CN110791467B

CN110791467B - Recombinant bacterium for producing acetylacetone and construction method and application thereof

Info

Publication number: CN110791467B
Application number: CN201810865467.0A
Authority: CN
Inventors: 赵广; 冯新军; 咸漠; 周怡斐; 高文杰
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2018-08-01
Filing date: 2018-08-01
Publication date: 2021-05-04
Anticipated expiration: 2038-08-01
Also published as: CN110791467A

Abstract

The invention discloses a recombinant bacterium for producing acetylacetone and a construction method and application thereof, belonging to the technical field of genetic engineering. The recombinant strain constructed by the invention can utilize glucose as a unique carbon source to synthesize acetylacetone, and the acetylacetone can be obtained by fermenting for 48 hours at a concentration of 53 mg/L.

Description

Recombinant bacterium for producing acetylacetone and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant bacterium for producing acetylacetone, and a construction method and application thereof.

Background

Acetylacetone is an organic compound, which is designated by the standard name 2, 4-pentanedione. Acetylacetone has wide application in a plurality of industries such as pharmaceutical industry, electronics, chemical industry, petroleum, materials, machinery and the like, for example, can be used as an intermediate for synthesizing medicaments for treating diabetes, synthesis of veterinary antibacterial medicaments, a petroleum cracking catalyst and the like. 90% of acetylacetone in China is used for the production of medicines and veterinary drugs, and 80% of acetylacetone in Japan is used for catalysts, solvents and the like of petrochemical reactions. Acetylacetone is used as a raw material, and the material can also react with substances such as guanidine and the like to synthesize the tetrazine high-nitrogen compound which is an energetic substance with excellent performance and has potential application prospect in the fields of energetic materials such as low-characteristic signal propellants, novel high-energy insensitive explosives and the like. In recent years, the situation of the downstream medicine export of acetylacetone is better and better, and the demand is rapidly increased.

Acetylacetone on the market is mainly obtained by chemical synthesis methods, such as acetone-acetic anhydride synthesis, ethyl acetoacetate-ketene synthesis, propyne-acetic acid synthesis, ethyl acetoacetate-acetic anhydride synthesis, and the like. The chemical synthesis method generally has the defects of higher cost, complex process, low yield, high energy consumption and the like, and does not meet the current requirements of low-carbon economy.

Disclosure of Invention

In order to solve the problems, the invention firstly provides a recombinant bacterium for synthesizing and producing acetylacetone by a biological method.

The recombinant bacterium overexpresses an acetylacetone lyase gene dke1, a methylglyoxal synthase gene mgsA and a triosephosphate isomerase gene tpiA, or overexpresses an acetylacetone lyase gene dke 1; the starting strain was E.coli.

In one embodiment of the invention, said gene dke1 encoding acetylacetonato lyase is derived from Acinetobacter johnsonii, the gene mgsA for methylglyoxal synthase is derived from E.coli, and the gene tpiA for triosephosphate isomerase is derived from E.coli; the coding acetylacetone lyase gene dke1 is a sequence synthesized after codon optimization, and the sequence is shown in SEQ ID NO. 1; the Gnen ID of the methylglyoxal synthase gene mgsA is 945574; the Gene ID of the triose phosphate isomerase Gene tpiA was 948409.

The invention also provides a method for constructing the recombinant bacterium, which comprises the following steps:

1) cloning an acetylacetone lyase gene dke1, carrying out enzyme digestion and connection on the obtained gene and a plasmid pACYCDuet-1, transferring the gene into E.coli DH5 alpha competent cells, screening positive clones, and extracting the plasmid to obtain a recombinant vector pACYCDuet-dke 1;

2) cloning a methylglyoxal synthase gene mgsA, carrying out enzyme digestion and connection on the obtained gene and the vector pACYCDuet-dke1 obtained in the step 1), transferring the gene into E.coli DH5 alpha competent cells, screening positive clones, and extracting plasmids to obtain a recombinant vector pACYCDuet-dke 1-mgsA;

3) cloning a triose phosphate isomerase gene tpiA, carrying out enzyme digestion and connection on the obtained gene and the vector pACYCDuet-dke1-mgsA obtained in the step 2), transferring the gene into an E.coli DH5 alpha competent cell, screening positive clones, and extracting a plasmid to obtain a recombinant vector pACYCDuet-dke 1-mgsA-tpiA;

4) transforming the recombinant vector obtained in the step 1) or the step 3) into a host Escherichia coli BL21(DE3) competent cell to obtain an Escherichia coli recombinant strain.

The invention also provides an application of the recombinant bacterium in fermentation production of acetylacetone, which comprises the following steps:

1) activating the recombinant bacteria to obtain a seed solution;

2) inoculating the seed liquid obtained in the step 1) to a fermentation medium containing chloramphenicol, and culturing to OD₆₀₀Obtaining a culture solution at 0.6-0.8;

3) adding an inducer into the obtained culture solution, and continuously culturing for 24-48 hours; and separating to obtain acetylacetone.

In one embodiment of the present invention, the culture conditions in step 2) are: 35 to 37 ℃ and 180 to 220rpm, preferably 37 ℃ and 180 rpm.

In one embodiment of the present invention, the carbon source of the fermentation medium in step 2) is glucose, the nitrogen source is an inorganic nitrogen source such as ammonium chloride or ammonium sulfate, and the other components are inorganic salts. The inoculation proportion of the seed liquid is that the volume ratio of the seed liquid to the fermentation medium is (1-2) to (100-130).

In one embodiment of the invention, the inducer in step 3) is isopropyl-beta-D-thiogalactoside (IPTG) and is added in an amount of 0.01-0.1 mM of final concentration.

In one embodiment of the invention, the culture conditions in step 3) are 30-33 ℃ and 180-220 rpm.

In one embodiment of the invention, the fermentation medium has the formula: 20g/L glucose, 15.2g/L disodium hydrogen phosphate dodecahydrate, 3g/L potassium dihydrogen phosphate, 0.5g/L sodium chloride, 1g/L ammonium chloride, 3g/L yeast powder, 0.5g/L magnesium sulfate and 0.1% (v/v) microelement mother liquor.

In one embodiment of the present invention, the formulation of the microelement mother liquor is: 6.0g/L ferrous sulfate heptahydrate, 2.0g/L boric acid, 2.0g/L manganese chloride tetrahydrate, 0.8g/L ammonium molybdate tetrahydrate and 0.2g/L copper sulfate pentahydrate.

In one embodiment of the present invention, the recombinant strain is activated and inoculated at an inoculum size of 1% into a fermentation medium containing 100. mu.g.mL-1 chloramphenicol, and the mixture is subjected to shaking culture at 37 ℃ and 180rpm until OD is reached₆₀₀When the temperature reached 0.6 ℃, the temperature was adjusted to 30 ℃, and 0.05mM IPTG was added for induction, the pH was adjusted to 7 with ammonia every 12h, and the fermentation was terminated 48h after the initial induction.

Advantageous effects

Aiming at the problems in the background technology, the invention uses Escherichia coli BL21(DE3) as a host strain, constructs and obtains engineering bacteria by over-expressing key synthetic genes of acetylacetone lyase gene dke1, methylglyoxal synthase gene mgsA and triosephosphate isomerase gene tpiA, and realizes the biosynthesis of acetylacetone for the first time by using glucose as a carbon source. The recombinant strain constructed by the invention can utilize glucose as a unique carbon source to synthesize acetylacetone, and the acetylacetone can be obtained by fermenting for 48 hours at a concentration of 53 mg/L.

Definitions and abbreviations

The following abbreviations or acronyms are used herein:

acetylacetonatolyase gene: dke1

Methylglyoxal synthase gene: mgsA

Triose phosphate isomerase gene: tpiA

Escherichia coli (Escherichia coli) E

"overexpression" or "overexpression" refers to the expression of a particular gene in an organism in large amounts, in excess of normal levels (i.e., wild-type expression levels), which can be achieved by enhancing endogenous expression or introducing a foreign gene.

Detailed Description

The invention is further elucidated below by way of examples. However, the present invention is not limited to the following examples.

The experimental procedures used in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The enzyme reagent is purchased from MBI Fermentas company, the kit for extracting plasmid and the kit for recovering DNA fragment are purchased from American OMEGA company, and the corresponding operation steps are carried out according to the product instruction; all media were formulated with deionized water unless otherwise indicated.

The formula of the culture medium is as follows:

1) seed liquid shake-flask culture medium

LB culture medium: 5g/L yeast powder, 10g/L NaCl, 10g/L peptone and the balance of water, sterilizing at 121 ℃ for 20 min.

2) Shake flask fermentation medium

Basic modified medium: 20g/L glucose, 15.2g/L disodium hydrogen phosphate dodecahydrate, 3g/L potassium dihydrogen phosphate, 0.5g/L sodium chloride, 1g/L ammonium chloride, 3g/L yeast powder, 0.5g/L magnesium sulfate and 0.1% (v/v) trace elements.

The formula of the microelement mother liquor comprises the following components: 6.0g/L ferrous sulfate heptahydrate, 2.0g/L boric acid, 2.0g/L manganese chloride tetrahydrate, 0.8g/L ammonium molybdate tetrahydrate and 0.2g/L copper sulfate pentahydrate.

In the actual culture process, to the culture medium can be added a certain concentration of antibiotics to maintain the stability of plasmid, such as 100 u g mL-1 chloramphenicol.

Example 1 construction of a recombinant bacterium for the biosynthetic production of acetylacetone by the biological method

1) Construction of vector pACYCDuet-dke1

The acetylacetonatolyase gene sequence derived from Acinetobacter johnsonii was codon-optimized and synthesized by Kinzhi, Suzhou. The synthesized gene is used as a template, PCR amplification is carried out to obtain primers 5'-CGGGATCCgATGGACTACTGCAACAAAAAACA-3' and 5'-CGGAATTCTTAAGCAGCTTCGTTTTTGGTAGC-3', and a recovery kit is utilized to recover a target fragment, wherein the size of the target fragment is 462bp, so that a dke1 gene fragment with the sequence shown in SEQ ID NO.1 is obtained.

The obtained dke1 gene fragment and plasmid pACYCDuet are cut by BamHI and EcoRI, the cut products are recovered and connected, the vector and dke1 gene fragment are connected for more than 6h at 16 ℃ according to the molar ratio of 1:5, the connection product is converted into E.coliDH5 alpha, and then the E.coliDH5 alpha is coated on an LB solid plate added with 100 mu g.mL-1 chloramphenicol, and positive clones are screened by PCR. After extraction of the recombinant plasmid pACYCDuet-dke1 from the positive clones, it was identified by restriction enzyme digestion and sequencing.

2) Construction of vector pACYCDuet-dke1-mgsA

In this example, the methylglyoxal synthase gene mgsA (Gnen ID: 945574) derived from Escherichia coli was obtained by PCR amplification using the genome of Escherichia coli as a template (primers: 5'-ATCGAGCTCGTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTGTACATAAGGAGATAATGGAACTGACGACTCGCAC-3' and 5'-AGCGTCGACTTACTTCAGACGGTCCGCGAG-3'), and the target fragment was recovered using a recovery kit, and the size of the target fragment was 459 bp.

Digesting the obtained mgsA gene fragment and the vector pACYCDuet-dke1 obtained in the step 1) by SacI and SalI, recovering digested products, connecting the vector and the mgsA gene fragment at a molar ratio of 1:5 at 16 ℃ for more than 6h, converting the connected product into E.coliDH5 alpha, coating the E.coliDH5 alpha on an LB solid plate added with 100 mu g.mL-1 chloramphenicol, and screening positive clones by PCR. After extracting the recombinant vector pACYCDuet-dke1-mgsA from the positive clone, the recombinant vector is identified by restriction enzyme digestion and sequencing.

3) Construction of the vector pACYCDuet-dke1-mgsA-tpiA

In this example, the triose phosphate isomerase Gene tpiA derived from Escherichia coli (Gene ID 948409) was obtained by PCR amplification using the Escherichia coli genome as a template (primers: 5'-GGGGTACCATGCGACATCCTTTAGTGATG-3' and 5'-CCGCTCGAGTTAAGCCTGTTTAGCCGCTTC-3'); and recovering the target fragment by utilizing a recovery kit, wherein the size of the target fragment is 768 bp.

The obtained tpiA gene fragment and the vector pACYCDuet-dke1-mgsA are cut by KpnI and XhoI, the cut products are recovered, the vector and the tpiA gene fragment are connected for more than 6 hours at 16 ℃ according to the molar ratio of 1:5 after connection, the connection products are transformed into E.coliDH5 alpha, and then the E.coliDH5 alpha is coated on an LB solid plate added with 100 mu g.mL-1 chloramphenicol, and positive clones are screened by PCR. After extracting the recombinant vector pACYCDuet-dke1-mgsA-tpiA from the positive clone, the recombinant vector is identified by restriction enzyme digestion and sequencing.

4) Transformation of

The vector pACYCDuet-dke1 was transformed into E.coli BL21(DE3) to obtain the recombinant bacterium 1 of interest.

And (3) transforming the pACYCDuet-dke1-mgsA-tpiA obtained in the step 3) into E.coli BL21(DE3) to obtain the target recombinant bacterium 2.

EXAMPLE 2 fermentative production of acetylacetone

The recombinant bacterium 1 of interest obtained in example 1 was spread on an LB solid plate containing 100. mu.g.mL-1 chloramphenicol; the coated plate was placed in a 37 ℃ incubator and cultured until single colonies grew.

Activating the obtained engineering strain monoclonal in LB culture to obtain seed liquid, inoculating the seed liquid into a 500mL baffle shake flask containing 100mL basic improved liquid culture medium (containing 100. mu.g.mL-1 chloramphenicol) according to the volume ratio of the seed liquid to the fermentation culture medium of 1:100, and performing shake culture at 37 ℃ and 180 rpm. OD₆₀₀When about 0.6 ℃ is reached, the temperature is adjusted to 30 ℃ and induction is carried out by adding IPTG to a final concentration of 0.05 mM. Adjusting the pH value to about 7 with ammonia water every 12h, and stopping fermentation 48h after primary induction.

EXAMPLE 3 fermentative production of acetylacetone

The recombinant bacterium 2 of interest obtained in example 1 was spread on an LB solid plate containing 100. mu.g.mL-1 chloramphenicol; the coated plate was placed in a 37 ℃ incubator and cultured until single colonies grew.

Activating the obtained engineering strain monoclonal in LB culture to obtain seed liquid, inoculating the seed liquid into 500mL baffle shake flask containing 100mL basic improved liquid culture medium (containing 100. mu.g.mL-1 chloramphenicol) according to the volume ratio of the seed liquid to the culture medium of 1:100, and performing shake culture at 37 ℃ and 180 rpm. OD₆₀₀When about 0.6 ℃ is reached, the temperature is adjusted to 30 ℃ and induction is carried out by adding IPTG to a final concentration of 0.05 mM. Adjusting the pH value to about 7 with ammonia water every 12h, and stopping fermentation 48h after primary induction.

Detection of the product (examples 2 and 3, respectively)

After fermentation, centrifuging 200ml fermentation liquor, collecting supernatant, extracting with diethyl ether at a ratio of 1:1, pouring the mixed solution into a separating funnel, standing, separating the upper diethyl ether layer, and adding anhydrous CaCl₂Dried and filtered through a 0.22 μm filter and the filtrate collected. And (3) qualitatively detecting the filtrate by using a liquid chromatography-mass spectrometer, and verifying that acetylacetone exists in the fermentation product.

Centrifuging the fermentation liquid at 10000rpm for 2min, filtering the supernatant with 0.22 μm water phase filter membrane to obtain a liquid phase automatic sample bottle, and quantitatively detecting with high performance liquid chromatography. The detection method comprises the following steps: agilent 1200HPLC, column: biorad Aminex HPX-87H, mobile phase: 5mM H2SO4, flow rate: 0.6ml/min, working temperature: 60 ℃, detector: 280nm UV detector.

Example 2 the product yield was found to be 35 mg/L; example 3 the final yield was found to be 53mg/L, 1.5 times that of example 2.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

SEQUENCE LISTING

<110> institute of bioenergy and Process in Qingdao, China academy of sciences

<120> recombinant bacterium for producing acetylacetone, construction method and application thereof

<130>

<160> 7

<170> PatentIn version 3.5

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<213> codon-optimized gene dke1 encoding acetylacetone lyase

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<213> example 11) primer 1

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<213> example 11) primer 2

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cggaattctt aagcagcttc gtttttggta gc 32

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<213> example 12) primer 1

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cataaggaga taatggaact gacgactcgc ac 92

<210> 5

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<213> example 12) primer 2

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agcgtcgact tacttcagac ggtccgcgag 30

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<213> example 13) primer 1

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ggggtaccat gcgacatcct ttagtgatg 29

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<213> example 13) primer 2

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ccgctcgagt taagcctgtt tagccgcttc 30

Claims

1. A recombinant bacterium for producing acetylacetone, which is characterized in that the recombinant bacterium overexpresses a gene encoding acetylacetone lyasedke1Methylglyoxal synthase genemgsAAnd triose phosphate isomerase genetpiAThe starting strain is Escherichia coli; the gene for coding the acetylacetone lyasedke1Gene derived from Acinetobacter johnsonii, methylglyoxal synthasemgsAGene of triose phosphate isomerase derived from Escherichia colitpiAFrom Escherichia coli; the gene of the acetylacetone lyasedke1Is a sequence synthesized after codon optimization, and the sequence is shown as SEQ ID NO. 1; gene of methylglyoxal synthasemgsAHas a Gene ID of 945574; gene of triose phosphate isomerasetpiAHas a Gene ID of 948409; the construction method of the recombinant strain comprises the following steps: 1) cloning of the Acetylacetone lyase Genedke1The obtained gene is cut and connected with plasmid pACYCDuet-1, and then the obtained gene is transferred intoE.coliScreening positive clones from DH5 alpha competent cells, extracting plasmids to obtain a recombinant vector pACYCDuet-dke1;

2) Cloning of the Gene of methylglyoxal synthasemgsAThe obtained gene and the vector pACYCDuet obtained in the step 1) -dke1Performing enzyme digestion, ligation and transformationE.coliScreening positive clones from DH5 alpha competent cells, extracting plasmids to obtain a recombinant vector pACYCDuet-dke1-mgsA;

3) Cloning of triose phosphate isomerase GenetpiAThe obtained gene and the vector pACYCDuet obtained in step 2)dke1-mgsAPerforming enzyme digestion, ligation and transformationE.coliScreening positive clones from DH5 alpha competent cells, extracting plasmids to obtain a recombinant vector pACYCDuet-dke1-mgsA-tpiA;

4) Transforming the recombinant vector obtained in step 1) or step 3) into a hostEscherichia coliBL21(DE3) competent cells to obtain recombinant Escherichia coli.

2. The application of the recombinant bacterium of claim 1 in the production of acetylacetone by fermentation, which is characterized by comprising the following steps:

1) activating the recombinant bacteria to obtain a seed solution;

3. Use according to claim 2, characterized in that the culture conditions of step 2) are: 35-37 ℃ and 180-220 rpm.

4. The use according to claim 2, wherein the carbon source of the fermentation medium of step 2) is glucose and the nitrogen source is an inorganic nitrogen source; the nitrogen source is one or two of ammonium chloride or ammonium sulfate; the inoculation proportion of the seed liquid is that the volume ratio of the seed liquid to the fermentation medium is (1-2) to (100-130).

5. The use according to claim 2, wherein the inducer of step 3) is isopropyl- β -D-thiogalactoside (IPTG) added at a final concentration of 0.01 to 0.1 mM.

6. Use according to claim 2, characterized in that: the culture conditions of the step 3) are 30-33 ℃ and 180-220 rpm.