CN114958700A - Escherichia coli engineering bacterium and application thereof - Google Patents

Abstract

The invention relates to an escherichia coli engineering bacterium and application thereof, and particularly relates to an escherichia coli gene deletion bacterium BL21 delta FadB, R and J, wherein the escherichia coli gene deletion bacterium is obtained by knocking out a FadB gene, a FadR gene and a FadJ gene from escherichia coli BL21, and the escherichia coli gene deletion bacterium BL21 delta FadB, R and J are used as transformation host bacteria of a recombinant plasmid pCDFDuet-1-MaMACS-PpFadE and a recombinant plasmid pET28a-SUMO-ctYdii to catalyze capric acid to generate trans-2-decenoic acid, so that the specificity is realized.

Description

A kind of Escherichia coli engineering bacteria and its application

This application is a divisional application with the application number of 202110211118.9, the application date of 2021.02.25, and the title of the invention "A mutant enzyme CYP153AM228L and its application in the synthesis of 10-hydroxy-2-decenoic acid".

technical field

The invention relates to an Escherichia coli engineering bacteria and application, and belongs to the technical field of biological fermentation.

Background technique

10-hydroxy-2-decenoic acid (10-HDA) is a monounsaturated fatty acid containing hydroxyl, and the molecular formula is C ₁₀ H ₁₈ O ₃ . So far, only royal jelly and propolis are found in nature, so it is also called royal jelly acid. Studies have shown that 10-HDA has many important physiological functions such as antibacterial, immune regulation, antioxidant, anti-tumor, and lowering blood sugar. The compound structure is as follows:

In view of the extensive and important application value of 10-HDA, the research to find efficient, convenient and low-cost production methods for 10-HDA has been widely paid attention to. At present, the main methods for obtaining 10-HDA are physical extraction and chemical synthesis. of which physical extraction

The source is single, and the content of 10-HDA in royal jelly is only 1.4%-2.4%, so the output is small and cannot meet the market demand. Although the chemical synthesis method can meet the needs of the industry, its operation steps are cumbersome and the chemical reagents have certain toxicity. Therefore, exploring an efficient, convenient and low-cost synthesis method for 10-HDA has important theoretical and application value for its large-scale development and utilization. In recent years, the production of 10-HDA by microbial fermentation synthesis has become a new target of researchers and the industry.

Chinese patent document CN109897870A (application number: 201910088897.0) discloses a method for preparing 10-hydroxy-2 decenoic acid by using decanoic acid as a raw material and using Escherichia coli engineering bacteria. The patent document utilizes decanoic acid to generate 10-HDA in one step.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides an Escherichia coli engineering bacteria and its application.

The application utilizes a two-step method to generate 10-HDA, and the fatty acyl-CoA dehydrogenase gene PpFadE, the fatty acyl-CoA synthase gene MaMACS, the acyl-CoA thioesterase gene ctydiI and the Chinese patent document CN109897870A (application number: 201910088897.0), the fatty acyl-CoA dehydrogenase gene MCAD, the fatty acyl-CoA synthase gene FadK, and the acyl-CoA thioesterase gene ydiI are not the same genes, and the alkane hydroxylase CYP153A in this application is to change the amino acid at position 228 from M. for L.

The present invention is aimed at the existing synthetic route constructed by a single engineering bacterium, and the 10-HDA synthesis efficiency is extremely low and cannot reach the industrialized level. Provided is a two-step method for producing 10-hydroxy-2-decenoic acid by using decanoic acid as a raw material and utilizing Escherichia coli engineering bacteria.

The technical scheme of the present invention is as follows:

An Escherichia coli gene-deficient strain BL21ΔFadB, R, J is obtained by knocking out FadB gene, FadR gene and FadJ gene in Escherichia coli BL21.

The Δ symbol means that a gene has been knocked out from the genome of the large intestine, FadB is enoyl-CoA hydratase, FadR is a protein operon, and FadJ is 3-hydroxyacyl-CoA dehydrogenase; the presence of these three enzymes The two-step method of the present invention catalyzes the production of 10-HDA from decanoic acid, so it is knocked out.

Application of Escherichia coli gene deletion bacteria BL21ΔFadB, R, J in the preparation of 10-hydroxy-2-decenoic acid.

The construction method of the above-mentioned Escherichia coli gene deletion bacteria BL21ΔFadB, R, J, comprises the following steps:

Ⅰ. Knock out the gene using RED recombination method, and construct gene deletion ΔFadRB strain (hereinafter referred to as ΔFadRΔFadB strain); for the construction method of this strain, refer to Chinese patent document CN110684794A;

Ⅱ Use the RED recombination method to knock out the gene and construct the E. coli gene-deficient bacteria BL21ΔFadB, R, and J, including the construction of the FadJ knockout box (the FadJ knockout box is abbreviated as Jk below); the FadJ knockout box is transformed into pkd46-ΔFadRΔFadB In competent cells, Escherichia coli gene deletion bacteria BL21ΔFadB, R, J were obtained.

Preferably according to the present invention, constructing the FadR knockout frame in step II includes the following steps:

Using the genome of Escherichia coli BL21 as a template, the upstream homology arm FadJ1 of the 3-hydroxyacyl-CoA dehydrogenase gene was amplified. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.25, and the nucleotide sequence of the downstream primer As shown in SEQ ID NO.26; using Escherichia coli BL21 genome as a template, amplify the downstream homology arm FadJ2 of 3-hydroxyacyl-CoA dehydrogenase gene, and the nucleotide sequence of the upstream primer is shown in SEQ ID NO.27 , the nucleotide sequence of the downstream primer is shown in SEQ ID NO.28; the pkd3 plasmid is used as a template to amplify the FRT-RKan-FRT gene fragment, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.29, the downstream primer The nucleotide sequence is shown in SEQ ID NO.30; then FadJ1, FRT-RKan-FRT and FadJ2 gene fragment multi-fragment seamless cloning, amplifying the knockout frame fragment of FadJ1-Kan-FadJ2, the core of the upstream primer The nucleotide sequence is shown in SEQ ID NO. 31, the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 32, and the FadJ knockout box is recovered from the purified gel (hereinafter, the FadJ knockout box is abbreviated as Jk).

Further preferably, the PCR amplification system is as follows, and the total system is 50 μL:

100μM upstream primer 2.0μL, 100μM downstream primer 2.0μL, template 2.0μL, 5U/μL phanta enzyme 25μL, ddH2O 19μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 1 min 15 s, 30 cycles; extension at 72 °C for 5 min.

Preferably according to the present invention, in step II, the FadJ knockout box is transformed into the pkd46-ΔFadRΔFadB recombinant bacteria, and finally obtain

Escherichia coli gene deletion bacteria BL21ΔFadB, R, J, including the following steps:

a. The plasmid pkd46 was transformed into ΔFadRΔFadB competent cells, and the obtained pkd46-ΔFadRΔFadB recombinant bacteria were prepared to prepare pkd46-ΔFadRΔFadB competent cells, and then the prepared pkd46-ΔFadRΔFadB competent cells were stored in 10% glycerol;

b. Transform the FadJ knockout box Jk into pkd46-ΔFadRΔFadB competent cells, and verify that the pkd46-Jk-BL21 recombinant strain confirms that after the knockout frame is transferred, pkd46 is eliminated at 42°C, and the Jk-ΔFadRΔFadB recombinant strain is obtained after screening;

c. The Jk-ΔFadRΔFadB recombinant strain was prepared to be competent to transform the pcp20 plasmid, and the Jk resistance and the pcp20 plasmid were eliminated at 42°C to obtain the ΔFadRBJ recombinant strain, which is the E. coli gene deletion strain BL21ΔFadB, R, J.

A method for preparing 10-hydroxy-2-decenoic acid by using decanoic acid as a raw material and utilizing Escherichia coli engineering bacteria resting cells, comprising the following steps:

(1) Construction of the optimized recombinant plasmid pCDFDuet-1-MaMACS-PpFadE, the optimized recombinant plasmid pET21b-CYP153A M228L-CPR _BM3 , and the optimized recombinant plasmid pET28a-SUMO-ctYdiI;

The nucleotide sequence of the expression gene of the CYP153A-CPR _BM3 fusion enzyme is shown in SEQ ID NO.13; the nucleotide sequence of the expression gene of the CYP153AM228L-CPR _BM3 fusion enzyme is shown in SEQ ID NO.14; fatty acyl CoA synthesis The nucleotide sequence of the enzyme gene MaMACS is shown in SEQ ID NO.15; the nucleotide sequence of the fatty acyl-CoA dehydrogenase gene PpFadE is shown in SEQ ID NO.16; the nucleus of the acyl-CoA thioesterase gene ctYdiI The nucleotide sequence is shown in SEQ ID NO.17;

The CYP153A M228L is a mutation of the amino acid at position 228 of the CYP153A enzyme from M to L.

(2) Use the recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdiI obtained in step (1) to transform the E. coli knockout strains to construct E. coli BL21ΔFadB, R, J, pCDFDuet-1-MaMACS -PpFadE, pET28a-SUMO-ctYdiI, the recombinant plasmid pET21b-CYP153AM228L-CPR _{BM3 was} transformed into Escherichia coli BL21 to construct Escherichia coli BL21 pET21b-CYP153AM228L-CPR _BM3 , two engineered bacteria were screened, induced and cultured to obtain induced cells ;

(3) The induced cells obtained in step (2) were cultured in the transformation medium to obtain resting cells, and then rested in Escherichia coli BL21ΔFadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdiI Add decanoic acid to the cells and culture to obtain trans-2-decenoic acid; add Escherichia coli BL21 pET21b-CYP153A M228L-CPR _BM3 resting cells to the reaction solution, and culture to obtain 10-hydroxy-2-decenoic acid.

Preferably according to the present invention, in the step (1), the construction of the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE includes the following steps:

Using Escherichia coli DH5a genome as a template, amplify the fatty acyl-CoA synthase gene MaMACS, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, Then, the pCDFDuet-1 plasmid was double digested with BamH I and Hind III, and then ligated with ligase to obtain the recombinant plasmid pCDFDuet-1-MaMACS;

The PCR amplification system is as follows, the total system is 25 μL:

100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5U/μL phanta enzyme 12.5 μL, ddH ₂ O 9.5 μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 50 s, 30 cycles; extension at 72 °C for 5 min.

Using Escherichia coli DH5a genome as a template, amplify the fatty acyl-CoA dehydrogenase gene PpFadE, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10 , and then the pCDFDuet-1-MaMACS plasmid was double digested with Nde I and Ava I, and ligated by ligase to obtain the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE;

The PCR amplification system is as follows, the total system is 25 μL:

100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5U/μL phanta enzyme 12.5 μL, ddH ₂ O 9.5 μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 40 s, 30 cycles; extension at 72 °C for 5 min.

Preferably according to the present invention, in the step (1), the construction of the recombinant plasmid pET28a-SUMO-ctYdiI includes the following steps:

Using Escherichia coli DH5a genome as a template, amplify the acyl-CoA thioesterase gene ctydiI, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.12. Then, the pET28a-SUMO plasmid and the acyl-CoA thioesterase gene ctydiI were double digested with BamHI and Xho I, respectively, and ligated with ligase to prepare the recombinant plasmid pET28a-SUMO-ctYdiI;

The PCR amplification system is as follows, the total system is 25 μL:

100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5U/μL phanta enzyme 12.5 μL, ddH ₂ O 9.5 μL.

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 15 s, 30 cycles; extension at 72 °C for 5 min.

Preferably according to the present invention, in the step (2), the screening is to insert the transformed Escherichia coli engineering bacteria into the corresponding concentrations of 50 μg/mL kanamycin, 100 μg/mL ampicillin and 40 μg/mL streptomycin In the LB liquid medium, shake screening and culture at 35-40 °C until the OD ₆₀₀ of the bacterial liquid is 0.8-1.2.

According to the preferred embodiment of the present invention, in the step (2), the induction culture is to cool the screened and cultured bacterial liquid to 18-20°C for 0.5-1 hour, then add IPTG to a concentration of 0.5-0.8mM, add oil Acid to reach a mass percentage concentration of 0.4-0.8%, add Tween 80 to a mass percentage concentration of 0.2-0.5%, continue inducing and culturing for 18-20 hours, separate cells, and prepare induced cells.

Further preferably, in the step (2), the inductive culture condition is to cool the bacterial liquid of the screening culture to 20 ° C for 1 hour, then add IPTG to make the IPTG concentration in the medium be 0.5mM, and add oleic acid to make the medium. The medium mass percentage concentration was 0.6%, Tween 80 was added to make the mass percentage concentration of Tween 80 in the medium to be 0.3%, and the induction culture was continued for 18 hours, and the cells were separated to obtain induced cells.

Further preferably, in the step (2), the induction culture condition is to cool the screened and cultured bacterial liquid to 20°C for 1 hour, then add FeCl ₃ with a final concentration of 0.5mM and 5-ALA with a final concentration of 0.5mM to continue the induction culture. After 18 hours, cells were isolated and induced cells were prepared.

Further preferably, in the step (2), the cells are separated by centrifugation at 5000 rpm for 15 minutes, the precipitate is collected, and then washed with saline with a concentration of 0.85% by mass.

Preferably according to the present invention, the transformation medium components in the step (3) include the following:

Glycerol is 0.8-1.2% by mass fraction, glucose is 0.3-0.5% by mass fraction, kana antibiotic is 40-60 μg/mL, ampicillin antibiotic 90-110 μg/mL, streptomycin antibiotic 30-50 μg/mL, the balance The solvent was potassium phosphate buffer at a concentration of 100 mM at pH 7.4.

Further preferably, glycerol is 1% by mass percentage, glucose is 0.4% by mass percentage, kana antibiotic 50 μg/mL, ampicillin 100 μg/mL, streptomycin antibiotic 40 μg/mL, and the remaining solvent is the concentration of pH7.4 100 mM potassium phosphate buffer.

Preferably according to the present invention, in step (3), decanoic acid is added to the resting cells to a concentration of 0.3-0.7g/L, and the reaction is carried out at 28-37°C for 7-10h to obtain trans-2-decenoic acid ; Add Escherichia coli BL21 pET21b-CYP153AM228L-CPR _BM3 resting cells to the reaction solution, and react at 28-37°C for 10-24h to obtain 10-hydroxy-2-decenoic acid.

Further preferably, in the step (3), the conversion concentration of capric acid is 0.5 g/L.

More preferably, in step (3), capric acid is added to the resting cells to a concentration of 0.5 g/L, and the reaction is carried out at 30° C. for 9 h to obtain trans-2-decenoic acid; Escherichia coli is added to the reaction solution. BL21 pET21b-CYP153A M228L-CPR _BM3 resting cells were reacted at 30 °C for 20 h to obtain 10-hydroxy-2-decenoic acid.

According to a preferred embodiment of the present invention, in the step (3), the transformation culture conditions are cultured at 29-31° C. for 8 hours.

Preferably according to the present invention, in the step (3), capric acid is dissolved in dimethyl sulfoxide.

beneficial effect

1. The present invention constructs the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE, the recombinant plasmid pET21b-CYP153AM228L-CPR _BM3 , and the recombinant plasmid pET28a-SUMO-ctYdiI to realize the high-efficiency expression of the 10-hydroxy-2-decenoic acid expression element, and through After special induction treatment, the engineering bacteria Escherichia coli BL21ΔFadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdiI resting cells were fermented to produce trans-2-decenoic acid with capric acid as raw material, and then the The engineering bacteria Escherichia coli BL21 pET21b-CYP153A M228L-CPR _BM3 resting cells were added to the above-mentioned reaction solution to continue the reaction, and finally 10-hydroxy-2-decenoic acid was obtained by fermenting raw decanoic acid as a raw material, so as to realize the process with low-value decanoic acid. Two-step production process of high value-added 10-hydroxy-2-decenoic acid 0.5g/L decanoic acid was fermented in shake flasks to produce 0.273g/L of 10-hydroxy-2-decenoic acid with a conversion rate of 54.6% .

2. The present invention significantly improves the conversion rate of 10-hydroxy-2-decenoic acid by optimizing the expression pathway of 10-hydroxy-2-decenoic acid, and optimizing each expression element in the pathway and the relevant conditions in the transformation process, thereby It makes the industrial production of 10-hydroxy-2-decenoic acid possible.

Description of drawings

Fig. 1 is a schematic diagram of a recombinant plasmid structure;

In the figure: A is the recombinant plasmid pET21b-CYP153A M228L-CPR _BM3 , B is the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE, and C is the recombinant plasmid pET28a-SUMO-ctYdiI.

Fig. 2 is the agarose gel electrophoresis picture of PCR amplification of CYP153A and CPR _BM3 gene products;

In the figure, M is the marker, the first row is the CYP153A gene, and the second row is the CPR _BM3 gene.

Fig. 3 is the agarose gel electrophoresis image of the target gene band of the recombinant plasmid pET21b-CYP153A M228L-CPR _BM3 amplified by PCR;

M in the figure is the marker, and 1-4 in the figure are the linearized bands of the recombinant plasmid pET21b-CYP153A M228L-CPR _BM3 .

Fig. 4 is agarose gel electrophoresis image of PCR amplification ctYdiI product;

In the figure, M is the marker, and 1-5 are the bands of the ctydiI gene.

Fig. 5 is Nde I, Xho I double digestion plasmid pET21b plasmid map;

In the figure, lane M is Marker; lanes 1-6 are the results of enzyme cleavage.

Fig. 6 is pET28a-SUMO, ctYdiI double digestion lipose gel electrophoresis image;

The first gel map is the sequence of the pET28a-SUMO double-enzyme-digested gene, and 1-3 in the figure are the bands after pET28a-SUMO double-enzyme digestion; 3 are the bands after double digestion with ctYdiI.

Figure 7 is an SDS-PAGE protein gel image;

The first glue is BL21ΔFadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdiI protein expression, MaMACS is 59KD, PpFadE is 45KD, ctYdiI is 27KD; the second glue is Escherichia coli BL21 The protein expression of pET21b-CYP153A M228L-CPRBM3, CYP153A M228L-CPRBM3 protein is 117KD.

Fig. 8 is the chromatogram of decanoic acid producing 10-hydroxy-2-decenoic acid;

In the figure, the retention time of 8.395min is trimethylsilanol phosphate, the retention time of 10.426min is trans-2-decenoic acid TMS derivative, the retention time of 11.564min is lauric acid TMS derivative, and the retention time of 12.744min is 10-hydroxyl Decanoic acid TMS derivative, retention time of 13.092min is 10-hydroxy-2-decenoic acid TMS derivative, retention time of 14.292min is palmitic acid TMS derivative, retention time of 15.564min is (Z)-oleic acid TMS derivative , the TMS is trimethylsilane.

Figure 9 is a mass spectrum of the intermediate product trans-2-decenoic acid.

Figure 10 is the mass spectrum of the final product 10-hydroxy-2-decenoic acid.

Figure 11 is an agarose gel electrophoresis image of colony PCR to verify ΔFadRΔFadB knockout FadJ product;

In the figure, lane M is Marker; lanes 1-2 are FadJ gene bands, and lanes 3-8 are FadJ knockout box fusion fragment gene bands.

Detailed ways

The content of the present invention will be further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited to this. The operation methods not described in detail in the examples are conventional operation methods known to those skilled in the art.

The reagents and medicines used in the present invention are all common commercial products.

Example 1

The fusion enzyme gene of alkane hydroxylase CYP153A of Marinobacter aquaeolei and Bacillus megaterium P450NADH reductase, namely CYP153A-CPR _BM3 fusion enzyme gene, CYP153AM228L-CPR _BM3 fusion enzyme gene, fatty acyl CoA dehydrogenase PCR amplification of the enzyme gene PpFadE, the fatty acyl-CoA synthase gene MaMACS, and the acyl-CoA thioesterase gene ctydiI.

According to the fusion enzyme genes of alkane hydroxylase CYP153A of Marinobacter aquaeolei and Bacillus megaterium P450NADH reductase, namely CYP153A gene, CPR _BM3 gene, CYP153AM228L-CPR _BM3 fusion enzyme gene, fatty acyl CoA dehydrogenase Enzyme gene PpFadE, fatty acyl-CoA synthase gene MaMACS, ester acyl-CoA thioesterase gene ctydiI design PCR amplification primers, and the nucleotide sequences of the upstream primers are respectively SEQ ID NO.1, 3, 5, 7, 9, 11 As shown, the nucleotide sequences of the downstream primers are shown in SEQ ID NO. 2, 4, 6, 8, 10, and 12;

Among them, the fusion enzyme gene of alkane hydroxylase CYP153A of Marinobacter aquaeolei and Bacillus megaterium P450NADH reductase, namely CYP153A-CPRBM3, CYP153AM228L-CPRBM3 fusion enzyme gene, fatty acyl-CoA dehydrogenase gene PpFadE , the nucleotide sequences of fatty acyl-CoA synthase gene PpFadE, ester acyl-CoA thioesterase gene ctYdiI are respectively shown in SEQ ID NO.13, 14, 15, 16, 17, and the corresponding expressed amino acid sequence is as SEQ ID NO. 18, 19, 20, 21, 22, the nucleotide sequence of CYP153A M228L is shown in SEQ ID NO.23, and the amino acid sequence of CYP153A M228L is shown in SEQ ID NO.24.

The described construction of the recombinant plasmid pET21b-CYP153A-CPR _BM3 includes the following steps:

Using the fusion enzyme gene of alkane hydroxylase CYP153A of Marinobacter aquaeolei and Bacillus megaterium (Bacillus megaterium) P450NADH reductase as template to carry out PCR amplification, the nucleotide sequence of the upstream primer of CYP153A is as follows Shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, the nucleotide sequence of the upstream primer of CPR _BM3 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3. As shown in SEQ ID NO.4, the pET21b plasmid was then double digested with Nde I and Xho I, and connected by a multi-segment seamless cloning kit to obtain a recombinant plasmid pET21b-CYP153A-CPR _BM3 ;

The PCR amplification system is as follows, the total system is 25 μL:

100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5U/μL phanta enzyme (a high-fidelity enzyme) 12.5 μL, ddH ₂ O 9.5 μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 45 s for CYP153A and CPR _BM3 for 55 s, 30 cycles; extension at 72 °C for 5 min.

PCR product recovery:

After PCR amplification, the length of the fragment was analyzed by 1% agarose gel electrophoresis. The results are shown in Figure 2. The target band was cut out according to the size of the fragment and recovered using the DNA gel recovery kit from Shanghai Sangon Bioengineering Co., Ltd. PCR product.

The described construction of the recombinant plasmid pET21b-CYP153A M228L-CPR _BM3 includes the following steps:

Using the constructed pET21b-CYP153A-CPR _BM3 fusion enzyme gene as a template to carry out PCR amplification, the nucleotide sequence of the upstream primer of CYP153AM228L-CPR _BM3 is as shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.5 ID No. 6; then, after removing the original template with Dpn I enzyme, it was transformed into the competent cell Escherichiacoli BL21 to make it circular, and the recombinant plasmid pET21b-CYP153A M228L-CPR _{BM3 was obtained} .

The PCR amplification system is as follows, the total system is 25 μL:

100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5U/μL phanta enzyme (a high-fidelity enzyme) 12.5 μL, ddH ₂ O 9.5 μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 100 s, 30 cycles; extension at 72 °C for 5 min.

PCR product recovery:

After PCR amplification, the length of the fragment was analyzed by 1% agarose gel electrophoresis. The results are shown in Figure 3. The plasmid of pET21b-CYP153A M228L-CPR _BM3 is shown in Figure 1, A, and the target band was cut according to the size of the fragment. PCR products were recovered using a DNA gel recovery kit from Shanghai Sangon Bioengineering Co., Ltd.

DpnI digestion template

DpnI digestion system:

PCR product 44μL

DpnI 1 μL

10X QuickCut Buffer 5μL

Reaction conditions: 37°C for 2h.

DpnI was inactivated at 70°C for 15 min after the completion of the reaction.

The described construction of the recombinant plasmid pCDFDuet-1-MaMACS includes the following steps:

Using Escherichia coli DH5a genome as a template, amplify the fatty acyl-CoA synthase gene MaMACS, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, Then the pCDFDuet-1 plasmid was double digested with Hind III and BamH I, and then ligated with ligase to obtain the recombinant plasmid pCDFDuet-1-MaMACS;

The PCR amplification system is as follows, the total system is 25 μL:

100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5U/μL phanta enzyme (a high-fidelity enzyme) 12.5 μL, ddH ₂ O 9.5 μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 50 s, 30 cycles; extension at 72 °C for 5 min.

PCR product recovery:

After PCR amplification, the length of the fragment was analyzed by 1% agarose gel electrophoresis, and the target band was cut out according to the size of the fragment, and the PCR product was recovered using a DNA gel recovery kit from Shanghai Sangon Bioengineering Co., Ltd.

The described construction of the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE includes the following steps:

Take Escherichia coli DH5a genome as a template, amplify fatty acyl CoA dehydrogenase gene PpFadE, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.10 , and then the pCDFDuet-1-MaMACS plasmid was double digested with Nde I and Ava I, and ligated by ligase to obtain the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE;

The PCR amplification system is as follows, the total system is 25 μL:

100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5U/μL phanta enzyme 12.5 μL, ddH ₂ O 9.5 μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 40 s, 30 cycles; extension at 72 °C for 5 min.

PCR product recovery:

After PCR amplification, the length of the fragment was analyzed by 1% agarose gel electrophoresis, and the target band was cut out according to the size of the fragment, and the PCR product was recovered using a DNA gel recovery kit from Shanghai Sangon Bioengineering Co., Ltd.

The described construction of recombinant plasmid pET28a-SUMO-ctYdiI includes the following steps:

Amplify the acyl-CoA thioesterase gene ctydiI, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.12, and then the pET28a-SUMO plasmid And the acyl-CoA thioesterase gene ctydiI was double digested with BamH I and Xho I respectively, and ligated by ligase to obtain the recombinant plasmid pET28a-SUMO-ctYdiI;

The PCR amplification system is as follows, the total system is 25 μL:

100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5U/μL phanta enzyme 12.5 μL, ddH ₂ O 9.5 μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 15 s, 30 cycles; extension at 72 °C for 5 min.

PCR product recovery:

After PCR amplification, the length of the fragment was analyzed by 1% agarose gel electrophoresis. The results are shown in Figure 4. The target band was cut out according to the size of the fragment and recovered using the DNA gel recovery kit from Shanghai Sangon Bioengineering Co., Ltd. PCR product.

Example 2

Recombinant plasmid vector pET21b, pCDFDuet-1, pCDFDuet-1-MaMACS, pET28a-SUMO and gene ctYdiI digestion

The pET21b plasmid was extracted and subjected to double digestion reaction. The reaction system was as follows:

Reaction conditions: 37°C for 6h.

The pET21b plasmid vector was double digested and purified by 1% agarose gel electrophoresis as shown in Figure 5, and the target fragment was recovered using a DNA gel recovery kit.

The pCDFDuet-1 plasmid was extracted and subjected to double-enzyme digestion reaction. The reaction system was as follows:

Reaction conditions: 37°C for 6h.

The pCDFDuet-1 plasmid vector was double digested and purified by 1% agarose gel electrophoresis, and the target fragment was recovered using a DNA gel recovery kit.

The pCDFDuet-1-MaMACS plasmid was extracted and subjected to double digestion reaction. The reaction system was as follows:

Reaction conditions: 37°C for 6h.

The pCDFDuet-1-MaMACS plasmid vector was double digested and purified by 1% agarose gel electrophoresis, and the target fragment was recovered using a DNA gel recovery kit.

The pET28a-SUMO plasmid and the acyl-CoA thioesterase ctYdiI PCR product were extracted and subjected to double digestion reaction. The reaction system was as follows:

Reaction conditions: 37°C for 6h.

The pET28a-SUMO plasmid and the acyl-CoA thioesterase ctYdiI PCR product were double digested and purified by 1% agarose gel electrophoresis as shown in Figure 6, and the target fragment was recovered using a DNA gel recovery kit.

Example 3

Recombinant plasmid pET21b-CYP153A-CPR _BM3 multi-fragment seamless cloning, recombinant plasmid pET28a-SUMO-ctYdiI ligation

The pET21b plasmid that has undergone double digestion is connected with the CYP153A, CPR _BM3 PCR products, and the ligation reaction system is as follows:

The above-mentioned ligation reaction system was thoroughly mixed, centrifuged for several seconds, the droplets on the tube wall were collected at the bottom of the tube, and ligated at 37°C for 30 min to obtain the recombinant plasmid pET21b-CYP153A-CPR _BM3 .

The digested pCDFDuet-1 plasmid was ligated with the MaMACS PCR product, and the ligation reaction system was as follows:

The above-mentioned ligation reaction system was thoroughly mixed, centrifuged for several seconds, the droplets on the tube wall were collected at the bottom of the tube, and the reaction was carried out at 37° C. for 37 minutes to obtain the recombinant plasmid pCDFDuet-1-MaMACS.

The digested pCDFDuet-1-MaMACS plasmid was ligated with the PpFadE PCR product, and the ligation reaction system was as follows:

The above ligation reaction system was fully mixed, centrifuged for several seconds, the tube wall was dropped to the bottom of the tube, and the reaction was carried out at 37°C for 37 minutes to obtain the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE as shown in Figure 1B.

The double digested pET28a-SUMO plasmid was ligated with the ctYdiI PCR product, and the ligation reaction system was as follows:

After fully mixing the above ligation reaction system, centrifuge for a few seconds, drop the tube wall to the bottom of the tube, and connect at 22°C for 2 hours to obtain the recombinant plasmid pET28a-SUMO-ctYdiI as shown in C in Figure 1.

Example 4

Transformation of recombinant plasmid pET21b-CYP153A-CPR _BM3 , recombinant plasmid pET21b-CYP153A M228L-CPR _BM3 , recombinant plasmid pCDFDuet-1-MaMACS-PpFadE, recombinant plasmid pET28a-SUMO-ctYdiI:

(1) Preparation of competent cells

①Pick a single colony of Escherichia coli BL21(DE3) (or pick a preserved strain) and inoculate it into 10ml of liquid LB medium, and cultivate overnight at 37°C and 210rpm;

② Take 5ml of bacterial liquid and inoculate it in 500ml of LB medium, and cultivate at 37°C and 210rpm until the bacterial liquid OD ₆₀₀ is about 0.375;

③Place the bacterial liquid on the ice-water mixture for 10min, and pre-cool a 50ml centrifuge tube at the same time;

④ Transfer the bacterial liquid to a centrifuge tube, centrifuge at 4°C and 3700 rpm for 10 min to collect the bacterial cells;

⑤ Add 10 mL of pre-cooled 0.1M CaCl ₂ solution to each centrifuge tube, resuspend the cells, then add 30 mL of pre-cooled 0.1 M CaCl ₂ solution, invert and mix, and let stand on ice for 20 min;

⑥ 4 ℃, 3700rpm centrifugation for 10min to collect the bacteria, add pre-cooled 0.1M CaCl ₂ solution containing 15% glycerol according to the volume ratio of the bacteria liquid in step ④ is 3:125, resuspend the bacteria to obtain competent cell;

⑦ Distribute the competent cells and freeze them at -80℃.

Competent cells of Escherichia coli gene deletion strain BL21 (ΔFadB, R, J) were prepared in the same way.

The construction method of Escherichia coli gene deletion bacteria BL21ΔFadB, R, J, including the following steps:

Ⅰ Knock out the gene by RED recombination method to construct a gene-deficient ΔFadRB strain, hereinafter referred to as a ΔFadRΔFadB strain; the construction method of this strain is referred to in Chinese patent document CN110684794A (application number: 201911038194.3).

Ⅱ Constructing the FadJ knockout box, the specific steps are as follows:

Using the genome of Escherichia coli BL21 as a template, the upstream homology arm FadJ1 of the 3-hydroxyacyl-CoA dehydrogenase gene was amplified. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.25, and the nucleotide sequence of the downstream primer As shown in SEQ ID NO.26; using Escherichia coli BL21 genome as a template, amplify the downstream homology arm FadJ2 of 3-hydroxyacyl-CoA dehydrogenase gene, and the nucleotide sequence of the upstream primer is shown in SEQ ID NO.27 , the nucleotide sequence of the downstream primer is shown in SEQ ID NO.28; the pkd3 plasmid is used as a template to amplify the FRT-RKan-FRT gene fragment, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.29, the downstream primer The nucleotide sequence is shown in SEQ ID NO.30; then FadJ1, FRT-RKan-FRT and FadJ2 gene fragment multi-fragment seamless cloning, amplifying the knockout frame fragment of FadJ1-Kan-FadJ2, the core of the upstream primer The nucleotide sequence is shown in SEQ ID NO.31, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.32, and the FadJ knockout frame is recovered from the purification gel (hereinafter, the FadJ knockout frame is abbreviated as Jk);

The PCR amplification system is as follows, the total system is 50 μL:

100μM upstream primer 2.0μL, 100μM downstream primer 2.0μL, template 2.0μL, 5U/μL phanta enzyme 25μL, ddH2O 19μL;

PCR amplification conditions are as follows:

Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 1 min 15 s, 30 cycles; extension at 72 °C for 5 min.

Ⅲ Transform the FadJ knockout box prepared in step Ⅱ into pkd46-ΔFadRΔFadB recombinant bacteria, and finally obtain Escherichia coli gene deletion bacteria BL21ΔFadB, R, J, and the specific steps are as follows:

a. The plasmid pkd46 was transformed into ΔFadRΔFadB competent cells, and the obtained pkd46-ΔFadRΔFadB recombinant bacteria were prepared to prepare pkd46-ΔFadRΔFadB competent cells, and then the prepared pkd46-ΔFadRΔFadB competent cells were stored in 10% glycerol;

b. Transform the FadJ knockout box Jk into pkd46-ΔFadRΔFadB competent cells, and verify that the pkd46-Jk-BL21 recombinant strain confirms that after the knockout frame is transferred, pkd46 is eliminated at 42°C, and the Jk-ΔFadRΔFadB recombinant strain is obtained after screening;

c. The Jk-ΔFadRΔFadB recombinant strain was prepared and transformed into the pcp20 plasmid, and the Jk resistance and pcp20 plasmid were eliminated at 42°C to obtain the ΔFadRBJ recombinant strain, namely the E. coli gene-deficient strain BL21ΔFadB, R, J.

Figure 11 is an agarose gel electrophoresis image of colony PCR to verify ΔFadRΔFadB knockout FadJ product;

In the figure, lane M is Marker; lanes 1-2 are FadJ gene bands, and lanes 3-8 are FadJ knockout box fusion fragment gene bands.

(2) Transformation of recombinant plasmids

①Add 10 μL of recombinant plasmid pET21b-CYP153A M228L-CPR _BM3 to 100 μL of freshly prepared E. coli BL21 competent cells, mix gently, ice bath for 30 minutes; 10 μL of recombinant plasmid pET21b-CYP153A-CPR _{BM3 was} added to 100 μL of freshly prepared In E. coli BL21 competent cells, mix gently, ice bath for 30 minutes; add 10 μL of recombinant plasmid pCDFDuet-1-MaMACS-PpFadE and recombinant plasmid pET28a-SUMO-ctYdiI to 100 μL of freshly prepared E. coli gene-deficient bacteria BL21ΔFadB, Competent cells of R and J.

② Heat shock at 42°C for 90s, then quickly cool in an ice bath for 3min;

③ The above-mentioned competent cells were inserted into 500 μL LB medium, 37 ° C, 200 rpm shaking culture for 60 min;

4. get 200 μL of above-mentioned bacterial liquid, spread on the LB solid medium with 100mg/mL, 50mg/mL, 40mg/mL ampicillin, kanamycin, streptomycin;

⑤Incubate in a 37℃ incubator for 30min, after the bacterial liquid is sucked dry, invert the plate and incubate at 37℃ for 12-16h.

(3) Identification of positive clones:

①Protein expression and solubility identification

Take 900 μL of the above bacterial solution, add IPTG with a final concentration of 0.32 mM, induce expression for 4 h, centrifuge at 12,000 rpm for 1 min, collect the bacterial cells, add 2 times the loading buffer, resuspend the bacterial cells, denature them in a water bath at 100 °C for 10 min, and use SDS- The protein expression was detected by PAGE, and the results were shown in Figure 7, which were shown as positive clones.

②Bacteria sample sequencing

The positive clones identified by the above two methods were sent to a sequencing company for sequencing to further prove the correctness of the constructed positive clones. ctYdiI; Escherichia coli BL21 pET21b-CYP153A M228L-CPR _BM3 ; Escherichia coli BL21 pET21b-CYP153A-CPR _BM3 ;

Example 5

Fermentation of E. coli BL21ΔFadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdiI and E. coli BL21 pET21b-CYP153A M228L-CPR _BM3 fusion enzyme engineering bacteria

(1) Strain activation: Escherichia coli BL21ΔFadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdiI in Example 4 were inoculated into 50 mL containing kanamycin and In liquid LB medium of streptomycin, shake culture at 37°C and 200rpm for 12h;

(2) Bacterial transfer: Take 3 mL of the above-mentioned activated strain and insert it into 300 mL of liquid medium containing kanamycin and streptomycin, and shake at 37 ° C and 200 rpm until the OD600 of the bacterial liquid is 1.0, then cool to 20 ° C to adapt After 1 hour, adding IPTG to make the IPTG concentration in the medium be 0.5 mM, adding oleic acid to make the oleic acid mass percent concentration in the medium 0.6%, adding Tween 80 to make the mass percent concentration of Tween 80 in the medium 0.3%, overnight induction culture;

(3) Collecting the bacterial cells: take 300 mL of the above bacterial liquid, centrifuge at 5000 rpm for 15 min at 4°C, and collect the bacterial cells;

(4) Wash the pellet three times with 0.85% physiological saline, and resuspend the bacteria in the transformation medium to prepare a bacterial suspension. The transformation medium contains 100 mM potassium phosphate buffer (pH 7.4), 1% glycerol, 0.4% glucose, 50 μg/mL kana antibiotic, 40 μg/mL streptomycin, and the total volume is 30 mL, 0.5g/L capric acid was added respectively to carry out the reaction. React at 30°C for 20h and sample at 9h.

Escherichia coli BL21 pET21b-CYP153A M228L-CPR _BM3 fusion enzyme engineering bacteria fermentation

(1) Activation of strains: E. coli BL21 pET21b-CYP153A M228L-CPR _BM3 in Example 4 was inoculated into 50 mL of liquid LB medium containing ampicillin at an inoculum of 1%, and cultured with shaking at 37° C. and 200 rpm. 12h;

(2) Bacterial transfer: Take 3 mL of the above-mentioned activated bacterial strain and insert it into 300 mL of liquid medium containing ampicillin, shake at 37 ° C and 200 rpm until the OD600 of the bacterial liquid is 1.0, cool down to 20 ° C and adapt to 1 hour, add IPTG The IPTG concentration in the medium was 0.5 mM, FeCl ₃ with a final concentration of 0.5 mM, and 5-ALA (5-aminolevulinic acid hydrochloride) with a final concentration of 0.5 mM were added to induce and culture for 18 hours. cell;

(3) Collecting the bacterial cells: take 300 mL of the above bacterial liquid, centrifuge at 5000 rpm for 15 min at 4°C, and collect the bacterial cells;

(4) Wash the pellet three times with 0.85% physiological saline, and resuspend the bacteria in the transformation medium to prepare a bacterial suspension. The transformation medium contained 100 mM potassium phosphate buffer (pH 7.4), and the volume of the bacteria was 3 mL after resuspending, which was added to the reaction system completed in the previous step, and sampled at 30° C. for 20 h. After the reaction was terminated, 1 mL of 0.4 mol/L HCl was added to terminate the reaction, and 0.1 g/L of lauric acid was added as an internal standard.

Fermentation broth silanization treatment: take 1 mL of fermentation broth into a 1.5 mL centrifuge tube, extract the fatty acids in the sample solution with 1.5 mL of ethyl acetate for each sample (750 μL each time), mix with a cyclone mixer for 60 s, at room temperature 4000 r/m Centrifuge for 10 min. The extract was evaporated to dryness, and the dried sample was redissolved in 0.5 mL of ethyl acetate (chromatographically pure), 0.5 mL of n-hexane (chromatically pure), and 100 μL of BSTFA-TMCS (99:1, v/v) derivatization reagent was added After 5 min at room temperature, incubate in an oven at 70 °C for 50 min.

Gas mass spectrometry detection of generated products: gas chromatography with helium as carrier gas, constant flow mode, injection volume of 1 μL, split injection, split ratio of 1:5, injection temperature of 250 °C, maintained at 50 °C for 1 min, at 15 °C / min to 250 °C and hold for 10 min. The product chromatograms are shown in Figure 8, Figure 9, and Figure 10.

Through the calculation and analysis of the chromatogram and mass spectrum of the product, it was concluded that decanoic acid produced 10-hydroxy-2-decenoic acid with high added value through a two-step method. L of 10-hydroxy-2-decenoic acid with a conversion of 54.6%

Comparative Example 1

The difference from the fermentation of the recombinant bacteria in Example 5 is that the E. coli BL21pET21b-CYP153A-CPR _BM3 obtained in Example 4 was used to replace the E. coli BL21 pET21b-CYP153A M228L-CPR _BM3 . L decanoic acid was fermented in shake flasks to produce 0.077 g/L of 10-hydroxy-2-decenoic acid with a conversion rate of 15.4%

Comparative Example 2

The difference between the transformation of the recombinant plasmid pET21b-CYP153A M228L-CPR _BM3 , the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE, and the recombinant plasmid pET28a-SUMO-ctYdiI and Example 4 is that the E. coli gene deletion bacteria BL21 (ΔFadB , R) competent cells, the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE, recombinant plasmid pET28a-SUMO-ctYdiI was transformed into the competent cells of freshly prepared Escherichia coli gene deletion bacteria BL21ΔFadB, R, to obtain Escherichia coli BL21ΔFadB, R, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdiI; others are the same.

The obtained Escherichia coli BL21ΔFadB, R, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdiI and Escherichia coli BL21 pET21b-CYP153A M228L-CPR _BM3 fusion enzyme engineering bacteria were fermented, and the fermentation method was the same as in Example 5, and the experimental results obtained Escherichia coli BL21ΔFadB, R, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdiI catalyzed decanoic acid and could not produce trans-2-decenoic acid, so 10-hydroxy-2-decenoic acid could not be produced.

In this experiment, decanoic acid was used as the substrate, and 10-HDA was generated by two-step biological cascade catalysis. In the previous laboratory, 10-HAD was synthesized by one-step method with decanoic acid as the substrate. The fatty acyl-CoA dehydrogenase obtained by optimization in this application The synthetic pathway constructed by gene PpFadE, fatty acyl-CoA synthase gene MaMACS, acyl-CoA thioesterase gene ctydiI and patent document CN109897870A fatty acyl-CoA dehydrogenase gene MCAD, fatty acyl-CoA synthase gene FadK, acyl-CoA thioester The enzyme gene ydiI is not the same gene. The P450 fusion enzyme in this paper changes the amino acid at position 228 from M to L. The production of 10-HDA can be significantly improved by the replacement of catalytic elements and the change of biosynthesis.

From the experimental results of Example 5 and Comparative Example 2, it can be seen that the present invention uses Escherichia coli gene deletion bacteria BL21ΔFadB, R, and J as the transformation host bacteria of recombinant plasmid pCDFDuet-1-MaMACS-PpFadE and recombinant plasmid pET28a-SUMO-ctYdiI, Catalytic decanoic acid to produce trans-2-decenoic acid with specificity, combined with Escherichia coli BL21 pET21b-CYP153AM228L-CPR _BM3 fusion enzyme engineering bacteria to synthesize 10-hydroxy-2-decenoic acid in two steps, and finally 10-hydroxy-2 -The conversion rate of decenoic acid reached 54.6%, which was significantly better than the effect of E. coli gene deletion bacteria BL21ΔFadB, R as the host bacteria.

SEQUENCE LISTING

<110> Qilu University of Technology

<120> A kind of Escherichia coli engineering bacteria and its application

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<170> PatentIn version 3.5

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tggtatgcct caggtaatcg cgatgaacgt aaatttgata atccggatca gtttattatc 1200

gatcgtaaag atgcacgcaa tcacatgtct tttggctatg gtgttcatcg ctgtatgggt 1260

aatcgtctgg ccgaattaca gctgcgtatt ctgtgggaag aaatcttaaa acgctttgat 1320

aatatcgaag ttgtggaaga accagaacgt gtgcagagca attttgttcg cggctatagc 1380

cgcttaatgg ttaaactgac acctaatagt atgggcggca ttccttcacc aagccgagag 1440

cagtcagcta aaaaagagcg caaaaccgta gaaaacgctc ataatacgcc gcttcttgtg 1500

ctatacggtt caaatatggg aacagccgaa ggaacggcgc gtgatttagc ggatattgcg 1560

atgagcaaag gattcgcacc gcaagtcgca acgcttgatt cccacgcagg aaaccttccg 1620

cgtgaaggag ctgttttaat tgtaacggct tcttataacg gtcatcctcc tgataacgca 1680

aaggaatttg ttgactggtt agaccaagcg tctgctgatg aagtaaaagg cgtgcgctac 1740

tccgtatttg gatgcggtga taaaaactgg gcgacaacgt atcaaaaagt gcctgctttt 1800

attgatgaaa ctcttgccgc taaaggggca gaaaacatag ctgaacgcgg tgaagcagat 1860

gcaagcgacg actttgaagg cacatacgaa gaatggcgtg aacacatgtg gagtgactta 1920

gcagcctact ttaacttaga cattgaaaac agcgaagaaa atgcgtctac gctttcactt 1980

caatttgtcg acagcgctgc ggacatgccg cttgcgaaaa tgcaccgtgc gttttcagca 2040

aacgtcgtag caagcaaaga gcttcaaaag ccaggcagtg cacgaagcac gcgtcatctt 2100

gaaattgaac ttccaaaaga agcttcttat caagaaggag atcatttagg tgttattcct 2160

cgcaactatg aaggaatagt aaatcgtgta gcaacaagat ttggtctaga tgcatcacag 2220

caaatccgtt tggaagctga agaagaaaaa ttagctcatt tgccactcgg taaaacagta 2280

tcagtagaag agcttctgca atacgtggag cttcaagatc ctgttacgcg cacgcagctt 2340

cgcgcaatgg ctgctaaaac agtctgcccg ccgcataaag tagagcttga agtcttgctt 2400

gaaaagcagg cgtacaaaga acaagtgctg gcaaaacgtt taacaatgct tgaactgctt 2460

gaaaaatatc cggcgtgtga aatggaattc agcgaattta tcgcacttct tccaagcatg 2520

cgtccgcgct attactcaat ttcttcatca cctcgtgtcg atgaaaaaca agcaagcatc 2580

acggtcagcg ttgtttcagg agaagcgtgg agcggatacg gagaatacaa aggaattgca 2640

tcgaactatc ttgccaatct gcaagaagga gatacgatta cgtgctttgt ttccacaccg 2700

cagtcaggat ttacgctgcc aaaaggccct gaaacaccac ttatcatggt aggaccggga 2760

acaggcgtcg cgccgtttag aggctttgtg caggctcgca agcagttaaa agaacaagga 2820

cagtcgcttg gagaagcgca tttatacttt ggctgccgtt cacctcatga agattatctg 2880

tatcaaaaag agcttgaaaa cgcccaaaat gaaggcatca ttacgcttca taccgctttt 2940

tctcgcgtac caaatcagcc gaaaacatac gttcaacacg tgatggaaca agacggcaag 3000

aaattgattg aacttcttga ccaaggagcg cacttctata tttgcggaga cggaagccaa 3060

atggcacctg acgttgaagc aacgcttatg aaaagctatg ctgaagttca ccaagtgagt 3120

gaagcagacg ctcgcttatg gctgcagcag ctagaagaaa agggccgata cgcaaaagac 3180

gtgtggggctg ggtaa 3195

<210> 15

<211> 1653

<212> DNA

<213> Artificial sequences

<400> 15

atgtcagata ccaccaccgc atttaccgtt ccagcggttg ccaaagcagt tgccgcagcc 60

attccggatc gcgaactgat tattcagggt gatcgtcgct atacctatcg tcaggttatt 120

gaacgctcaa atagattagc tgcatattta cattcacagg gcttaggctg tcataccgaa 180

cgcgaagcct tagcaggtca tgaagttggc caggatctgc tgggcctgta tgcatataat 240

ggcaatgaat ttgtggaagc cttactgggt gcctttgcag ctagggtggc cccgtttaat 300

gtgaattttc gctatgttaa atcagaatta cattatctgt tagccgatag tgaagcaacc 360

gccctgattt atcatgcagc ctttgccccg cgtgtggcag aaattctgcc ggaactgccg 420

cgtctgcgcg tgctgattca gattgcagat gaatcaggta atgaattact ggatggtgca 480

gttgattatg aagatgcctt agcaagcgtg agcgcccagc cgccgccggt tcgtcattgt 540

ccggatgatc tgtatgttct gtataccggc ggtaccaccg gtatgccgaa aggcgtttta 600

tggcgtcagc atgatatttt tatgacctct tttggcggtc gtaatctgat gaccggcgaa 660

cctagctcta gtattgatga aattgttcag cgcgcagcct caggtccggg taccaaatta 720

atgattctgc cgccgttaat tcatggtgcc gcacagtgga gtgttatgac cgcaattacc 780

accggccaga ccgtggtgtt tccgaccgtt gtggatcatc tggatgcaga agatgtggtt 840

cgtaccattg aacgtgaaaa agttatggtt gtgaccgtgg tgggcgatgc aatggcacgt 900

ccgttagttg ccgcaattga aaaaggtatt gccgatgtgt cttcactagc cgtggtagcc 960

aatggcggcg ccctgctgac cccgtttgtt aaacagcgtc tgattgaagt gctgccgaat 1020

gcagtggttg tggatggtgt tggctcaagc gaaaccggtg cacagatgca tcacatgtct 1080

acccccggcg ccgtggcaac cggtaccttt aatgcaggtc cggatacctt tgttgccgca 1140

gaggacctga gcgccatttt accgccgggc catgaaggca tgggctggtt agcccagcgc 1200

ggctatgttc cgttaggcta taaaggcgat gcagccaaaa ccgccaaaac ctttccggtt 1260

attgatggcg tgcgctatgc agttccgggt gatcgcgcac gtcatcatgc agatggtcat 1320

attgaactgc tgggtcgcga tagtgtttgt attaatagcg gcggcgaaaa aatttttgtg 1380

gaagaagtgg aaaccgcaat tgcctcacat ccggcagttg ccgatgtggt tgtggcaggt 1440

cgtccgagtg aacgctgggg tcaggaagtt gttgcagttg tggccctgag cgatggtgca 1500

gcagtggatg caggcgaact gattgcacat gcctctaatt ccctggctcg ctataaactg 1560

ccgaaagcca ttgtgtttcg cccggttatt gaacgctctc cgagcggtaa agccgattat 1620

cgttgggcac gcgaacaggc agttaatggt taa 1653

<210> 16

<211> 1227

<212> DNA

<213> Artificial sequences

<400> 16

gattttgcct atagtccgaa agttcaggca ttacgtgaac gtgtgaccgc ctttatggat 60

gcacatgtgt atccagccga agcagtgttt gaacgccagg ttgccgaagg tgatcgctgg 120

cagccgaccg ccattatgga agaactgaaa gccaaagcac gcgccgaagg cctgtggaat 180

ctgtttctgc cggaatcaga atatggtgca ggtctgtcta atctggaata tgcaccgtta 240

gcagaaatta tgggccgtag cttactgggc ccggaaccgt ttaattgtag tgccccggat 300

accggtaata tggaagtttt agttcgctat ggtagcgaag cccagaaacg tcagtggtta 360

gaaccgttac tgcgcggtga aattcgtagt gcctttgcaa tgaccgaacc ggatgttgca 420

tctagcgatg ccaccaatat ggcagcaacc gcaattcgcg atggcgatca gtgggttatt 480

aatggtcgca aatggtggac ctcaggcgcc tgtgatccgc gttgtaaagt gatgattttt 540

atgggcttaa gcgatccgga aggcccgcgt catcagcagc attcaatggt gctggttccg 600

accgatacgc caggtgttaa aattgtgcgt ccgctgccgg tgtttggcta tgatgatgcc 660

ccgcatggtc atgccgaagt gctgtttgaa aatgttcgtg ttccgtatga aaatgttatt 720

ttaggcgaag gtcgcggctt tgaaattgca cagggtcgtc tggggcctgg tcgtattcat 780

cattgtatgc gctctattgg catggcagaa cgcgcattag aactgatgtg taaacgctca 840

gttgaacgta ccgcctttgg tcgtccgtta gcacgtctgg gcggtaatgt ggataaaatt 900

gcagattctc gcatggaaat tgatatggca cgcttactga ccttaaaagc cgcctatatg 960

atggataccg tgggtaataa agttgcacgc tctgaaattg cacagattaa agttgtggcc 1020

ccgaatgtgg ccctgaatgt tattgatcgt gccattcaga ttcatggcgg cgcaggcgtg 1080

agcggcgatt ttccgttagc ctatatgtat gccatgcagc gtaccctgcg tttagcagat 1140

ggtccggatg aagttcatcg cgcagccatt ggcaaatatg aaattggtaa atatgttccg 1200

gttgaaatgc tgcgctcagg tcgttaa 1227

<210> 17

<211> 423

<212> DNA

<213> Artificial sequences

<400> 17

ggatccatga tttggcagcg caccgccacc ctggatgcac tgaatgcaat gggtgcgaat 60

aatatggtgg gcctgctgga tattcgcttt acccgtctgg atgataatga aattgaagca 120

accatgccgg ttgatcatcg tacccatcaa cctttcggtt tactgcatgg cggcgcaagc 180

gtggtgttag ccgaaacctt aggtagtgtt gcaggctatc tgtgtaccga aggcgaacag 240

aatattgtgg gcttagaagt taatgccaat catttacgct cagtgcgtag cggtcgcgtg 300

cgcggcgtgt gtcgcgcagt tcatgtgggt cgtcgtcatc aggtttggca gattgaaatt 360

tttgatgaac aggatcgctt atgttgtagc tctcgtctga ccaccgccgt tgtgtaactc 420

gag 423

<210> 18

<211> 1064

<212> PRT

<213> Artificial sequences

<400> 18

Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile

1 5 10 15

Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu

20 25 30

Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro

35 40 45

Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala

50 55 60

Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln

65 70 75 80

Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr

85 90 95

Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu

100 105 110

Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro

115 120 125

Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe

130 135 140

Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln

145 150 155 160

Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg

165 170 175

Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe

180 185 190

Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala

195 200 205

Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp

210 215 220

Ser Asp Arg Met Ala Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala

225 230 235 240

Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe

245 250 255

Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu

260 265 270

Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys

275 280 285

Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu

290 295 300

Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu

305 310 315 320

Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys

325 330 335

Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr

340 345 350

Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly

355 360 365

Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser

370 375 380

Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile

385 390 395 400

Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His

405 410 415

Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp

420 425 430

Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro

435 440 445

Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val

450 455 460

Lys Leu Thr Pro Asn Ser Met Gly Gly Ile Pro Ser Pro Ser Arg Glu

465 470 475 480

Gln Ser Ala Lys Lys Glu Arg Lys Thr Val Glu Asn Ala His Asn Thr

485 490 495

Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr

500 505 510

Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln

515 520 525

Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala

530 535 540

Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala

545 550 555 560

Lys Glu Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys

565 570 575

Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr

580 585 590

Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys

595 600 605

Gly Ala Glu Asn Ile Ala Glu Arg Gly Glu Ala Asp Ala Ser Asp Asp

610 615 620

Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Leu

625 630 635 640

Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Glu Asn Ala Ser

645 650 655

Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala

660 665 670

Lys Met His Arg Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu

675 680 685

Gln Lys Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu

690 695 700

Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro

705 710 715 720

Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Ala Thr Arg Phe Gly Leu

725 730 735

Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala

740 745 750

His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr

755 760 765

Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala

770 775 780

Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Val Leu Leu

785 790 795 800

Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met

805 810 815

Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu

820 825 830

Phe Ile Ala Leu Leu Pro Ser Met Arg Pro Arg Tyr Tyr Ser Ile Ser

835 840 845

Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val

850 855 860

Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala

865 870 875 880

Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe

885 890 895

Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Gly Pro Glu Thr

900 905 910

Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly

915 920 925

Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly

930 935 940

Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu

945 950 955 960

Tyr Gln Lys Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu

965 970 975

His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln

980 985 990

His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln

995 1000 1005

Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro

1010 1015 1020

Asp Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Glu Val His Gln

1025 1030 1035

Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu

1040 1045 1050

Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly

1055 1060

<210> 19

<211> 1064

<212> PRT

<213> Artificial sequences

<400> 19

Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile

1 5 10 15

Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu

20 25 30

Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro

35 40 45

Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala

50 55 60

Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln

65 70 75 80

Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr

85 90 95

Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu

100 105 110

Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro

115 120 125

Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe

130 135 140

Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln

145 150 155 160

Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg

165 170 175

Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe

180 185 190

Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala

195 200 205

Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp

210 215 220

Ser Asp Arg Leu Ala Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala

225 230 235 240

Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe

245 250 255

Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu

260 265 270

Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys

275 280 285

Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu

290 295 300

Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu

305 310 315 320

Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys

325 330 335

Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr

340 345 350

Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly

355 360 365

Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser

370 375 380

Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile

385 390 395 400

Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His

405 410 415

Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp

420 425 430

Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro

435 440 445

Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val

450 455 460

Lys Leu Thr Pro Asn Ser Met Gly Gly Ile Pro Ser Pro Ser Arg Glu

465 470 475 480

Gln Ser Ala Lys Lys Glu Arg Lys Thr Val Glu Asn Ala His Asn Thr

485 490 495

Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr

500 505 510

Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln

515 520 525

Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala

530 535 540

Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala

545 550 555 560

Lys Glu Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys

565 570 575

Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr

580 585 590

Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys

595 600 605

Gly Ala Glu Asn Ile Ala Glu Arg Gly Glu Ala Asp Ala Ser Asp Asp

610 615 620

Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Leu

625 630 635 640

Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Glu Asn Ala Ser

645 650 655

Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala

660 665 670

Lys Met His Arg Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu

675 680 685

Gln Lys Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu

690 695 700

Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro

705 710 715 720

Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Ala Thr Arg Phe Gly Leu

725 730 735

Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala

740 745 750

His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr

755 760 765

Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala

770 775 780

Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Val Leu Leu

785 790 795 800

Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met

805 810 815

Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu

820 825 830

Phe Ile Ala Leu Leu Pro Ser Met Arg Pro Arg Tyr Tyr Ser Ile Ser

835 840 845

Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val

850 855 860

Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala

865 870 875 880

Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe

885 890 895

Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Gly Pro Glu Thr

900 905 910

Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly

915 920 925

Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly

930 935 940

Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu

945 950 955 960

Tyr Gln Lys Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu

965 970 975

His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln

980 985 990

His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln

995 1000 1005

Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro

1010 1015 1020

Asp Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Glu Val His Gln

1025 1030 1035

Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu

1040 1045 1050

Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly

1055 1060

<210> 20

<211> 550

<212> PRT

<213> Artificial sequences

<400> 20

Met Ser Asp Thr Thr Thr Ala Phe Thr Val Pro Ala Val Ala Lys Ala

1 5 10 15

Val Ala Ala Ala Ile Pro Asp Arg Glu Leu Ile Ile Gln Gly Asp Arg

20 25 30

Arg Tyr Thr Tyr Arg Gln Val Ile Glu Arg Ser Asn Arg Leu Ala Ala

35 40 45

Tyr Leu His Ser Gln Gly Leu Gly Cys His Thr Glu Arg Glu Ala Leu

50 55 60

Ala Gly His Glu Val Gly Gln Asp Leu Leu Gly Leu Tyr Ala Tyr Asn

65 70 75 80

Gly Asn Glu Phe Val Glu Ala Leu Leu Gly Ala Phe Ala Ala Arg Val

85 90 95

Ala Pro Phe Asn Val Asn Phe Arg Tyr Val Lys Ser Glu Leu His Tyr

100 105 110

Leu Leu Ala Asp Ser Glu Ala Thr Ala Leu Ile Tyr His Ala Ala Phe

115 120 125

Ala Pro Arg Val Ala Glu Ile Leu Pro Glu Leu Pro Arg Leu Arg Val

130 135 140

Leu Ile Gln Ile Ala Asp Glu Ser Gly Asn Glu Leu Leu Asp Gly Ala

145 150 155 160

Val Asp Tyr Glu Asp Ala Leu Ala Ser Val Ser Ala Gln Pro Pro Pro

165 170 175

Val Arg His Cys Pro Asp Asp Leu Tyr Val Leu Tyr Thr Gly Gly Thr

180 185 190

Thr Gly Met Pro Lys Gly Val Leu Trp Arg Gln His Asp Ile Phe Met

195 200 205

Thr Ser Phe Gly Gly Arg Asn Leu Met Thr Gly Glu Pro Ser Ser Ser

210 215 220

Ile Asp Glu Ile Val Gln Arg Ala Ala Ser Gly Pro Gly Thr Lys Leu

225 230 235 240

Met Ile Leu Pro Pro Leu Ile His Gly Ala Ala Gln Trp Ser Val Met

245 250 255

Thr Ala Ile Thr Thr Gly Gln Thr Val Val Phe Pro Thr Val Val Asp

260 265 270

His Leu Asp Ala Glu Asp Val Val Arg Thr Ile Glu Arg Glu Lys Val

275 280 285

Met Val Val Thr Val Val Gly Asp Ala Met Ala Arg Pro Leu Val Ala

290 295 300

Ala Ile Glu Lys Gly Ile Ala Asp Val Ser Ser Leu Ala Val Val Ala

305 310 315 320

Asn Gly Gly Ala Leu Leu Thr Pro Phe Val Lys Gln Arg Leu Ile Glu

325 330 335

Val Leu Pro Asn Ala Val Val Val Asp Gly Val Gly Ser Ser Glu Thr

340 345 350

Gly Ala Gln Met His His Met Ser Thr Pro Gly Ala Val Ala Thr Gly

355 360 365

Thr Phe Asn Ala Gly Pro Asp Thr Phe Val Ala Ala Glu Asp Leu Ser

370 375 380

Ala Ile Leu Pro Pro Gly His Glu Gly Met Gly Trp Leu Ala Gln Arg

385 390 395 400

Gly Tyr Val Pro Leu Gly Tyr Lys Gly Asp Ala Ala Lys Thr Ala Lys

405 410 415

Thr Phe Pro Val Ile Asp Gly Val Arg Tyr Ala Val Pro Gly Asp Arg

420 425 430

Ala Arg His His Asp Ala Gly His Ile Glu Leu Leu Gly Arg Asp Ser

435 440 445

Val Cys Ile Asn Ser Gly Gly Glu Lys Ile Phe Val Glu Glu Val Glu

450 455 460

Thr Ala Ile Ala Ser His Pro Ala Val Ala Asp Val Val Val Ala Gly

465 470 475 480

Arg Pro Ser Glu Arg Trp Gly Gln Glu Val Val Ala Val Val Ala Leu

485 490 495

Ser Asp Gly Ala Ala Val Asp Ala Gly Glu Leu Ile Ala His Ala Ser

500 505 510

Asn Ser Leu Ala Arg Tyr Lys Leu Pro Lys Ala Ile Val Phe Arg Pro

515 520 525

Val Ile Glu Arg Ser Pro Ser Gly Lys Ala Asp Tyr Arg Trp Ala Arg

530 535 540

Glu Gln Ala Val Asn Gly

545 550

<210> 21

<211> 408

<212> PRT

<213> Artificial sequences

<400> 21

Asp Phe Ala Tyr Ser Pro Lys Val Gln Ala Leu Arg Glu Arg Val Thr

1 5 10 15

Ala Phe Met Asp Ala His Val Tyr Pro Ala Glu Ala Val Phe Glu Arg

20 25 30

Gln Val Ala Glu Gly Asp Arg Trp Gln Pro Thr Ala Ile Met Glu Glu

35 40 45

Leu Lys Ala Lys Ala Arg Ala Glu Gly Leu Trp Asn Leu Phe Leu Pro

50 55 60

Glu Ser Glu Tyr Gly Ala Gly Leu Ser Asn Leu Glu Tyr Ala Pro Leu

65 70 75 80

Ala Glu Ile Met Gly Arg Ser Leu Leu Gly Pro Glu Pro Phe Asn Cys

85 90 95

Ser Ala Pro Asp Thr Gly Asn Met Glu Val Leu Val Arg Tyr Gly Ser

100 105 110

Glu Ala Gln Lys Arg Gln Trp Leu Glu Pro Leu Leu Arg Gly Glu Ile

115 120 125

Arg Ser Ala Phe Ala Met Thr Glu Pro Asp Val Ala Ser Ser Asp Ala

130 135 140

Thr Asn Met Ala Ala Thr Ala Ile Arg Asp Gly Asp Gln Trp Val Ile

145 150 155 160

Asn Gly Arg Lys Trp Trp Thr Ser Gly Ala Cys Asp Pro Arg Cys Lys

165 170 175

Val Met Ile Phe Met Gly Leu Ser Asp Pro Glu Gly Pro Arg His Gln

180 185 190

Gln His Ser Met Val Leu Val Pro Thr Asp Thr Pro Gly Val Lys Ile

195 200 205

Val Arg Pro Leu Pro Val Phe Gly Tyr Asp Asp Ala Pro His Gly His

210 215 220

Ala Glu Val Leu Phe Glu Asn Val Arg Val Pro Tyr Glu Asn Val Ile

225 230 235 240

Leu Gly Glu Gly Arg Gly Phe Glu Ile Ala Gln Gly Arg Leu Gly Pro

245 250 255

Gly Arg Ile His His Cys Met Arg Ser Ile Gly Met Ala Glu Arg Ala

260 265 270

Leu Glu Leu Met Cys Lys Arg Ser Val Glu Arg Thr Ala Phe Gly Arg

275 280 285

Pro Leu Ala Arg Leu Gly Gly Asn Val Asp Lys Ile Ala Asp Ser Arg

290 295 300

Met Glu Ile Asp Met Ala Arg Leu Leu Thr Leu Lys Ala Ala Tyr Met

305 310 315 320

Met Asp Thr Val Gly Asn Lys Val Ala Arg Ser Glu Ile Ala Gln Ile

325 330 335

Lys Val Val Ala Pro Asn Val Ala Leu Asn Val Ile Asp Arg Ala Ile

340 345 350

Gln Ile His Gly Gly Ala Gly Val Ser Gly Asp Phe Pro Leu Ala Tyr

355 360 365

Met Tyr Ala Met Gln Arg Thr Leu Arg Leu Ala Asp Gly Pro Asp Glu

370 375 380

Val His Arg Ala Ala Ile Gly Lys Tyr Glu Ile Gly Lys Tyr Val Pro

385 390 395 400

Val Glu Met Leu Arg Ser Gly Arg

405

<210> 22

<211> 237

<212> PRT

<213> Artificial sequences

<400> 22

Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro

1 5 10 15

Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser

20 25 30

Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu

35 40 45

Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg

50 55 60

Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp

65 70 75 80

Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile

85 90 95

Gly Gly Ser Gly Ser Met Ile Trp Gln Arg Thr Ala Thr Leu Asp Ala

100 105 110

Leu Asn Ala Met Gly Ala Asn Asn Met Val Gly Leu Leu Asp Ile Arg

115 120 125

Phe Thr Arg Leu Asp Asp Asn Glu Ile Glu Ala Thr Met Pro Val Asp

130 135 140

His Arg Thr His Gln Pro Phe Gly Leu Leu His Gly Gly Ala Ser Val

145 150 155 160

Val Leu Ala Glu Thr Leu Gly Ser Val Ala Gly Tyr Leu Cys Thr Glu

165 170 175

Gly Glu Gln Asn Ile Val Gly Leu Glu Val Asn Ala Asn His Leu Arg

180 185 190

Ser Val Arg Ser Gly Arg Val Arg Gly Val Cys Arg Ala Val His Val

195 200 205

Gly Arg Arg His Gln Val Trp Gln Ile Glu Ile Phe Asp Glu Gln Asp

210 215 220

Arg Leu Cys Cys Ser Ser Arg Leu Thr Thr Ala Val Val

225 230 235

<210> 23

<211> 1410

<212> DNA

<213> Artificial sequences

<400> 23

atgccgacgt taccacgtac ctttgatgac attcagtctc gcttaatcaa tgctacaagt 60

cgtgtggttc caatgcagcg tcagattcag ggtctgaaat ttctgatgag tgccaaacgc 120

aaaacctttg gtccacgtcg cccaatgccg gaatttgtgg aaacacctat cccggatgtt 180

aatacattag ccttagagga cattgatgtg agtaatccgt ttctgtatcg ccagggccag 240

tggcgcgcat attttaaacg cttacgcgat gaagctccag ttcattatca gaaaaatagc 300

ccatttggtc cgttttggag cgtgacccgc tttgaggaca ttctgtttgt ggataaatca 360

catgatctgt ttagcgccga accacagatc atcttaggtg atcctccgga aggcctgtca 420

gtggaaatgt ttattgcgat ggaccctcct aaacatgatg tgcagcgctc tagtgttcag 480

ggtgtggttg cccctaaaaa tctgaaagaa atggaaggcc tgattcgtag tcgtacgggc 540

gatgtgttag attcattacc gacggataaa ccgtttaatt gggttcctgc ggtgagcaaa 600

gaactgacgg gtagaatgct ggctacctta ctggattttc cgtatgaaga acgtcataaa 660

ctggttgaat ggagcgatcg cttggccggt gcggcaagtg ctacgggcgg cgaatttgcg 720

gatgaaaatg ctatgtttga tgatgcggca gatatggcac gctctttttc tcgcctgtgg 780

cgcgataaag aagcccgccg tgcagcaggc gaagaaccgg gctttgattt aatctcactg 840

ctacagtcta ataaagaaac caaggatctg atcaatcgtc ctatggaatt tattggcaat 900

ctgaccctgc tgattgtggg cggtaatgat acgacccgca atagcatgtc aggcggctta 960

gttgccatga atgaatttcc tcgtgaattt gaaaaactga aagccaaacc ggaactgatt 1020

ccgaatatgg tgagcgaaat tattcgttgg cagacaccac tggcctatat gcgccgcatt 1080

gccaaacagg atgttgaact gggcggtcag accatcaaaa aaggtgatcg cgttgttatg 1140

tggtatgcct caggtaatcg cgatgaacgt aaatttgata atccggatca gtttattatc 1200

gatcgtaaag atgcacgcaa tcacatgtct tttggctatg gtgttcatcg ctgtatgggt 1260

aatcgtctgg ccgaattaca gctgcgtatt ctgtgggaag aaatcttaaa acgctttgat 1320

aatatcgaag ttgtggaaga accagaacgt gtgcagagca attttgttcg cggctatagc 1380

cgcttaatgg ttaaactgac acctaatagt 1410

<210> 24

<211> 470

<212> PRT

<213> Artificial sequences

<400> 24

Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile

1 5 10 15

Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu

20 25 30

Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro

35 40 45

Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala

50 55 60

Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln

65 70 75 80

Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr

85 90 95

Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu

100 105 110

Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro

115 120 125

Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe

130 135 140

Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln

145 150 155 160

Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg

165 170 175

Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe

180 185 190

Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala

195 200 205

Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp

210 215 220

Ser Asp Arg Leu Ala Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala

225 230 235 240

Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe

245 250 255

Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu

260 265 270

Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys

275 280 285

Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu

290 295 300

Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu

305 310 315 320

Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys

325 330 335

Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr

340 345 350

Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly

355 360 365

Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser

370 375 380

Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile

385 390 395 400

Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His

405 410 415

Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp

420 425 430

Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro

435 440 445

Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val

450 455 460

Lys Leu Thr Pro Asn Ser

465 470

<210> 25

<211> 45

<212> DNA

<213> Artificial sequences

<400> 25

tcgagctccg tcgacaagct tatggaaatg acatcagcgt ttacc 45

<210> 26

<211> 38

<212> DNA

<213> Artificial sequences

<400> 26

gtccacggag aattcatctc taatgctgtg ctgacgcc 38

<210> 27

<211> 22

<212> DNA

<213> Artificial sequences

<400> 27

ctgtactgga agccgcttat gg 22

<210> 28

<211> 44

<212> DNA

<213> Artificial sequences

<400> 28

gtggtggtgg tggtgctcga gttattgcag gtcagttgca gttg 44

<210> 29

<211> 25

<212> DNA

<213> Artificial sequences

<400> 29

gagatgaatt ctccgtggac ctgca 25

<210> 30

<211> 39

<212> DNA

<213> Artificial sequences

<400> 30

ataagcggct tccagtacag ggtaccgagc tcggatccg 39

<210> 31

<211> 24

<212> DNA

<213> Artificial sequences

<400> 31

atggaaatga catcagcgtt tacc 24

<210> 32

<211> 23

<212> DNA

<213> Artificial sequences

<400> 32

ttattgcagg tcagttgcag ttg 23

Claims (10)

1. Escherichia coli gene deletion bacteria BL21 delta FadB, R and J are obtained by knocking out FadB genes, FadR genes and FadJ genes from Escherichia coli BL 21.

2. The use of the escherichia coli gene deletion bacterium BL21 Δ FadB, R and J as defined in claim 1 in the preparation of 10-hydroxy-2-decenoic acid.

3. The method for constructing escherichia coli gene-deleted bacteria BL21 delta FadB, R and J as claimed in claim 1, comprising the following steps:

knocking out genes by using an RED recombination method, and constructing a gene deletion delta FadRB strain;

II, knocking out genes by using an RED recombination method, and constructing escherichia coli gene deletion bacteria BL21 delta FadB, R and J, wherein the construction specifically comprises constructing a FadJ knock-out frame; and (3) transforming the FadJ knockout frame into pkd 46-delta FadR delta FadB competent cells to prepare escherichia coli gene deletion bacteria BL21 delta FadB, R and J.

4. The method of claim 3, wherein the step ii of constructing the FadR knockout box comprises the steps of:

taking a genome of escherichia coli BL21 as a template, amplifying an upstream homologous arm FadJ1 of the 3-hydroxyacyl-CoA dehydrogenase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.25, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 26; taking a genome of escherichia coli BL21 as a template, amplifying a downstream homologous arm FadJ2 of the 3-hydroxyacyl-CoA dehydrogenase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.27, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 28; taking pkd3 plasmid as a template, amplifying an FRT-RKan-FRT gene fragment, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.29, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 30; then, carrying out multi-fragment seamless cloning on FadJl, FRT-RKan-FRT and FadJ2 gene fragments, amplifying a knockout frame fragment of FadJ1-Kan-FadJ2, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.31, the nucleotide sequence of a downstream primer is shown as SEQ ID No.32, and recovering purified glue to obtain a FadJ knockout frame;

preferably, the PCR amplification system is as follows, and the total system is 50 μ L:

mu.L 100. mu.M forward primer 2.0. mu.L, mu.L 100. mu.M reverse primer 2.0. mu.L, template 2.0. mu.L, 5U/. mu.L phanta enzyme 25. mu.L, ddH ₂ O19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 deg.C for 15s, annealing at 60 deg.C for 15s, extension at 72 deg.C for 1min for 15s, and circulating for 30 times; extension at 72 ℃ for 5 min.

5. The construction method as claimed in claim 3, wherein the FadJ knockout frame is transformed into pkd46- Δ FadR Δ FadB recombinant bacteria in the step II, and finally the Escherichia coli gene deletion bacteria BL21 Δ FadB, R and J are obtained, comprising the following steps:

a. transforming the plasmid pkd46 into a delta FadR delta FadB competent cell to obtain a pkd 46-delta FadR delta FadB recombinant strain, preparing a pkd 46-delta FadR delta FadB competent cell, and preserving the prepared pkd 46-delta FadR delta FadB competent cell by using glycerol with the mass concentration of 10%;

b. the FadJ knockout frame Jk is transformed into pkd 46-delta FadR delta FadB competent cells, after the confirmation of the knockout frame transfer of the pkd46-Jk-BL21 recombinant bacteria is verified, pkd46 is eliminated at 42 ℃, and the Jk-delta FadR delta FadB recombinant bacteria are obtained after screening;

c. preparing a competent transformation pcp20 plasmid from the Jk-delta FadR delta FadB recombinant strain, eliminating Jk resistance and the pcp20 plasmid at 42 ℃ to obtain a delta FadRBJ recombinant strain, namely escherichia coli gene deletion strain BL21 delta FadB, R and J.

6. An engineering bacterium of Escherichia coli BL21 delta FadB, R, J, pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdii is constructed by transforming recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdii into the Escherichia coli gene deletion bacterium BL21 delta FadB, R and J in claim 1;

the nucleotide sequence of the fatty acyl CoA synthetase gene MaMACS is shown as SEQ ID NO. 15; the nucleotide sequence of the fatty acyl CoA dehydrogenase gene PpFadE is shown as SEQ ID NO. 16; the nucleotide sequence of the ester acyl-CoA thioesterase gene ctYdii is shown in SEQ ID NO. 17.

7. The engineered Escherichia coli BL21 Δ FadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdii of claim 6, wherein the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE is constructed, comprising the steps of:

using Escherichia coli DH5a genome as a template, amplifying fatty acyl CoA dehydrogenase gene PpFadE, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.9, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.10, then carrying out double enzyme digestion on pCDFDuet-1-MaMACS plasmid by Nde I and Ava I, and connecting by ligase to prepare a recombinant plasmid pCDFDuet-1-MaMACS-PpFadE;

the PCR amplification system was as follows, 25. mu.L total:

mu.L 100. mu.M forward primer 1.0. mu.L, mu.L 100. mu.M reverse primer 1.0. mu.L, template 1.0. mu.L, 5U/. mu.L phanta enzyme 12.5. mu.L, ddH ₂ O 9.5μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15S, annealing at 60 ℃ for 15S, extension at 72 ℃ for 40S, and circulation for 30 times; extending for 5min at 72 ℃;

preferably, the recombinant plasmid pET28a-SUMO-ctYdii is constructed by the following steps:

using a genome of escherichia coli DH5a as a template, amplifying an ester acyl coenzyme A thioesterase gene ctyydiI, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.11, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.12, then performing double enzyme digestion on pET28a-SUMO plasmid and an ester acyl coenzyme A thioesterase gene ctyydiI respectively by BamHI and Xho I, and performing ligase ligation to prepare a recombinant plasmid pET28 a-SUMO-ctydiI;

the PCR amplification system was as follows, 25. mu.L total:

mu.L 100. mu.M forward primer 1.0. mu.L, mu.L 100. mu.M reverse primer 1.0. mu.L, template 1.0. mu.L, 5U/. mu.L phanta enzyme 12.5. mu.L, ddH ₂ O 9.5μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

8. A method for preparing trans-2-decenoic acid by using decanoic acid as a raw material comprises the following steps:

(1) the engineering bacteria of Escherichia coli BL21 delta FadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdii of claim 6, which are screened, induced and cultured to obtain induced cells;

(2) culturing the induced cells prepared in the step (1) by a transformation culture medium to prepare resting cells, and then adding capric acid into the resting cells of escherichia coli BL21 delta FadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdii to culture to prepare trans-2-decenoic acid.

9. The method according to claim 8, wherein in the step (1), the engineering bacteria of Escherichia coli BL21 Δ FadB, R, J, pCDFDuet-1-MaMACS-PpFadE, pET28a-SUMO-ctYdii are inoculated into LB liquid medium containing 50 μ g/mL kanamycin, 100 μ g/mL ampicillin and 40 μ g/mL streptomycin at corresponding concentrations, and screened and cultured at 35-40 ℃ with shaking until OD is reached ₆₀₀ 0.8 to 1.2;

preferably, in the step (1), after the bacteria solution after the screening culture is cooled to 18-20 ℃ and adapted for 0.5-1 hour, IPTG is added to the concentration of 0.5-0.8 mM, oleic acid is added to the concentration of 0.4-0.8% by mass, tween 80 is added to the concentration of 0.2-0.5% by mass, the induction culture is continued for 18-20 hours, and cells are separated to prepare induced cells;

preferably, in the step (1), the inducing culture conditions are that after the bacterial liquid of the screening culture is cooled to 20 ℃ to adapt for 1 hour, IPTG is respectively added to ensure that the concentration of IPTG in the culture medium is 0.5mM, oleic acid is added to ensure that the concentration of Tween in the culture medium is 0.6 percent, Tween 80 is added to ensure that the concentration of Tween in the culture medium is 0.3 percent, the inducing culture is continued for 18 hours, and cells are separated to prepare the inducing cells;

preferably, in the step (1), the cells are separated by centrifuging at 5000rpm for 15min, collecting the precipitate, and then washing with 0.85% by mass of saline.

10. The method of claim 8, wherein the transformation medium components in step (2) comprise the following:

0.8-1.2% of glycerol by mass fraction, 0.3-0.5% of glucose by mass fraction, 40-60 μ g/mL of kanamycin antibiotic, 90-110 μ g/mL of ampicillin, 30-50 μ g/mL of streptomycin antibiotic, and the balance of a 100mM potassium phosphate buffer solution with pH7.4 as a solvent;

preferably, glycerol is 1% by mass, glucose is 0.4% by mass, kanamycin antibiotic is 50. mu.g/mL, ampicillin is 100. mu.g/mL, streptomycin antibiotic is 40. mu.g/mL, and the balance is 100mM potassium phosphate buffer, pH 7.4;

preferably, capric acid is added into the resting cells in the step (2) to the concentration of 0.3-0.7g/L, and the reaction is carried out for 7-10h at the temperature of 28-37 ℃ to prepare trans-2-decenoic acid;

preferably, in the step (2), the concentration of converted decanoic acid is 0.5 g/L;

preferably, capric acid is added into the resting cells in the step (2) until the concentration is 0.5g/L, and the reaction is carried out for 9h at the temperature of 30 ℃ to prepare trans-2-decenoic acid;

preferably, in the step (2), the transformation culture condition is culture at 29-31 ℃ for 8 hours;

preferably, in the step (2), the decanoic acid is dissolved in dimethyl sulfoxide.

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