CN110684794B - Method for preparing alpha, beta unsaturated fatty acid by using fatty acid as raw material and utilizing escherichia coli engineering bacteria - Google Patents

Abstract

The invention relates to a method for preparing alpha, beta unsaturated fatty acid by using escherichia coli engineering bacteria, which comprises the steps of overexpressing FadE, FadD and ydi genes on the path, knocking out FadR and FadB genes at the same time, blocking the influence of beta oxidation and the regulation of a cell regulator, optimizing a trans-2-decenoic acid expression path, obtaining the escherichia coli engineering bacteria, and remarkably improving the conversion rate of the trans-2-decenoic acid by using the engineering bacteria through optimizing relevant conditions in the conversion process, so that the method can be further used for producing 10-hydroxyl-2-decenoic acid.

Description

Method for preparing alpha, beta unsaturated fatty acid by using fatty acid as raw material and utilizing escherichia coli engineering bacteria

Technical Field

The invention relates to a method for preparing alpha, beta unsaturated fatty acid by using escherichia coli engineering bacteria, belonging to the technical field of biological fermentation.

Background

α, β unsaturated fatty acids (α, β -unsaturated fatty acids) are unsaturated fatty acids having an unsaturated double bond at the α, β carbon. Unsaturated fatty acids (such as oleic acid, linoleic acid, erucic acid, etc.) are mainly used for preparing ore flotation agents, oil field chemicals and dimer acids and trimer acids for producing coatings.

In the case of trans-2-decenoic acid, trans-didecenoic acid is a compound in royal jelly components, and is used for synthesizing 10HDA, synthetic flavor for food (trans-2-decenoic acid ethyl ester) and pharmaceutical intermediate (trans-2-decenoic acid derivative)

The existing method for obtaining the alpha, beta unsaturated fatty acid is mainly a chemical synthesis method. The synthesis method is mainly used for constructing or developing alpha and beta-unforurated carbonyl compounds, and functional groups are constructed through aldehyde condensation, wittig type reaction or dehydrogenation of the carbonyl compounds.

Alpha, beta unsaturated fatty acid is used as a reaction intermediate, is difficult to obtain and is complex to prepare, so that the current situation that the yield is small and the market demand cannot be met is caused, at present, the production of trans-didecanoic acid in large scale is not available in the market, and the price is high and is about 3 ten thousand/kg.

Therefore, the method for synthesizing the alpha, beta unsaturated fatty acid with high efficiency, convenience and low cost is explored, and has important theoretical and application values for large-scale development and utilization of the alpha, beta unsaturated fatty acid. A reaction sequence in a cell is designed by utilizing biocatalysis synthesis, three key enzymes are identified and expressed in a combined manner in a cascade sequence, the expression maturity of the key enzymes is improved in three steps, two genes which obstruct the cascade reaction are further knocked out, and side reactions are blocked, so that a way independent of natural generation is achieved, and alpha, beta-UCAs are generated by fatty acid through a mixture of the enzymes.

The related art of producing alpha, beta unsaturated fatty acid by fermentation of cheap raw materials such as capric acid has not been reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for preparing alpha, beta unsaturated fatty acid by using fatty acid as a raw material and utilizing escherichia coli engineering bacteria.

The technical scheme of the invention is as follows:

a method for preparing alpha, beta unsaturated fatty acid by using resting cells of Escherichia coli engineering bacteria by taking fatty acid as a raw material comprises the following steps:

(1) constructing a recombinant plasmid petDuet-FadE and a recombinant plasmid pet28 a-sumo-Ydii;

the nucleotide sequence of the expression gene of the acyl-coenzyme dehydrogenase FadE is shown as SEQ ID NO. 19; the nucleotide sequence of the ester acyl CoA thioesterase gene ydiI is shown as SEQ ID NO. 20;

(2) constructing a petDuet-FadE-FadD plasmid by using the recombinant plasmid petDuet-FadE prepared in the step (1);

the nucleotide sequence of the fatty acyl CoA synthetase gene FadD is shown as SEQ ID NO. 21;

(3) knocking out genes by using an RED recombination method, and constructing a delta FadRB strain with gene deletion (hereinafter, engineering bacteria for knocking out FadR and FadB simultaneously are abbreviated as delta FadRB);

(4) and (2) co-transforming the recombinant plasmids pet28a-sumo-Ydii and pet Duet-FadE-FadD (hereinafter, pet28a-sumo-Ydii and pet Duet-FadE-FadD are abbreviated as YED) prepared in the step (1) and (2) into a strain with the gene deletion of delta FadRB to prepare a strain of the recombinant strain YED-delta FadRB.

Preferably, in the step (1), the recombinant plasmid petDuet-FadE is constructed, which comprises the following steps:

using a genome of escherichia coli BL21 as a template to amplify ester acyl coenzyme dehydrogenase FadE, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.1, the nucleotide sequence of a downstream primer is shown as SEQ ID No.2, then carrying out enzyme digestion and purification on petDuet plasmid by using Hind111, and carrying out seamless cloning on the petDuet plasmid and the gene of the ester acyl coenzyme dehydrogenase FadE to prepare a recombinant plasmid petDuet-FadE;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

Preferably, in the step (1), the construction of the recombinant plasmid pet28a-sumo-Ydii comprises the following steps:

using a genome of escherichia coli DH5a as a template, amplifying an ester acyl CoA thioesterase gene ydiI, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.3, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.4, then performing double digestion on a pet28a-sumo plasmid and the ester acyl CoA thioesterase gene ydiI respectively by using BamH I and Xho I, and connecting by ligase to prepare a recombinant plasmid pet28 a-sumo-ydi;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

According to a further preferred embodiment of the present invention, the above ligase ligation condition is ligation at 22 ℃ for 10min.

Preferably, in the step (2), the specific steps for constructing the petDuet-FadE-FadD plasmid are as follows:

taking a genome of escherichia coli BL21 as a template, amplifying a fatty acyl CoA synthetase gene FadD, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.5, the nucleotide sequence of a downstream primer is shown as SEQ ID No.6, carrying out enzyme digestion and purification on the recombinant plasmid petDuet-FadE prepared in the step (1) by using Xho1, and carrying out seamless cloning on the recombinant plasmid petDuet-FadE-FadD and the gene of the fatty acyl CoA synthetase gene FadD to prepare a recombinant plasmid petDuet-FadD;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

Preferably, in the step (3), the RED homologous recombination method is adopted for constructing the Δ FadRB strain with gene deletion, and comprises constructing a FadR knockout frame, constructing a FadB knockout frame and respectively transforming the FadR knockout frame and the FadB knockout frame into pkd46-BL 21.

According to a further preferred embodiment of the present invention, the specific steps for constructing the FadR knockout box are as follows:

taking a genome of escherichia coli BL21 as a template, amplifying an upstream homologous arm FadR1 of an operon gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.7, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 8; taking a genome of escherichia coli BL21 as a template, amplifying a downstream homologous arm FadR2 of an enoyl-CoA hydratase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.9, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 10; amplifying an FRT-RKan-FRT gene fragment by using a pkd3 plasmid as a template, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.11, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 12; then overlap PCR with FadR1 and FRT-RKan-FRT gene fragment, recover the amplification product, then overlap PCR with FadR2 again; recovering the final purified gel to obtain a FadR knockout box (the FadR knockout box is abbreviated as Rk below);

the PCR amplification system was as follows, with a total of 50. mu.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, ddH2O 19. mu.L;

the PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

According to a further preferred embodiment of the present invention, the specific steps for constructing the FadB knockout box are as follows:

taking a genome of escherichia coli BL21 as a template, amplifying an upstream homologous arm FadB1 of an enoyl-CoA hydratase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.13, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 14; taking a genome of escherichia coli BL21 as a template, amplifying a downstream homology arm FadB2 of an enoyl-CoA hydratase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.15, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 16; amplifying an FRT-BKan-FRT gene fragment by using pkd3 plasmid as a template, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.17, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 18; overlapping pcr with FadB1 and FRT-BKan-FRT gene fragment, recovering glue, overlapping pcr with FadB 2; recovering the final purified gel to obtain a FadB knockout box (the FadB knockout box is abbreviated as Bk below);

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

According to a further preferred embodiment of the present invention, the specific steps for transforming the FadR knockout frame and the FadB knockout frame into pkd46-BL21, respectively, are as follows:

a. the plasmid pkd46 is transformed into BL21 competence to obtain pkd46-BL21 recombinant bacteria, and CaCl is used₂The pkd46-BL21 is prepared to be competent by treatment;

b. the FadR knockout frame Rk is transformed into a pkd46-BL21 competence, after confirming that the pkd46-Rk-BL21 recombinant bacteria transfer the knockout frame, the pkd46 is eliminated at 42 ℃, and Rk-BL21 recombinant bacteria are obtained after screening;

c. preparing a Rk-BL21 recombinant strain into a competent transformation pcp20 plasmid, eliminating Rk resistance and the pcp20 plasmid at 42 ℃ to obtain a delta FadR recombinant strain, and preparing the delta FadR competent strain for later use;

d. transforming the plasmid pkd46 into delta FadR competence to obtain pkd 46-delta FadR recombinant bacteria, and treating with CaCl2 to prepare pkd 46-delta FadR competence;

e. the FadB knockout frame Bk is transformed into pkd 46-delta FadR competence, after confirming that the pkd46-Bk-BL21 recombinant bacteria are transformed, pkd46 is eliminated at 42 ℃, and Bk-delta FadR recombinant bacteria are obtained after screening;

f. preparing the Bk-delta FadR recombinant bacteria into a competent transformation pcp20 plasmid, and eliminating Bk resistance and the pcp20 plasmid at 42 ℃ to obtain the delta FadRB recombinant bacteria.

Preferably, in the step (4), the specific steps for constructing the recombinant strain YED-delta FadRB are as follows:

taking the strain of the delta FadRB in the step (3) and using CaCl₂And (2) treating to prepare competence, and co-transforming the recombinant plasmids pet28a-sumo-Ydii and pet Duet-FadE-FadD prepared in the steps (1) and (2) into gene deletion delta FadRB competence to prepare the YED-delta FadRB recombinant strain.

Preferably, the method for screening positive strains in step (4) comprises the following steps: and (3) performing sequencing identification on the strains with positive identification results by adopting colony PCR identification and protein expression and solubility identification.

An engineering bacterium of Escherichia coli is prepared by the above method.

A method for preparing alpha, beta unsaturated fatty acid by using the escherichia coli engineering bacteria comprises the following steps:

fermenting and culturing the recombinant escherichia coli, collecting thalli after induction expression, ultrasonically breaking thalli cells, adding decanoic acid into broken bacteria liquid, and producing trans-2-decenoic acid by using a cell-free system at 35-40 ℃.

Preferably, according to the present invention, the induced expression is: inoculating the activated recombinant escherichia coli into 100mL of liquid culture medium containing corresponding antibiotics, and performing shaking culture at 35-40 ℃ until bacterial liquid OD₆₀₀When the concentration is 0.8-1.2, IPTG with the final concentration of 0.5-0.8 mM, 0.5-0.8% of oleic acid and 0.2-0.5% of Tween 80 are added, and the temperature is reduced to 18-22 ℃ for overnight culture.

More preferably, the induced expression is: inoculating the activated recombinant Escherichia coli into 100mL liquid culture medium containing kanamycin and ampicillin, and performing shaking culture at 37 deg.C to obtain bacterial liquid OD₆₀₀At 1.0, IPTG was added to a final concentration of 0.64mM0.6 percent of oleic acid and 0.3 percent of Tween 80, and the temperature is reduced to 18 ℃ for overnight culture.

Preferably, according to the invention, the concentration of decanoic acid conversion is 4 g/L.

Preferably, according to the invention, the decanoic acid is dissolved in dimethyl sulfoxide.

Advantageous effects

1. The invention discovers an expression way of trans-2-decenoic acid for the first time, simultaneously knocks out FadR and FadB genes by over-expressing FadE, FadD and ydi genes on the way, blocks the influence of beta oxidation and the regulation of a cell regulator, optimizes the expression way of the trans-2-decenoic acid, obviously improves the conversion rate of the trans-2-decenoic acid by optimizing relevant conditions in the conversion process, and can be further used for producing 10-hydroxy-2-decenoic acid;

2. the invention adopts the principle of double expression plasmid cascade reaction to construct the large intestine engineering bacteria, realizes the high-efficiency assembly of the trans-2-decenoic acid expression element, can produce the trans-2-decenoic acid by fermenting the decanoic acid serving as a raw material through a broken fluid cell-free system after induction treatment, and realizes the process of producing the high-added-value trans-2-decenoic acid by using the low-value decanoic acid.

Drawings

FIG. 1 is a construction diagram of metabolic pathways of engineering bacteria;

FIG. 2 is an agarose gel electrophoresis of the PCR amplified ydi product, where M is marker and lanes 1-5 are ydi;

FIG. 3 is an agarose gel electrophoresis of the FadE, fadD products of PCR amplification, where M is marker and lanes 1-7 are FadE; lanes 8-14 are FadD;

FIG. 4 is an agarose gel electrophoresis of a colony PCR-verified BL21 knock-out FadR product, wherein lane M is Marker; lanes 1-6 are FadR insertion Carner resistance validation results, lanes 7-8 are raw controls;

FIG. 5 is an agarose gel electrophoresis of a colony PCR-verified Δ FadR knock-out FadB product, wherein lane M is Marker; lanes 1-9 are the FadB insert Carner resistance validation results, lane 13 is the original control;

FIG. 6 is an agarose gel electrophoresis of the colony PCR verified pet28a-sumo-ydi plasmid product, wherein lane M is Marker; lanes 1-8 are plasmid validation results;

FIG. 7 is an agarose gel electrophoresis of a colony PCR-verified petdut-fadE plasmid product, wherein lane M is Marker; lanes 1-9 are plasmid validation results;

FIG. 8 is a chromatogram for measuring gas quality of fermentation product, wherein the first is a control group, the second to the fourth is an experimental group, the fifth is a trans-didecenoic acid standard sample, and the chromatographic peak in the square frame is a trans-didecenoic acid peak;

FIG. 9 is a mass spectrum of trans-didecenoic acid from a library comparison;

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited thereto. The procedures not described in detail in the examples are all conventional procedures known to those skilled in the art.

Source of biological material

The plasmids described in the examples were purchased from vast Ling plasmid GmbH, and were all general commercial products;

coli were purchased from Novozan Biotechnology Ltd and were all products on the general market;

example 1: PCR amplification and plasmid construction of escherichia coli ester acyl-CoA thioesterase genes ydiI, FadE and FadD.

Designing PCR amplification primers according to Escherichia coli FadE, ydiI and FadD, wherein the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO.1, 3 and 5, and the nucleotide sequences of the downstream primer are respectively shown as SEQ ID NO.2, 4 and 6;

wherein, the nucleotide sequences of the Escherichia coli FadE, the Escherichia coli ydiI and the Escherichia coli FadD are respectively shown in SEQ ID NO.19, 20 and 21.

Construction of a recombinant plasmid containing petDuet-FadE, comprising the following steps:

using a genome of escherichia coli BL21 as a template to amplify ester acyl coenzyme dehydrogenase FadE, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.1, the nucleotide sequence of a downstream primer is shown as SEQ ID No.2, then carrying out enzyme digestion and purification on petDuet plasmid by using Hind111, and carrying out seamless cloning on the petDuet plasmid and the gene of the ester acyl coenzyme dehydrogenase FadE to prepare a recombinant plasmid petDuet-FadE;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL。

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

The construction of the recombinant plasmid pet28a-sumo-Ydii comprises the following steps:

using a genome of escherichia coli DH5a as a template, amplifying an ester acyl CoA thioesterase gene ydiI, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.2, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.3, then performing double digestion on a pet28a-sumo plasmid and the ester acyl CoA thioesterase gene ydiI respectively by using BamH I and Xho I, and connecting by ligase to prepare a recombinant plasmid pet28 a-sumo-ydi;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extending for 5min at 72 ℃;

the above ligase ligation conditions were 22 ℃ ligation for 10min.

The specific steps for constructing the petDuet-FadE-FadD plasmid are as follows:

taking a genome of escherichia coli BL21 as a template, amplifying a fatty acyl CoA synthetase gene FadD, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.4, the nucleotide sequence of a downstream primer is shown as SEQ ID No.5, carrying out enzyme digestion and purification on the recombinant plasmid petDuet-FadE prepared in the step (1) by using Xho1, and carrying out seamless cloning on the recombinant plasmid petDuet-FadE-FadD and the gene of the fatty acyl CoA synthetase gene FadD to prepare a recombinant plasmid petDuet-FadD;

the PCR amplification system was as follows, with a total of 50. mu.L:

100 μ M Forward primer 2.0mu.L, 100. mu.M downstream primer 2.0. mu.L, template 2.0. mu.L, 5U/. mu.L phanta enzyme 25. mu.L, ddH₂O 19μL。

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

And (3) recovering a PCR product:

after the PCR amplification, the length of the fragment was analyzed by 1% agarose gel electrophoresis, and the result is shown in FIG. 2 and FIG. 3, where 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 Biotechnology engineering Co., Ltd.

Example 2: construction of a Strain with Gene deletion Δ FadRB

(1) The specific steps for constructing the gene deletion delta FadRB strain are as follows:

the plasmid pkd46 is transformed into BL21 competence to construct recombinant bacterium pkd46-BL 21. After being cultured and screened, the recombinant bacterium pkd46-BL21 utilizes CaCl₂The treatment is made to be pkd46-BL21 competent;

secondly, amplifying an upstream homologous arm FadR1 of an operon gene by using a colon bacillus BL21 genome as a template, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.7, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 8; taking a genome of escherichia coli BL21 as a template, amplifying a downstream homologous arm FadR2 of an enoyl-CoA hydratase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.9, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 10; amplifying an FRT-RKan-FRT gene fragment by using a pkd3 plasmid as a template, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.11, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 12;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH2O 19. mu.L.

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

After the PCR amplification is finished, the length of the fragment is analyzed through 1% agarose gel electrophoresis, a target band is cut according to the size of the fragment, and PCR products, namely FadR1, FadR2 and FRT-Kan-FRT of the upstream and downstream homologous arms at the two ends of FadR, are recovered by using a DNA gel recovery kit of Shanghai biological engineering Co.

And overlapping the upstream homology arm FadR1 and the FRT-Kan-FRT two-section fragment by using overlapping PCR to construct an overlapping fragment of FadR 1-Kan. The nucleotide sequence of the upstream primer is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 12;

the overlap PCR amplification system was as follows, 50. mu.L total:

the first step is as follows:

FadR1 fragment 4.0. mu.L, FRT-Kan-FRT 4.0. mu.L, 5U/. mu.L phanta enzyme 12.5. mu.L, ddH₂O 19μL。

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 5 times; extension at 72 ℃ for 5 min.

The second step is that:

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, ddH₂O 19μL。

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

After the PCR amplification is finished, the length of the fragment is analyzed through 1% agarose gel electrophoresis, a target band is cut according to the size of the fragment, and a PCR product, namely an overlapped fragment of FadR1-Kan, is recovered by using a kit.

A knockout frame for FadR1-Kan-FadR2 was constructed by double overlapping the overlapping fragment of FadR1-Kan with the downstream homology arm FadR2 using overlap PCR. The nucleotide sequence of the upstream primer is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 10;

the overlap PCR amplification system was as follows, 50. mu.L total:

the first step is as follows:

FadR1-Kanthe overlapping fragment of (1) 4.0. mu.L, FadR24.0. mu.L, 5U/. mu.L phanta enzyme 12.5. mu.L, ddH₂O 19μL。

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 5 times; extension at 72 ℃ for 5 min.

The second step is that:

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, ddH₂O 19μL。

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

After the PCR amplification is finished, the length of the fragment is analyzed through 1% agarose gel electrophoresis, a target band is cut according to the size of the fragment, and a kit is used for recovering a PCR product, namely a knockout frame of FadR1-Kan-FadR 2.

Transforming the FadR1-Kan-FadR2(Rk) knockout frame into the pkd46-BL21 competence prepared in the step (1), and culturing and screening the strain to obtain a pkd46-Rk-BL21 recombinant strain;

thirdly, taking the pkd46-Rk-BL21 recombinant bacteria prepared in the second step, culturing overnight at 42 ℃, removing the pkd46 plasmid, streaking and purifying bacterial colonies on a flat plate, selecting a single bacterial colony, respectively dotting the flat plate with the Kana monoclonal antibody, the Kana and the ampicillin double antibody, selecting bacterial colonies which only grow on the Kana monoclonal antibody flat plate but do not grow on the Kana and the ampicillin double antibody flat plate, and removing the Rk-BL21 recombinant bacteria of the pkd46 plasmid;

fourthly, culturing the Rk-BL21 recombinant strain prepared in the third step, and utilizing CaCl₂Processing to make Rk-BL21 competent; and transforming the pcp20 plasmid into Rk-BL21 competence, and obtaining the Rk-pcp20-BL21 recombinant strain after culturing and screening.

Fifthly, culturing the Rk-pcp20-BL21 recombinant bacteria prepared in the step (iv) at 42 ℃ overnight, eliminating the kanamycin resistance and the pcp20 plasmid of a knockout frame, streaking and purifying colonies on a flat plate, selecting single colonies, respectively dotting the kanamycin monoclonal antibody, the ampicillin monoclonal antibody and a non-resistant flat plate, selecting colonies which only grow on the non-resistant flat plate but do not grow on the kanamycin and the ampicillin flat plates, and making the colonies into competent bacteria for later use in order to remove the delta FadR recombinant bacteria of the kanamycin resistance and the pcp20 plasmid, wherein the result is shown in figure 4;

sixthly, the pkd46 plasmid is transformed into a strain competence of gene deletion delta FadR to construct a recombinant bacterium pkd 46-delta FadR. After culturing and screening, the recombinant bacterium pkd 46-delta FadR utilizes CaCl₂The treatment made pkd46- Δ FadR competent;

seventhly, adopting Escherichia coli BL21 genome as a template, amplifying an upstream homology arm FadB1 of an operator gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.13, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 14; taking a genome of escherichia coli BL21 as a template, amplifying a downstream homology arm FadB2 of an enoyl-CoA hydratase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.15, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 16; amplifying an FRT-RKan-FRT gene fragment by using a pkd3 plasmid as a template, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.17, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 18;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL。

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

After the PCR amplification is finished, the length of the fragment is analyzed through 1% agarose gel electrophoresis, a target band is cut according to the size of the fragment, and PCR products, namely FadB1, FadB2 and FRT-Kan-FRT of homologous arms at the upper and lower ends of the FadB are recovered by using a DNA gel recovery kit of Shanghai biological engineering Co., Ltd.

And overlapping FadB1 of the upstream homology arm with two segments of FRT-Kan-FRT by using overlapping PCR to construct an overlapping segment of FadB 1-Kan. The nucleotide sequence of the upstream primer is shown as SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 18;

the overlap PCR amplification system was as follows, 50. mu.L total:

the first step is as follows:

FadB1 fragment 4.0 μ L, FRT-Kan-FRT 4.0 μ L, 5U/. mu.L phanta enzyme 12.5 μ L, ddH₂O 19μL。

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 5 times; extension at 72 ℃ for 5 min.

The second step is that:

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, ddH₂O 19μL。

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

After the PCR amplification is finished, the length of the fragment is analyzed through 1% agarose gel electrophoresis, a target band is cut according to the size of the fragment, and a PCR product, namely an overlapped fragment of FadB1-Kan, is recovered by using a kit.

And overlapping the overlapping fragment of FadB1-Kan with FadB2 of a downstream homology arm twice by using overlapping PCR to construct a knockout frame of FadB1-Kan-FadB 2. The nucleotide sequence of the upstream primer is shown as SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 16;

the overlap PCR amplification system was as follows, 50. mu.L total:

the first step is as follows:

overlapping fragment of FadB1-Kan 4.0. mu.L, FadB24.0. mu.L, 5U/. mu.L phanta enzyme 12.5. mu.L, ddH₂O 19μL。

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 5 times; extension at 72 ℃ for 5 min.

The second step is that:

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, ddH₂O 19μL。

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

After the PCR amplification is finished, the length of the fragment is analyzed through 1% agarose gel electrophoresis, a target band is cut according to the size of the fragment, and a PCR product, namely a knockout frame of FadB1-Kan-FadB2, is recovered by using the kit.

Transforming the FadB1-Kan-FadB2(Bk) knockout frame into pkd 46-delta FadR competence prepared in the step (c), and culturing and screening the strain to obtain a pkd 46-Bk-delta FadR recombinant strain;

eighthly, taking the pkd 46-Bk-delta FadR recombinant bacteria prepared in the step seventhly, carrying out overnight culture at 42 ℃, removing a pkd46 plasmid in order to remove the plasmid, carrying out streak purification on a plate, selecting a plate with single colony for respectively dotting Kana monoclonal antibody, Kana and ampicillin on the plate, selecting a bacterial colony which only grows on the Kana monoclonal antibody plate but does not grow on the Kana and ampicillin plates, and removing the Bk-delta FadR recombinant bacteria of the pkd46 plasmid;

ninthly culturing the Bk-delta FadR recombinant bacteria prepared in the step III by using CaCl₂Processing to make Bk-delta FadR competence; and (3) transforming the pcp20 plasmid into Bk-delta FadR competence, and obtaining the Bk-pcp 20-delta FadR recombinant bacteria after culturing and screening.

C, culturing the Bk-pcp 20-delta FadR recombinant bacteria prepared in the step ninthly at 42 ℃ overnight, eliminating the kanamycin resistance and the pcp20 plasmid of the knockout frame, streaking and purifying bacterial colonies on a plate, picking out single bacterial colonies, respectively dotting the kanamycin resistance, the ampicillin and a plate without resistance, selecting bacterial colonies which only grow on the plate without resistance but do not grow on the kanamycin and the ampicillin, and removing the delta FadRB recombinant bacteria of the kanamycin resistance and the pcp20 plasmid, wherein the results are shown in fig. 4 and fig. 5;

example 3: the recombinant plasmids pet28a-sumo-Ydii and petDuet-FadE-FadD are respectively transformed into a strain with gene deletion delta FadRB to prepare a strain of the recombinant bacteria YED-delta FadRB;

(1) preparation of competent cells

Picking a single colony (or picking and storing a strain) of Escherichia coli YED-delta FadRB and inoculating the single colony to 1ml of liquid LB culture medium, and carrying out overnight culture at 37 ℃ and 200 rpm;

② inoculating 1ml of bacterial liquid into 50ml of LB culture medium, culturing at 37 ℃ and 200rpm until the bacterial liquid OD₆₀₀About 0.5;

thirdly, placing the bacterial liquid on the ice-water mixture for 10min, and precooling a 10ml centrifuge tube;

transferring the bacterial liquid into a centrifugal tube, centrifuging at 4 ℃ and 5000rpm for 10min, and collecting thalli;

fifthly, adding 5mL of precooled 0.1M CaCl into each centrifuge tube₂Resuspending the cells in the solution, and adding 0.5mL of precooled 0.1M CaCl₂The solution is reversed and mixed evenly, and is kept stand for 20min on ice;

sixthly, the thalli is collected by centrifugation for 10min at the temperature of 4 ℃ and the rpm of 5000, the supernatant is discarded, and 0.5mL of precooled 0.1M CaCl containing 15 percent of glycerin is added₂Suspending the thallus in the solution to prepare competent cells;

seventhly, subpackaging the competent cells and freezing and storing at the temperature of minus 80 ℃.

(2) Transformation of recombinant plasmids

Adding 10 mu L of recombinant plasmid pet28a-sumo-Ydii and recombinant plasmid petDuet-FadE-FadD into 100 mu L of freshly prepared competent cells, gently mixing uniformly, and carrying out ice bath for 30 min;

② heat-shocking at 42 ℃ for 90s, then rapidly placing in an ice bath for cooling for 3 min;

inoculating the competent cell into LB culture medium of 500. mu.L, and culturing at 37 deg.C and 200rpm for 60 min;

fourthly, 200 mu L of the bacterial liquid is taken and coated on LB solid culture medium with 50mg/mL kanamycin and ampicillin;

fifthly, placing the culture box at 37 ℃ for 30min, inverting the plate after the bacterial liquid is sucked dry, and culturing the plate at 37 ℃ for 16 h.

(3) Identification of positive clones:

bacterial colony PCR identification

The single colony cultured as described above was picked up to 1mL of LB medium containing kanamycin and ampicillin, cultured at 37 ℃ for 8 hours with shaking at 200rpm, 1. mu.L of the bacterial solution was aspirated, and colony PCR was performed according to a 20. mu.L PCR reaction system, and the results are shown in FIG. 3, in which the band of interest was present and the band was single, indicating that the colony was a positive clone, and in FIGS. 6 and 7.

② protein expression and solubility identification

Taking 900 mu L of the bacterial liquid, adding IPTG with the final concentration of 0.32mM, inducing expression for 4h, centrifuging at 12000rpm for 1min, collecting thalli, adding 2 times of loading buffer solution, re-suspending the thalli, carrying out water bath denaturation at 100 ℃ for 10min, detecting protein expression by SDS-PAGE, and displaying positive clones.

Sequencing of the bacterial sample

The positive clones identified by the above two methods are sent to a sequencing company for sequencing, and the correctness of the constructed positive clones is further proved.

Example 4: recombinant strain YED-delta FadRB engineering strain fermentation

(1) Activating strains: the positive recombinant E.coli of example 3 was inoculated in 50mL of a liquid LB medium containing kanamycin and ampicillin at an inoculum size of 1%, and cultured at 37 ℃ for 12 hours with shaking at 200 rpm;

(2) transferring thalli: inoculating 1mL of the above activated strain into 100mL of liquid medium containing kanamycin and ampicillin, and shake-culturing at 37 deg.C and 200rpm to OD₆₀₀When the concentration is 1.0, adding IPTG to ensure that the concentration of IPTG in the culture medium is 0.64mM, adding oleic acid to ensure that the concentration of oleic acid in the culture medium is 0.6 percent by mass, adding Tween 80 to ensure that the concentration of Tween 80 in the culture medium is 0.3 percent by mass, and cooling to 18 ℃ for overnight induction culture;

(3) and (3) collecting thalli: collecting 500mL of the above bacterial solution, centrifuging at 4200rpm at 4 ℃ for 15min, collecting the thallus, adding 30mL of 20mM phosphate buffer (pH7.0) and resuspending the thallus;

(4) ultrasonic disruption of bacterial cells: performing ultrasound for 4s at intervals of 6s and 400W for 20 min;

(5) adding 4g/L of capric acid into the broken bacteria liquid, and culturing at 37 ℃ for 12h to obtain fermentation liquid.

Carrying out methyl esterification treatment on fermentation liquor: taking 900ul fermentation liquor in a 2ml centrifuge tube, adding 900ul ethyl acetate, performing vortex extraction for 10min, centrifuging, and taking out supernatant. Adding 180ul of silanization reagent solution under the protection of nitrogen, oscillating and mixing uniformly, taking out after 30min of water bath at 70 ℃, cooling to room temperature, and passing through an ultrafiltration membrane for later use.

GC-MS detection of the product: the gas chromatography-mass spectrometry uses helium as carrier gas, in a constant flow mode, the sample injection volume is 1ul, the split-flow sample injection is carried out, and the split-flow ratio is 1: 5, the sample injection temperature is 250 ℃, the temperature is kept at 50 ℃ for 1min, and the temperature is increased to 250 ℃ at 15 ℃/min and kept for 10min. The chromatogram of the product is shown in FIG. 8, wherein the peak in the box is trans-2-decenoic acid, and the mass spectrum of trans-2-decenoic acid is shown in FIG. 9.

Comparative example 1

The method as described in example 4, except that the engineered bacteria were constructed without the knock-out of FadR and FadB genes, after the overexpression of the ydiI, FadE and FadD genes, the fermentation experiment verifies that the product trans-didecenoic acid is not detected, presumably, the FadR operon gene obstructs the beta oxidation pathway of fatty acid, and the FadB gene expression leads the trans-didecenoic acid to be metabolized and consumed through the beta oxidation cycle pathway.

Comparative example 2

The method described in example 1 was performed except that the pBb series plasmid single promoter was used to express the three genes of ydiI, FadE, FadD in tandem, and then fermentation was performed according to the method described in example 4, and it was assumed that trans-didecenoic acid was not detected in the experimental product, and it was difficult to express the three genes using a single promoter.

Conclusion

As can be seen from the results of the above examples and comparative examples, the engineered Escherichia coli transformed by the method of the present invention has a pathway for effectively synthesizing trans-2-decenoic acid, and the implementation manner of the synthesis pathway is specific, and when the pathway is constructed by other conventional manners, the uncertainty of an expression system may affect the obtainment of the final trans-2-decenoic acid product.

Sequence listing

<110> university of Qilu Industrial science

<120> a method for preparing alpha, beta unsaturated fatty acid by using fatty acid as raw material and using escherichia coli engineering bacteria

<160> 21

<170> SIPOSequenceListing 1.0

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<211> 48

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

taagaaggag atataccatg gatgatgatt ttgagtattc tcgctacg 48

<210> 2

<211> 41

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

ggtgatggct gctgcccatg gctacgcggc ttcaactttc c 41

<210> 3

<211> 36

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

cggatccatg atatggaaac gaaaaatcac cctgga 36

<210> 4

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

cctcgagtca caaaatagcg gtcgtcaa 28

<210> 5

<211> 41

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

agatatacat atggcagatc tatgaagaag gtttggctta a 41

<210> 6

<211> 40

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

gccgatatcc aattgagatc ttcaggcttt attgtccact 40

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

atgatgaggg cgagtttgta ccag 24

<210> 8

<211> 43

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

taggaacttc gttgccatgt tgaatggtca accagccatc tcg 43

<210> 9

<211> 31

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

aggaacttcg tattggtcgt cactatttcg c 31

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

ttatcgtcgc aatataactt aa 22

<210> 11

<211> 31

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

caacatggca acgaagttcc tattctctag a 31

<210> 12

<211> 37

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

acgaccaata cgaagttcct atactttcta gagaata 37

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

atgctttaca aaggcgacac cctg 24

<210> 14

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

ataggaactt cgatgatttc cagcgcactg tca 33

<210> 15

<211> 39

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

agtataggaa cttctgcaga aagattaccg cgatgccat 39

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

ttaagccgtt ttcaggtcgc caa 23

<210> 17

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

gaaatcatcg aagttcctat tctctaga 28

<210> 18

<211> 32

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

tctgcagaag ttcctatact ttctagagaa ta 32

<210> 19

<211> 2445

<212> DNA

<213> Escherichia Coli (E Coli.)

<400> 19

atgatgattt tgagtattct cgctacggtt gtcctgctcg gcgcgttgtt ctatcaccgc 60

gtgagcttat ttatcagcag tctgattttg ctcgcctgga cagccgccct cggcgttgct 120

ggtctgtggt cggcgtgggt actggtgcct ctggccatta tcctcgtgcc atttaacttt 180

gcgcctatgc gtaagtcgat gatttccgcg ccggtatttc gcggtttccg taaggtgatg 240

ccgccgatgt cgcgcactga gaaagaagcg attgatgcgg gcaccacctg gtgggagggc 300

gacttgttcc agggcaagcc ggactggaaa aagctgcata actatccgca gccgcgcctg 360

accgccgaag agcaagcgtt tctcgacggc ccggtagaag aagcctgccg gatggcgaat 420

gatttccaga tcacccatga gctggcggat ctgccgccgg agttgtgggc gtaccttaaa 480

gagcatcgtt tcttcgcgat gatcatcaaa aaagagtacg gcgggctgga gttctcggct 540

tatgcccagt ctcgcgtgct gcaaaaactc tccggcgtga gcgggatcct ggcgattacc 600

gtcggcgtgc caaactcatt aggcccgggc gaactgttgc aacattacgg cactgacgag 660

cagaaagatc actatctgcc gcgtctggcg cgtggtcagg agatcccctg ctttgcactg 720

accagcccgg aagcgggttc cgatgcgggc gcgattccgg acaccgggat tgtctgcatg 780

ggcgaatggc agggccagca ggtgctgggg atgcgtctga cctggaacaa acgctacatt 840

acgctggcac cgattgcgac cgtgcttggg ctggcgttta aactctccga cccggaaaaa 900

ttactcggcg gtgcagaaga tttaggcatt acctgtgcgc tgatcccaac caccacgccg 960

ggcgtggaaa ttggtcgtcg ccacttcccg ctgaacgtac cgttccagaa cggaccgacg 1020

cgcggtaaag atgtcttcgt gccgatcgat tacatcatcg gcgggccgaa aatggccggg 1080

caaggctggc ggatgctggt ggagtgcctc tcggtaggcc gcggcatcac cctgccttcc 1140

aactcaaccg gcggcgtgaa atcggtagcg ctggcaaccg gcgcgtatgc tcacattcgc 1200

cgtcagttca aaatctctat tggtaagatg gaagggattg aagagccgct ggcgcgtatt 1260

gccggtaatg cctacgtgat ggatgctgcg gcatcgctga ttacctacgg cattatgctc 1320

ggcgaaaaac ctgccgtgct gtcggctatc gttaagtatc actgtaccca ccgcgggcag 1380

cagtcgatta ttgatgcgat ggatattacc ggcggtaaag gcattatgct cgggcaaagc 1440

aacttcctgg cgcgtgctta ccagggcgca ccgattgcca tcaccgttga aggggctaac 1500

attctgaccc gcagcatgat gatcttcgga caaggagcga ttcgttgcca tccgtacgtg 1560

ctggaagaga tggaagcggc gaagaacaat gacgtcaacg cgttcgataa actgttgttc 1620

aaacatatcg gtcacgtcgg tagcaacaaa gttcgcagct tctggctggg cctgacgcgc 1680

ggtttaacca gcagcacgcc aaccggcgat gccactaaac gctactatca gcacctgaac 1740

cgcctgagcg ccaacctcgc cctgctttct gatgtctcga tggcagtgct gggcggcagc 1800

ctgaaacgtc gcgagcgcat ctcggcccgt ctgggggata ttttaagcca gctctacctc 1860

gcctctgccg tgctgaagcg ttatgacgac gaaggccgta atgaagccga cctgccgctg 1920

gtgcactggg gcgtacaaga tgcgctgtat caggctgaac aggcgatgga tgatttactg 1980

caaaacttcc cgaaccgcgt ggttgccggg ctgctgaatg tggtgatctt cccgaccgga 2040

cgtcattatc tggcaccttc tgacaagctg gatcataaag tggcgaagat tttacaagtg 2100

ccgaacgcca cccgttcccg cattggtcgc ggtcagtacc tgacgccgag cgagcataat 2160

ccggttggct tgctggaaga ggcgctggtg gatgtgattg ccgccgaccc aattcatcag 2220

cggatctgta aagagctggg taaaaacctg ccgtttaccc gtctggatga actggcgcac 2280

aacgcgctgg tgaaggggct gattgataaa gatgaagccg ctattctggt gaaagctgaa 2340

gaaagccgtc tgcgcagtat taacgttgat gactttgatc cggaagagct ggcgacgaag 2400

ccggtaaagt tgccggagaa agtgcggaaa gttgaagccg cgtag 2445

<210> 20

<211> 411

<212> DNA

<213> Escherichia Coli (E Coli.)

<400> 20

atgatatgga aacgaaaaat caccctggaa gcactgaatg ctatggggga aggaaacatg 60

gtgggattgc tggatattcg ctttgaacat attggtgatg acacccttga agcgacaatg 120

ccagtagact cacggacaaa gcagcctttc gggttgctgc atggaggtgc atctgtggta 180

ctggccgaaa gtatcggttc cgttgccggt tatttatgta ccgaaggtga gcaaaaagtg 240

gttggtctgg aaatcaatgc taaccacgtc cgctcggcac gagaagggcg ggtgcgcggc 300

gtatgcaaac cgttgcatct cggttcgcgt caccaggtct ggcagattga aatcttcgat 360

gagaaagggc gtttgtgctg ttcgtcacga ttgacgaccg ctattttgtg a 411

<210> 21

<211> 1683

<212> DNA

<213> Escherichia Coli (E Coli.)

<400> 21

ggctttattg tccactttgc cgcgcgcttc gtcacgtaat tctcgtcgca aaatttttcc 60

gacgttagat ttcggtaact catcacgaaa ctccaccagc ttcggtactt tgtagcccgt 120

gagctgacgg cggcaaaagg tcaccagtga ctcttcggta agcgatggat cttttttcac 180

tacgaagatt ttcaccgctt caccactgga gccggaaggt acgccaacag ccgcgacttc 240

ctgtacgcca ggatgctgca tgacgacatc ttcaatctcg ttgggataga cgttaaaacc 300

ggaaaccaga atcatgtctt ttttacgatc gacaatgcgc aggaaccctt cttcatccat 360

caccgcgatg tcgccggtgt gtaaccagcc atttttgatg atctcatctg tagcatccgg 420

acgctgccag taacccagca tcacctgcgg tcctttgaca caaagctcac ccggttgacc 480

cggtggtact tcattatcat catcatccac cagtttggct tccgtcgacg gcaccggcaa 540

accgatgcta ccactatgat aatcaatatc atatgggtta acgctgacca gcggcgcaca 600

ctcggtaagg ccatagcctt ccagcagata ctgtcctgtc agtttcaccc aacgctctgc 660

caccacttgc tgcactggca tccctccgcc tgcggaaaga tgcagactgg agaaatccag 720

ctgctggaac tctttattgt tcagcaacgc attgaacaag gtgttaacgc ccgtgatagc 780

ggtaaacgga tatttcgcta actcttttac caaccctgga atatcgcgcg ggttagtgat 840

aagcaggttc tgcccaccca gttcgataaa cagcaggcag ttaatggtca gggcaaaaat 900

gtgatacagc ggcagcgccg tcaccaccag ctctttgccc ggatgcaaca gcggaccata 960

ggtcgcgtta acctgttcca ggttcgccag catattgcgg tgagtcagca tcgcgccttt 1020

cgccacacca gtggtgccgc cggtgtattg cagaaaagct aaatcttccg gcaccagttc 1080

gggtttgacg tactgcatcc ggtagccgtt atgcagtgcg ctacgaaatg aaatggcatc 1140

tggcagatgg tatttcggca ccaaacgctt gatgtattta acaacgaaat tgactaccgt 1200

gccttttgcc gtagatagct gatcgcccat acgggtcaga attacgtgct gaacggcggt 1260

tttatcaacc actttttcca gtgtgtgagc aaagttagac acgataacaa tcgccgatgc 1320

gccgctatcg ttaagctgat gctcaagctc acgcggggta tacaacgggt taacgtttac 1380

gacgatcatc ccggcacgca aaatgccaaa cagcgccacc ggatattgca ataaattagg 1440

catcatcaac gcaacgcgat cgcctttctt cagccccaac ccttgttgca aataagcggc 1500

aaacgcgcga ctgcgttctt ccagcttgcg gaaggtcatt acctccccca tattcacaaa 1560

cgcaggctga tcggcgtagc gcgcgaccga ctgctcaaac atatctacca gagattgata 1620

acggtcaggg ttgatctccg tcggaacgtc cgcgggataa cggttaagcc aaaccttctt 1680

caa 1683

Claims (14)

1. A method for preparing alpha, beta unsaturated fatty acid by utilizing escherichia coli engineering bacteria is characterized by comprising the following steps:

fermenting and culturing engineering bacteria of escherichia coli, collecting bacteria after induction expression, ultrasonically breaking cells of the bacteria, adding decanoic acid into broken bacteria liquid, and producing trans-2-decenoic acid by using a cell-free system at 35-40 ℃;

the construction method of the escherichia coli engineering bacteria comprises the following steps:

(1) constructing a recombinant plasmid petDuet-FadE and a recombinant plasmid pet28 a-sumo-Ydii;

the nucleotide sequence of the acyl-coenzyme dehydrogenase FadE gene is shown in SEQ ID NO. 19; the nucleotide sequence of the ester acyl CoA thioesterase gene Y diI is shown in SEQ ID NO. 20;

(2) constructing a petDuet-FadE-FadD plasmid by using the recombinant plasmid petDuet-FadE prepared in the step (1);

the nucleotide sequence of the fatty acyl CoA synthetase gene FadD is shown as SEQ ID NO. 21;

(3) knocking out FadR and FadB genes by using an RED recombination method, and constructing a gene-deleted strain FadRB;

(4) and (2) co-transforming the recombinant plasmid pet28a-sumo-Ydii and petDuet-FadE-FadD prepared in the step (1) and the step (2) into a gene deletion strain Δ FadRB to prepare a recombinant strain YED- Δ FadRB.

2. The method according to claim 1, wherein the step (1) of constructing the recombinant plasmid petDuet-FadE comprises the steps of:

using a genome of escherichia coli BL21 as a template to amplify an ester acyl coenzyme dehydrogenase FadE gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.1, the nucleotide sequence of a downstream primer is shown as SEQ ID No.2, then carrying out enzyme digestion and purification on petDuet plasmid by HindIII, and carrying out seamless cloning on the petDuet plasmid and the gene of the ester acyl coenzyme dehydrogenase FadE to prepare a recombinant plasmid petDuet-FadE;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

3. The method of claim 1, wherein the step (1) of constructing the recombinant plasmid pet28a-sumo-ydi comprises the steps of:

using Escherichia coli DH5a genome as template, amplifying ester acyl CoA thioesterase gene Y diI, the nucleotide sequence of upstream primer is shown as SEQ ID NO.3, the nucleotide sequence of downstream primer is shown as SEQ ID NO.4, then using pet28a-sumo plasmid and ester acyl CoA thioesterase gene Y diI respectivelyBamH I andXho carrying out double enzyme digestion respectively, and connecting by ligase to prepare a recombinant plasmid pet28 a-sumo-Ydii;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

4. The method of claim 3, wherein the ligase ligation conditions are ligation at 22 ℃ for 10min.

5. The method of claim 1, wherein in the step (2), the specific steps for constructing the petDuet-FadE-FadD plasmid are as follows:

taking a genome of escherichia coli BL21 as a template, amplifying a fatty acyl CoA synthetase gene FadD, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.5, the nucleotide sequence of a downstream primer is shown as SEQ ID No.6, carrying out enzyme digestion and purification on the recombinant plasmid petDuet-FadE prepared in the step (1) by using Xho1, and carrying out seamless cloning on the recombinant plasmid petDuet-FadE-FadD and the fatty acyl CoA synthetase gene FadD to prepare a recombinant plasmid petDuet-FadD;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

6. The method of claim 1, wherein in the step (3), the remote Recombinant Expression (RED) homologous recombination method is adopted for constructing the fatR strain, and the method comprises constructing a FadR knockout frame, constructing a FadB knockout frame, and transforming the FadR knockout frame and the FadB knockout frame into pkd46-BL21 respectively.

7. The method of claim 6, wherein the specific steps for constructing the FadR knockout box are as follows:

taking a genome of escherichia coli BL21 as a template, amplifying an upstream homologous arm FadR1 of an operon gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.7, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 8; taking a genome of escherichia coli BL21 as a template, amplifying a downstream homologous arm FadR2 of an enoyl-CoA hydratase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.9, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 10; amplifying an FRT-RKan-FRT gene fragment by using a pkd3 plasmid as a template, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.11, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 12; then carrying out overlap PCR on FadR1 and FRT-RKan-FRT gene segments, recovering amplification products, and then carrying out overlap PCR on FadR2 again; finally, recovering the purified glue to obtain a FadR knockout frame;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

8. The method of claim 6, wherein the specific steps of constructing the FadB knockout box are as follows:

taking a genome of escherichia coli BL21 as a template, amplifying an upstream homologous arm FadB1 of an enoyl-CoA hydratase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.13, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 14; taking a genome of escherichia coli BL21 as a template, amplifying a downstream homology arm FadB2 of an enoyl-CoA hydratase gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No.15, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 16; amplifying an FRT-BKan-FRT gene fragment by using pkd3 plasmid as a template, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.17, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 18; then carrying out overlapped PCR on the FadB1 and the FRT-BKan-FRT gene segment, and carrying out overlapped PCR on the recovered gel and FadB2 again after the overlapped PCR is finished; finally, recovering the purified glue to obtain a FadB knockout frame;

the PCR amplification system was as follows, with a total of 50. mu.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, ddH₂O 19μL；

The PCR amplification conditions were as follows:

pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 15s, and circulation for 30 times; extension at 72 ℃ for 5 min.

9. The method of claim 6, wherein the specific steps of transforming the FadR and FadB knockout boxes into pkd46-BL21, respectively, are as follows:

a. the plasmid pkd46 is transformed into BL21 competence to obtain pkd46-BL21 recombinant bacteria, and CaCl is used₂The pkd46-BL21 is prepared to be competent by treatment;

b. the FadR knockout frame Rk is transformed into a pkd46-BL21 competence, after confirming that the pkd46-Rk-BL21 recombinant bacteria transfer the knockout frame, the pkd46 is eliminated at 42 ℃, and Rk-BL21 recombinant bacteria are obtained after screening;

c. preparing competent transformation pcp20 plasmid from Rk-BL21 recombinant bacteria, eliminating Rk resistance at 42 ℃, obtaining fatR recombinant bacteria, and preparing fatR competent for standby;

d. transforming the plasmid pkd46 into a fatR competent state to obtain a pkd46 fatR recombinant strain, and adding CaCl₂Treatment to prepare pkd 46. Δ FadR competence;

e. the FadB knockout frame Bk is transformed into a pkd 46-fatR competence, after the verification of the pkd46-Bk-BL21 recombinant bacteria confirms that the knockout frame is transformed, the pkd46 is eliminated at 42 ℃, and the Bk-fatR recombinant bacteria are obtained after screening;

f. preparing competent transformation pcp20 plasmid from the Bk-fatR recombinant strain, eliminating Bk resistance at 42 ℃, obtaining the fatRB recombinant strain from the pcp20 plasmid.

10. The method according to claim 1, wherein in step (4), the specific steps of constructing the recombinant bacterium YED _ Δ FadRB are as follows:

treating the strain of the fatRB in the step (3) with CaCl2 to prepare competence, and co-transforming the recombinant plasmids pet28a-sumo-Ydii and petDuet-FadE-FadD prepared in the steps (1) and (2) into the strain with the fatRB gene deletion to prepare the YED-fatRB recombinant strain.

11. The method of claim 1, wherein said inducible expression is:

inoculating the activated recombinant escherichia coli into 100mL of liquid culture medium containing corresponding antibiotics, and performing shaking culture at 35-40 ℃ until bacterial liquid OD₆₀₀When the concentration is 0.8-1.2, IPTG with the final concentration of 0.5-0.8 mM, 0.5-0.8% of oleic acid and 0.2-0.5% of Tween 80 are added, and the temperature is reduced to 18-22 ℃ for overnight culture.

12. The method of claim 11, wherein said inducible expression is: inoculating the activated recombinant Escherichia coli into 100mL liquid culture medium containing kanamycin and ampicillin, and performing shaking culture at 37 deg.C to obtain bacterial liquid OD₆₀₀When the concentration is 1.0, IPTG with the final concentration of 0.64mM, 0.6% of oleic acid and 0.3% of Tween 80 are added, and the temperature is reduced to 18 ℃ for overnight culture.

13. The method of claim 1, wherein the decanoic acid has a reaction concentration of 4 g/L.

14. The method of claim 1, wherein the decanoic acid is dissolved in dimethyl sulfoxide.

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