reaction, and the results are shown in Table 2 through high performance liquid chromatography detection.

Example 10

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 10: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.3 as a template and taking the nucleotide sequenceThe monooxygenase gene HpaBC shown as SEQ ID NO.7 is taken as a gene fragment 1, the monooxygenase gene HpaBC shown as SEQ ID NO.9 in nucleotide sequence is taken as a gene fragment 2, and the cell biomass is 18g/m³The addition amount of 4-coumaric acid is 10.5g/L, the addition amount of ammonium formate is 5g/L, the reaction temperature is 40 ℃, samples are sampled, filtered and diluted after 6 hours of reaction, and the results are shown in Table 2 after high performance liquid chromatography detection.

Example 11

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 11: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.3 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.7 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.9 as a gene fragment 2, and ensuring that the cell biomass is 20g/m³The addition amount of 4-coumaric acid is 10g/L, the addition amount of ammonium formate is 5g/L, the reaction temperature is 40 ℃, samples are sampled, filtered and diluted after 6 hours of reaction, and the results are shown in Table 2 through high performance liquid chromatography detection.

Example 12

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 12: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.4 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.5 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.11 as a gene fragment 2, and taking the cell biomass as 18g/m³The addition amount of 4-coumaric acid is 10g/L, the addition amount of ammonium formate is 5g/L, the reaction temperature is 22 ℃, samples are sampled, filtered and diluted after 6 hours of reaction, and the results are shown in Table 2 through high performance liquid chromatography detection.

Example 13

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 13: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.4 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.8 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.10 as a gene fragment 2, and taking the cell biomass as 18g/m³The addition amount of 4-coumaric acid is 10g/L, the addition amount of ammonium formate is 5.3g/L, the reaction temperature is 25 ℃, samples are sampled, filtered and diluted after 6 hours of reaction, and the results are shown in Table 2 after high performance liquid chromatography detection.

Example 14

The procedure of the steps for the construction of the recombinant strain sHG05 and the preparation of caffeic acid of the example was used in this example 14: taking a plasmid pCDuet with a nucleotide sequence shown as SEQ ID NO.1 as a template, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.5 as a gene fragment 1, taking a monooxygenase gene HpaBC with a nucleotide sequence shown as SEQ ID NO.11 as a gene fragment 2, and taking the cell biomass as 18g/m³The adding amount of the 4-coumaric acid is 10g/L, the adding amount of the ammonium formate is 5g/L, the reaction temperature is 27 ℃, samples are sampled, filtered and diluted after 6.2h of reaction, and the results are shown in Table 2 through high performance liquid chromatography detection.

TABLE 2

	Residual quantity g/L of coumaric acid	Caffeic acid yield g/L	Conversion rate of caffeic acid		Residual quantity g/L of coumaric acid	Caffeic acid yield g/L	Conversion rate of caffeic acid
								Example 1	0.1	0.981	89.31%	Example 8	0.013	10.907	99.29%
Example 2	0.08	1.003	91.29%	Example 9	0.1	10.791	98.24%
								Example 3	0.996	0.987	89.85%	Example 10	0.5	10.9	94.50%
Example 4	0.003	1.089	99.14%	Example 11	0.175	10.706	97.46%
								Example 5	0.009	3.27	99.23%	Example 12	0.8	10.028	91.29%
Example 6	0.008	7.093	99.34%	Example 13	0.4	10.53	95.86%
								Example 7	0.017	10.905	99.27%	Example 14	0.2	10.682	97.24%

From the above examples, examples 1 to 14 and table 2, it can be found that the heavy material constructed by the present inventionThe group strain sHG05 can be used as biological enzyme, can be well applied to catalyze 4-coumaric acid to prepare caffeic acid, provides sufficient internal coenzyme for the reaction process, and improves the yield of caffeic acid; meanwhile, from example 4 to example 8, the discovery that the cell biomass is 8-18 g/m in the biotransformation that the recombinant strain sHG05 catalyzes 4-coumaric acid to generate caffeic acid³The concentration of the 4-coumaric acid is 1-10 g/L, the concentration of the ammonium formate is 1-5 g/L, the reaction temperature is 25-40 ℃, and the reaction time is 2-6 hours, so that the 4-coumaric acid is efficiently converted into the caffeic acid, the yield of the caffeic acid product is more than 99%, the yield of the caffeic acid is high, the residual quantity of the 4-coumaric acid substrate is greatly reduced, the purity of the caffeic acid is also effectively improved, and further, the cell biomass is preferably 8-18 g/m³The concentration of the 4-coumaric acid is 1-10 g/L, the concentration of the ammonium formate is 1-5 g/L, the reaction temperature is 25-40 ℃, and the reaction time is 2-6 h, so that the preparation method and the catalytic application of the strain provided by the invention are used as the environment of the biological catalytic reaction, the separation and purification of the caffeic acid are facilitated, and the problems that in the prior art, the separation and purification of the next step are influenced due to more substrate residues, low caffeic acid purity and low yield in the biological preparation process of the caffeic acid are solved.

Finally, it should be noted that the above embodiments are only used for illustrating and not limiting the technical solutions of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the present invention without departing from the spirit and scope of the present invention, and all modifications or partial substitutions should be covered by the scope of the claims of the present invention.

Sequence listing

<110> Nanjing Hegu Biotechnology Ltd

<120> bacterial strain for improving caffeic acid yield and catalytic preparation method

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ggaactccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact 900

tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt 960

tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg 1020

atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg 1080

aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt 1140

tggaacctct tacgtgccga tcaacgtctc aagagggccg cggcaaagcc gtttttccat 1200

aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac 1260

ccgacaggac tataaagata ccaggcgttt cccctggcgg ctccctcgtg cgctctcctg 1320

ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca 1380

cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc 1440

cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa 1500

agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt 1560

gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc 1620

aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg 1680

caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga 1740

agatcatctt attaatcaga taaaatattt ctagatttca gtgcaattta tctcttcaaa 1800

tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc 1860

cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc 1920

ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag 1980

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 2040

ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt 2100

gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag 2160

caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt 2220

atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc 2280

gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc 2340

ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg 2400

ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag 2460

acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc 2520

gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat 2580

gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac 2640

agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc 2700

gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac 2760

caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg 2820

cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag 2880

ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc 2940

ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga 3000

gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa 3060

ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt 3120

gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaaatta atacgactca 3180

ctata 3185

<210> 2

<211> 2877

<212> DNA

<213> Artificial sequence

<400> 2

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atgggcagca gccattgagg ggttttttgc tgaaacctca ggcatttgag 120

aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa 180

gcggctattt aacgaccctg ccctgaaccg acgaccgggt catcgtggcc ggatcttgcg 240

gcccctcggc ttgaacgaat tgttagacat tatttgccga ctaccttggt gatctcgcct 300

ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct 360

tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc 420

tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac 480

caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag 540

ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca 600

aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc 660

aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg 720

cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg 780

tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc 840

gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc 900

accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg 960

tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct 1020

gatagttgag tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat 1080

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 1140

aaaaataaac aaatagctag ctcactcggt cgctacgctc cgggcgtgag actgcggcgg 1200

gcgctgcgga cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg 1260

tgaggcaaaa cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca 1320

accacggtta agcagttccc caactgactt aaccttcgat caaaccacct ccccaggtgg 1380

ttttttcgtt tacagggcaa aagattacgc gcagaaaaaa aggatctcaa gaagatcctt 1440

tgatcttttc tactgaaccg ctctagattt cagtgcaatt tatctcttca aatgtagcac 1500

ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc cccgcgccca 1560

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 1620

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 1680

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 1740

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 1800

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 1860

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 1920

atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 1980

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 2040

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 2100

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 2160

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 2220

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 2280

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 2340

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 2400

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 2460

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 2520

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 2580

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 2640

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 2700

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 2760

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 2820

tctcgacgct ctcccttatg cgactcctgc attaggaaat taatacgact cactata 2877

<210> 3

<211> 4588

<212> DNA

<213> Artificial sequence

<400> 3

ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag 60

gagatatacc atgggcagca gccgtctact agcgcagctt aattaaccta ggctgctgcc 120

accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 180

ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat 240

taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 300

cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 360

aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 420

ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 480

ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 540

caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 600

cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 660

taacgtttac aatttctggc ggcacgatgg catgagatta tcaaaaagga tcttcaccta 720

gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 780

gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 840

ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 900

atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 960

agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 1020

ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 1080

tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 1140

ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 1200

caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 1260

gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 1320

atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 1380

accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgaaggtaa ctggcttcag 1440

cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 1500

gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 1560

cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 1620

gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 1680

caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 1740

aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 1800

tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 1860

gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 1920

ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 1980

atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 2040

cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg 2100

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt 2160

acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 2220

gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 2280

tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 2340

ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt 2400

cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca 2460

gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt 2520

tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga 2580

aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 2640

gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 2700

gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 2760

ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 2820

acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 2880

agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 2940

cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg 3000

cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg 3060

cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg 3120

gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 3180

gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc 3240

ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg 3300

cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg 3360

tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc 3420

attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca 3480

ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc 3540

gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc 3600

gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc 3660

agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt 3720

gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca 3780

atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga 3840

agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc 3900

acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg 3960

tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt 4020

tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc 4080

gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca 4140

ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga 4200

ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc 4260

gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt 4320

gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag 4380

tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa 4440

gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc 4500

tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga 4560

tcccgcgaaa ttaatacgac tcactata 4588

<210> 4

<211> 3011

<212> DNA

<213> Artificial sequence

<400> 4

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atgggcagca gccatactag cgcagcttaa ttaacctagg ctgctgccac 120

cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 180

gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc 240

ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgacaag 300

ctgacgaccg ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga acccctattt 360

gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac 420

tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt 480

tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca 540

agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc 600

ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt 660

gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc 720

tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg 780

agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg 840

cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat 900

acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta 960

cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc 1020

atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc 1080

gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga 1140

gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa 1200

gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg aagcatttat 1260

cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 1320

ggcatgcagc gctcttccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg 1380

cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt tatactgaaa gaacagattt 1440

tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa gagttggtag ctcagcgaac 1500

cttgagaaaa ccaccgttgg tagcggtggt ttttctttat ttatgagatg atgaatcaat 1560

cggtctatca agtcaacgaa cagctattcc gttactctag atttcagtgc aatttatctc 1620

ttcaaatgta gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc 1680

atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga 1740

gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct 1800

ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 1860

ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag 1920

ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg 1980

ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc 2040

ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt 2100

aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac 2160

gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc 2220

ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag 2280

acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc 2340

caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact 2400

gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc 2460

ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg 2520

ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat 2580

cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg 2640

cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc 2700

cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac 2760

tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg 2820

ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac 2880

cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc 2940

gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac 3000

gactcactat a 3011

<210> 5

<211> 1563

<212> DNA

<213> Serratia plymuthica AS9

<400> 5

atgaaaccag aagactttcg tgccgacagc aaacgcccgt tcaccggcgc cgagtacctg 60

aaaagcctgc aggatggccg cgaaatctat atttacggcg agcgggtgaa ggacgtcacc 120

acccacccgg cgttccgcaa tgcggcggct tcggtcggcc aattgtacga cgcgctgcac 180

gatccggtca gccaggatcg cctgtgctgg aataccgaca ccggcaacgg cggctacacc 240

cacaagttct tccgctacgc tcgcagcccg gaagaaatgc gccaacagcg tgacgccatc 300

gccgactggt cgcgccagac ctacggctgg atggggcgca ccccggacta caaggcggcc 360

ttcggctgcg ctttgggggc ctatccggag ttttacgggc agttcgccga taatgcccgc 420

cattggtaca aacgcattca ggaaaccggt ctgtacttta accacgccat cgtcaatccg 480

ccgattgacc gccacaaacc ggtcaacgaa gtgaaagacg tttacatcca ggtggagaaa 540

gagaccgacg ccggcattat cgtcagcggc gccaaggtgg tggcgaccaa ctcggcgctg 600

acccactaca actttatcgg cttcggctcc gcccaggtga tgggcgataa cccggatttt 660

gcgctgatgt ttgtggcgcc gatggacgcc gaaggggtga agcttatctc ccgcgcctcc 720

tatgagctgg tggccggcgc gaccggatcg ccgttcgatt acccgctctc gagccgtttt 780

gacgagaacg atgcaatcct gattatggat cacgtgctga tcccctggga aaacgtgctg 840

atctaccgcg atttcgatcg ttgtcgccgt tggagcaccc agggcggctt cgcccggttg 900

ttcccgctgc aggcctgcgt gcgtttggcg gtgaagatgg actttatcac cgcgctgctg 960

caaaagagcc tgtcctgcac cggcgtgctg gagttccgcg gcgtgcaggc cgatctcggc 1020

gaagtggtgg cctggcgcaa cctgttctgg tcgctgagcg atgcgatgtg cgcggagtcc 1080

acaaaatggg aaaacggcgc ctatctgccg gattccgccg cgttgcagac ctaccgcgtg 1140

atggcgccga tggcctacac caaggtgaag cacatcatcg aaaagaacgt caccagcggg 1200

ctgatttatc tgccgtccag cgtgcgcgac atgaacaacc cggagatcga caaatacctg 1260

gctcgctacg tgcgcggatc cgatgggatg gatcacgttg agcgcatcaa gatcctcaag 1320

ctgatgtggg atgcgatcgg cagcgagttc ggcggccgtc atgagctgta tgagatcaac 1380

tatgccggca gtcaggatga gatccgcctg cagtgtctgc gccacgcgca gggatccggc 1440

accatggatc agatgatgca gatggtcgac aagtgcctgt ccgattacga tcagcacggc 1500

tggaaagtgc cgcacctgca taacaatggc gatattaatc agctggataa tctgctgaag 1560

taa 1563

<210> 6

<211> 1515

<212> DNA

<213> Deinococcus radiodurans

<400> 6

atgaccaccg aattcaaggg ccagaccatt ccccccatgc acacgggcgg ccagggcgcc 60

gtcaccgggc agcgatttct cgaccgcctg cgtcagaacc cgccgacgct gtatatcgac 120

gggcagcggg tggccgaccc caccacccac cccagcacca gaaacatgtg ccagtcgctc 180

gcgggcctct acgacctgca atttcaggaa gacctgaaag aaacgctgac ctacgaggac 240

ggcggcaagc gctacgcccg ctcgttcatg gtgccgcgca ccaaagacga cctgcgcctg 300

attgccgagt cgcaccgcat tcgcgccaat tacggcctgg gctttctggg ccgtgcgccc 360

gactacatga acgccaacgt gatggcggcg ggcgccgggg ccgagtattt caacgggtgc 420

agcgcggcgg tgcccggcga ccccaagcgc gattttgccg ccaacatgcg ccgctattac 480

gagtatgtgc agggcaacga cctgtgcctg acccacgcgc tgaccaaccc gcaggtcaac 540

cgcagcaaga tggcctccga actgcccgac ccgtacatcg cgctgggcat cgtggaggaa 600

acggacgaag gcgtgatcgt gcgcggcgcc cgcatgatgg cgacgctgcc gattgccgac 660

gaaatcctga ttttccccag tacggtgctc aaggaaaacg ccgacaagag ccgctacgcg 720

atgggtttcg ggattcctac caatacgccc ggcctcagct ttcagtgccg cgagccgatt 780

gacgtgggcc gcgaccccga agaccatccc ctcagcagcc gcttcgacga acaggacgcc 840

ttcgtcatct tcgacgacgt gctggtgccc tgggagcgca ttttcttgct ctacgacgtg 900

gaactggcga acaaggccta cgccggcacc gacgccgtac tgcacatggc ctatcaggtg 960

gtcaacctca aaatcgccaa gaccgaggcg ttcctgggca ccgcgcagag catcgtgaac 1020

gccatcggca gcggcgggtt tcagcatgtg caggccaaaa tcgccgaaat catcatcatg 1080

ctcgaaatca tgaaggcgct ggaagtcgcc gcccgcgagc aggccgagcc gaacgactac 1140

ggtgtgatga cccccgcccg tgccccgctg gacgccgccc gcaactacta ccccgccaac 1200

cacgcccggc tgcccgagct gctgcaacta ctgggggcgt ccggaatcat catgatgccg 1260

agcaaggccg accgcgaagg cccgctgggg ccgcaaatcg ccaagtacct gcaaaccggc 1320

aacgccaacg ccgacgaacg cctgcgcctc tttcgcctcg cctgggacat gtccatgagc 1380

agtttcgcgg gccgccagga actctacgag cgctacttct tcggcgaccc ggtgcggatg 1440

cactcggcgc tgtacgaggt ctacgacagc agcgaagcgg tggcgcggat tggggagttt 1500

ttgcagcgga agtga 1515

<210> 7

<211> 516

<212> DNA

<213> Serratiamarcescens

<400> 7

atgtctcagg aaaatgaaca gcggctgcgc tttcgcgacg cgatggccag cctgtcggcg 60

gcggtgaata tcgtcaccac cgacggcccg gcggggcgct gcggcatcac cgccacggcg 120

gtgtgctcgg tgaccgatac gccgccgacg ctgctggtgt gcatcaaccg caacagcgcg 180

atgaacccgg tatttcagga aaatcgcagg ctgtgcgtca acgtgctcaa ccacgaacag 240

gaactgatgg cgcgccactt cgccggcatg accggcgtca gcatggaaga tcgtttcagg 300

ctggaagagt ggcagcttgg cgcgttgggg cagcctgtat tgcgcaatac cctggccagc 360

ctggaagggg agatcgagca gatccagagc atcggcactc accagatgta cctggtgcag 420

atcaagcaga tcgcgctgag cgaagccggc aacggcctga tctacttcaa gcgcaacttc 480

cattcggtga tccaccagat ggcggtgccg gcctga 516

<210> 8

<211> 1563

<212> DNA

<213> Klebsiellaoxytoca

<400> 8

atgaaacctg aaaatttccg tgcagacgct aaacgcccgt taactggcga agagtacctg 60

aagagcctgc aggacggccg cgaaatctat atctacggcg aacgcgttaa ggacgtgacc 120

acccacccgg cgttccgcaa cgccgccgcc tccgtcgccc agatgtacga tgcgctgcat 180

aaacccgagc tgcaggatac cctgtgctgg ggcaccgata ccggcagcgg cggctatacc 240

cacaagttct tccgcgtcgc gaaaagcgcc gacgatctgc gccagcagcg cgacgccatc 300

gctgaatggt cgcgcctgag ctacggctgg atgggccgta ccccggacta caaagccgca 360

ttcggctgcg cgctcggcgc aaacccggcg ttttatggtc agtttgaaca gaacgcccgt 420

aactggtaca cccgcatcca ggaaaccggc ctctacttta accacgccat cgtcaacccg 480

ccgatcgacc gccataaacc agccgacgaa gtgaaagacg tctatatcaa gctggagaaa 540

gagaccgatg ccgggatcat cgtcagcggc gcgaaagtgg tggccaccaa ctcggcgctg 600

acccactaca atatgattgg cttcggctcg gcgcaggtga tgggcgaaaa cccggacttc 660

gcgctgatgt ttgtcgcgcc gatggatgcc gaaggcgtta agcttatctc ccgcgcctcc 720

tatgaactgg tcgccggagc aaccggatcg ccgtacgact atccgctctc cagccgcttc 780

gacgaaaacg atgcgatcct ggtgatggat aatgtgctga tcccatggga aaacgtgctg 840

atctatcgcg acttcgatcg ctgccgccgc tggacgatgg aaggcggttt cgcccgcatg 900

tatccgctgc aggcctgcgt gcgtctggcc gtgaagctcg actttatcac cgccctgctc 960

aagcgctcgc tggagtgcac cggcaccctg gagttccgcg gcgtacaggc ggatctcggc 1020

gaagtggtag cctggcgcaa tatgttctgg gcgctgagcg actcgatgtg cgccgaagcc 1080

acgccgtggg ttaacggcgc gtatctgccg gatcacgccg cgctgcaaac ctatcgcgtg 1140

atggcgccga tggcctacgc caaaatcaaa aacattattg aacgcaacgt caccagcggc 1200

ctgatctatc tgccgtccag cgcccgcgat ctcaacaatc cgcagatcaa cgaatatctg 1260

gcgaaatacg tgcgcggatc gaacggcatg gatcacgtcg agcgtatcaa gatcctcaaa 1320

ttgatgtggg acgccatcgg cagcgagttt ggtgggcgtc acgaactgta cgaaatcaac 1380

tactcaggta gccaggatga aattcgcctg cagtgcctgc gtcaggcgca gagctccggc 1440

aacatggaca aaatgatggc gatggtggat cgctgcctgt ccgagtacga ccagaacggc 1500

tggacggtgc cgcatctgca caacaatacc gatatcaaca tgctggataa gctgctgaag 1560

taa 1563

<210> 9

<211> 2318

<212> DNA

<213> Neurospora crassa

<400> 9

ggggccgcca gactagcagg caaagctaga ggtaccttga tggatgcagc tctcgttgtg 60

atgggaggta agtaggagcg cgaggaaaag aggggctcaa gtgaatgaga gaagagaccc 120

gggctgattc agttccttgg tctttttttg aagatataaa tcatggacat ttcccgtctt 180

gtcctcatct actacatcac cactcttcac tcaacaacag acactctact ataaagacgt 240

tcctctcgtc tttcatccat ccactataac accaaacttt tctatcacat aaacaaaaca 300

accgacaaaa tggtacgttt agcgcgctcc gttgcttctt ctccctcttc cttcacttcg 360

gtccttccac agtgtttctc aaacatcatc acccccgaga gcgtccagcc ttcgacctag 420

tgtccagggc tactcctcga cgggacttca ccagtgtcgg cagcagcaac tccccgcaag 480

tgtacaattg ctaacaacat gacgcgaaca ggtcaaggtt cttgctgttc tctacgacgg 540

tggcaagcac ggcgaggagg taagatgaac attttccaca tccaacttac tccactctgt 600

catggagtgg ccatccactt tccttggcat atccgccaaa tgtctccatc accattgtcc 660

atgatcgatc aaaagcccag ccctatgagc tcaagagccc aaaaaaagaa gagcgctgag 720

aaagatgaga caacctgagc tacatctctc atgcgactga aagagacgga acggacacaa 780

atggcggggt agacatggct cgctcaaagg catcaccgca actgtttacc cgtcatgttg 840

aatgcttgag gtcacggacc gggtttcggg atccattcgg ggatgtgggg cgccaccccg 900

acctgctttg gcgtcgaacc cggcggttgg cttccggctt ggcatagcgt gggttgttgg 960

cttggctttt atggacatgg caatggagac attggatgac acgggaaata tgggagaaaa 1020

caccaagaag gcaatggcta acaattgtca actcatcagg ttcccgagct tctcggaacc 1080

atccagaacg agcttggtct ccgcaagtgg ctcgaggatc agggccacac cctcgtcacc 1140

acctgcgaca aggacggcga gaactctacc tttgacaagg agctcgagga tgccgagatc 1200

atcatcacca ctcccttcca ccccggctac ctcactgctg agcgtctcgc ccgcgccaag 1260

aagctcaagc ttgctgtcac tgccggtatc ggttccgacc acgtcgacct caatgccgcc 1320

aacaagacca acggcggtat caccgtcgcc gaggttaccg gctccaacgt cgtctccgtt 1380

gctgagcacg ttctcatgac catcctcgtc ctcgtccgca acttcgttcc cgcccacgag 1440

cagatccagg agggccgctg ggacgtcgcc gaggccgcca agaacgagtt cgatctcgag 1500

ggcaaggttg tcggtaccgt cggtgtcggc cgtatcggtg agcgtgtcct ccgccgtctc 1560

aagcccttcg actgcaagga gctcctctac tacgactacc agcccctttc cgccgagaag 1620

gaggctgaga tcggctgccg ccgcgtcgcc gacctcgagg agatgctcgc ccagtgcgac 1680

gtcgtcacca tcaactgccc ccttcacgag aagacccagg gtctcttcaa caaggagctc 1740

atctccaaga tgaagaaggg ctcgtggctc gtcaacactg cccgtggcgc catcgtcgtc 1800

aaggaggacg tcgccgaggc cctcaagtcc ggccacctcc gcggctacgg cggtgacgtc 1860

tggttccccc agcccgcccc tcaggaccac cccctccgct acgccaagaa ccccttcggc 1920

ggcggcaacg ccatggtccc ccacatgtcc ggcacctcgc tcgacgccca gaagcgttac 1980

gctgctggca ccaaggccat catcgagagc tacctctccg gcaagcacga ctacaggcct 2040

gaggacctca tcgtctacgg cggtgactac gccaccaagt cttatggtga gcgcgagcgc 2100

gccaaggctg ctgctgctgc tgccaagtct gcttaaggag ggcatcagca agcagcaagc 2160

acttcttttt tttttctttt gcatttttgc atgattaaat agcgattttt gggggctttc 2220

ttttgggata tatatctggt tttgggtatg aagcaataaa gatcgtggaa tatcaatgat 2280

catgacctga cctaacaata atcgttccca ttgtgatg 2318

<210> 10

<211> 2135

<212> DNA

<213> Tribolium castaneum

<400> 10

aacgaaggtg tttttttcca ccatgactac tgccggaaag gtaatttttt cataattatg 60

ttcagattaa ccgttttgat ccctcagcct ataacttgca aagctgcagt tgcttggggg 120

cccccgagaa gacctaaaac tcgaaaccat cgaagtggcc cctcctaaga gtcacgaagt 180

ccgaattaaa attatttaca gtgcagtttg tcatacagat gcttacaccc tcagtggcca 240

agatcccgag ggattattcc ctgtcatctt aggccacgag ggcggtggaa ttgtggaaag 300

tgtgggagag ggagttacaa acgttcagcc aggtatttta caattgctca ttgatttcta 360

attaattgaa atgaaaaggt gaccatgtca ttccgttata cattccccaa tgtggcgaat 420

gcaaattttg caagtcgccc aaaaccaact tgtgccaaaa aatcagggtg acacagggac 480

aaggggtgat gcccgatggt accagtagat tcacgtgcaa tggtaaaaaa gtgtaccatt 540

ttatgggttg ctccaccttt agcgagtata ccgttgttgc cgacatctca gttgctaaag 600

taagtgataa caaaggtgaa ggtcaccagt attaatcatt gttatcaagg tgaatcccaa 660

tgccgaactt gacaaaattt gtctgttggg ttgtggcgtc ccaactggat atggagcagc 720

cttaaatacc gccaaagttg aaaaagggag cacctgcgcg gtttggggct tgggggctgt 780

gggccttgcc gtcgggattg gatgcaaggc tgcgggggcg tccaaaatca tcggagtcga 840

tgtcaatcca gacaaattca aaattggtaa aaacacaatt ttcagtaaaa cctctacaat 900

ccgaacctaa ttttattgac attaagtgta taaatagtac ctataaaact atgccgggtg 960

tttcagtttg gtattgtaac ttatttattt aaaataatta taacaaatca cataccgggt 1020

gatgagtttc acataccaca tatatcgtta tatgtttatt aacgtctatc agtaggatat 1080

ttagttttca agtgtttcat cgataatgca tggaatacag gtacgatcaa tttgaaatca 1140

tttctttttt atttgttgtt tattaccgtt accaactgac acaaaattcc gtagacattt 1200

tttttattat taaaatgaga aaaaaaagag ggtgcaaatt gtaacagata gcataaaata 1260

gtcgttttat gagggttata aaacttgttg tttttgagat tattttttac tacttaaatt 1320

ttaaaatata aatttccctt ctatgtagcc taccgtgatt ttgataattt gacatataat 1380

tttctagcca taaatttaca ccaattgttt caaaaaactg atatccgtcg aagttacttg 1440

agaatgctac ttctttatat catcaatttc agtactgttt tcaaactttt attacttttt 1500

gagatacgct atacaaatct agcaattttg catctcgtag tatttagttc aaatatcgca 1560

ttatttattg ttgcaatgaa ttcgtagcca aagacttcgg cttcaccgac ttcgtgaacc 1620

ccaaagacta cgaaaagccg attcaggaag tgttggctga aaagacagac ggtggtctcg 1680

acttcacttt cgaatgtgtt gggaatgttc agactatgag agcagcgttg gagtcatgtc 1740

acaagggttg gggggtctcc acaattgtgg gggttgctgg tgcagggcaa gaaataagca 1800

ctagaccttt ccagctcgta acagggaggg tgtggaaagg aacggctttt ggagggtgga 1860

aaagccgcga tagtgttccg aaactagttg aggaatattg ccataaacct caaatcatgc 1920

cgcttgacaa gtttatcacg cataatttga aatttggtca aattaacgaa gcgttccatc 1980

ttatgcacaa aggggaaagg taagttcttt tattgtgtga tacttgactt attatttttt 2040

tagtattcga actgttgttg caattgctgg aagtgcgtaa agtaaacaca ttgtcaatac 2100

gaaaatattt taattaataa aaatggaaaa attaa 2135

<210> 11

<211> 1136

<212> DNA

<213> Mycobacterium intracellulare

<400> 11

atggcgaaat gcgtgatggt gctgtatccg gatccggtgg atggctatcc gccggcgtat 60

gcgcgcgata gcattccgac cattcatggc tatccacccc gagcaccatt gattttaccc 120

cgggcgaact gctgggctgc gtgagcggcg cgctgggcct gcgcaaattt tttgaagatg 180

cgggccatga actggtggtg acgagcgata aagatggccc ggatagcgaa tttgaacgcg 240

cgctgccgga tgcggaaatt gtgattagtc agccgttttg gccggcgtat ctgaccaaag 300

aacgcattgc gaaagcgccg aaactgaaac tggcgctgac cgcgggcatt ggcagcgatc 360

atgtggatct ggatgcggcg aaagaacgcg gcattaccgt ggcggaagtg acctatagca 420

acagcattag cgtggcggaa catgcggtga tgcagattct ggcgctggtg cgcaactttg 480

tgccgagcca tcgctgggcg gtggagggcg gctggaacat tgcggattgc gtggaacgcg 540

cgtatgatct ggaaggcatg gatgtgggcg tgattgcggc gggccgcatt ggccgtgccg 600

tgctgcgccg tctggcgcca tttgatgtga acctgcatta taccgatacc cgccgcctgg 660

cgccggaggt ggaaaaagaa ctgaacgtga cctttcatcc gaccgtgcaa gaactggtgc 720

gcgcggtgga tgtggtgagc attcatagcc cgctgtatgc ggatacccgc gcgatgtttg 780

atgaaaaact gattagcacc atgcgccgcg gcagctatat tgtgaacacc gcgcgcgcgg 840

aagaaaccgt gccggaagcg attgcggatg cgctgcgcag cggtcagctg ggcggctatg 900

cgggcgatgt gtggtatccg cagccgccac cggttgcgca tccgtggcgc accatgccga 960

acaacgcgat gaccccgcat gttagcggca ccaccctgag cgcgcaagcg cgctatgcgg 1020

cgggcacccg cgaaattctg gaaagctggt ttgcgggcac cccgattcgc ccggaatatc 1080

tgattgtgga aggcggccgc ctggcgggca ccggcgcgct gagctatcag aaataa 1136

Claims (9)

1. A preparation method of a strain for improving the yield of caffeic acid is characterized by comprising the following specific preparation steps:

s1, construction of plasmid pHG04

Using plasmid as a template, carrying out gene amplification by polymerase chain reaction, digesting by DpnI (methylated template digestive enzyme) to obtain a linearized vector, marking the linearized vector as a linearized vector 1, synthesizing a monooxygenase gene HpaBC through a whole gene, marking the monooxygenase gene HpaBC as a gene fragment 1, connecting the gene fragment 1 at the enzyme cutting site of the linearized vector 1 by a connecting kit to obtain a circular plasmid, and marking the circular plasmid as pHG 04;

s2 construction of recombinant plasmid pHG05

Taking the circular plasmid pHG04 constructed in the step S1 as a template, performing gene amplification through polymerase chain reaction, digesting by DpnI to obtain a linearized vector, marking the linearized vector as a linearized vector 2, synthesizing dehydrogenase gene FDH through a whole gene, marking the dehydrogenase gene FDH as a gene fragment 2, connecting the enzyme cutting sites of the linearized vector 2 with the gene fragment 2 through a connecting kit to obtain a recombinant circular plasmid, and marking the recombinant circular plasmid as pHG 05;

s3 construction of recombinant Strain sHG05

Adding 1 μ l of the circular recombinant plasmid pHG05 constructed in the step S2 into 100 μ l of BL21 (DE 3) competent cells, carrying out ice bath for 30min, placing in a water bath for hot compress for heat shock treatment, immediately carrying out ice bath for 2min, adding 1ml of LB liquid culture medium, placing at 37 ℃ for culture for 1h to recover thalli, finally uniformly coating the cells containing the circular recombinant plasmid pHG05 on an agarose culture dish, and picking a single colony to obtain a recombinant strain, wherein the recombinant strain is marked as sHG 05; the formula of the LB liquid culture medium: in each liter of culture medium, 10g of tryptone, 5g of yeast extract, 10g of NaCl and the balance of water, wherein the pH is controlled to be 5.5-6.5;

s4 cell culture

And (4) adding glycerol into the recombinant strain sHG05 constructed in the step S3 to enable the final concentration of the recombinant strain sHG05 to be 15-40%, and placing the recombinant strain in a refrigerator at the temperature of-80 ℃ for preservation.

2. The method of claim 1, wherein the water bath temperature is 42 ℃ and the hot compress time is 60S in step S3.

3. The method of claim 1, wherein the plasmid in step S1 is one of plasmid pADuet, plasmid pCDuet, plasmid pEDuet or plasmid pRDuet; wherein the nucleotide sequence of the plasmid pADuet is shown as SEQ ID NO.1, the nucleotide sequence of the plasmid pCDuet is shown as SEQ ID NO.2, the nucleotide sequence of the plasmid pEDuet is shown as SEQ ID NO.3, and the nucleotide sequence of the plasmid pRDuet is shown as SEQ ID NO. 4.

4. The method for preparing a strain capable of improving caffeic acid production according to claim 1, wherein the nucleotide sequence of the monooxygenase gene HpaBC of step S1 is any one of the sequences shown in SEQ ID No. 5-SEQ ID No. 8.

5. The method for preparing a strain capable of improving caffeic acid production according to claim 1, wherein the nucleotide sequence of the dehydrogenase gene FDH in step S2 is any one of the sequences shown in SEQ ID No. 9-SEQ ID No. 11.

6. A catalytic process for the preparation of caffeic acid using the strain prepared according to claim 1, characterized by the following specific catalytic steps:

s. 1, fermentation culture

Activating the recombinant strain sHG05 preserved in the glycerol into an LB liquid culture medium according to the inoculum size of 1% in volume ratio, carrying out overnight culture at 37 ℃, and transferring the overnight-cultured recombinant strain sHG05 into 50ml of TB fermentation culture medium according to the inoculum size of 1% in volume ratio; the formula of the LB liquid culture medium: in each liter of culture medium, 10g of tryptone, 5g of yeast extract, 10g of NaCl and the balance of water, wherein the pH is controlled to be 5.5-6.5; the formula of the TB fermentation medium comprises the following components: in each liter of culture medium, 12g of tryptone, 24g of yeast extract, 4ml of glycerol and the balance of water, wherein the pH is controlled to be 5.5-6.5;

s. 2, inducible expression

When the OD600 of the cell reaches 0.2-1.2, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.1-0.3 mM, and carrying out induced expression for 12-24 h at 20-40 ℃;

s. 3, bioconversion

After the induction expression is finished, centrifuging for 3-15 min under the conditions that the temperature is 4 ℃ and the rotating speed is 2000-8000 rpm, taking cell sediment, placing the cell sediment into 10ml of phosphate buffer solution, adding 4-coumaric acid and ammonium formate, then carrying out biocatalytic reaction, and then measuring the content of caffeic acid by adopting high performance liquid chromatography.

7. The catalytic process of claim 6, wherein the cell biomass in step S.s3 is 8-18 g/m³The concentration of the 4-coumaric acid is 1-10 g/L, and the concentration of the ammonium formate is 1-5 g/L.

8. The catalytic method of claim 6, wherein the biocatalytic reaction temperature in step S.s3 is 25-40 ℃ and the reaction time is 2-6 hours.

9. The catalytic process for the preparation of caffeic acid of claim 6, wherein the formulation of phosphate buffer in step s.s 3: mixing solution A and solution B according to the volume ratio of 39:61, and adjusting the pH value to 7.0 to obtain a phosphate buffer solution, wherein the solution A is NaH with the concentration of 0.2mM₂PO₄·2H₂O solution, wherein the B solution is Na with the concentration of 0.2mM₂HPO₄·12H₂And (4) O solution.

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