CN112553221B

CN112553221B - Bifunctional thiolase gene MvaE, expression vector, recombinant strain and application thereof

Info

Publication number: CN112553221B
Application number: CN202011258653.1A
Authority: CN
Inventors: 周佳; 田宝霞; 李相前
Original assignee: Huaiyin Institute of Technology
Current assignee: Yangzhou Mataris Biotechnology Co ltd
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2023-01-31
Anticipated expiration: 2040-11-11
Also published as: CN112553221A

Abstract

The invention discloses a bifunctional thiolase geneMvaE. The invention also discloses a bifunctional thiolase, an expression cassette, a recombinant vector, a recombinant bacterium or cell and application thereof. The invention also discloses a method for producing fatty acid. The difunctional thiolase gene MvaE can utilize rich culture medium containing glucose, glycerol or other carbon sources as raw materials under the facultative anaerobic condition, so that the C content in host bacteria can be remarkably increased _14:0 ，C _16:0 And C _18:0 The fatty acid content, the magnitude of increase varies from 27% to 213% depending on the host strain.

Description

Bifunctional thiolase gene MvaE, expression vector, recombinant strain and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a bifunctional thiolase gene MvaE, an expression vector, a recombinant strain and application thereof.

Background

Fatty acids are important platform chemicals that can be widely used in the synthesis of biofuels, organic detergents, rubber compounds, plastic products, textile chemicals, and food additives. The traditional fatty acid acquisition routes include direct extraction and separation from animal and plant cells and chemical synthesis from petroleum products. The methods have the defects of low efficiency, high cost, high land occupancy rate, unfriendly environment and the like. Compared with the conventional method, the microbial synthesis method of fatty acid has inherent advantages in cost, land occupancy rate and environmental protection, and is considered to be the most potential method capable of replacing the conventional method. However, the key technology which restricts the microbial synthesis efficiency of fatty acid is not completely solved. The yield and the type of the microbial synthesized fatty acid are closely related to the optimization of a fatty acid metabolic pathway and the catalytic property of key enzyme.

Biosynthesis of fatty acids is achieved primarily by two metabolic pathways, namely through the forward synthesis of fatty acids and the reverse cycle of fatty acid beta-oxidation. Some thiolases can promote a reverse cycle of beta-oxidation of fatty acids, causing the product of beta-oxidation of fatty acids, acetyl-coa, to flow back along the beta-oxidation pathway of fatty acids in a reverse direction to fatty acid formation. Studies have shown that different types of thiolases can affect the efficiency of fatty acid synthesis and the extension length of the carbon chain. Endogenous thiolase, dehydrogenase and a series of exogenous thioesterases are co-expressed in escherichia coli, so that reverse cycle synthesis of fatty acid and fatty alcohol with different chain lengths can be promoted by fatty acid beta-oxidation, and short-chain enrichment is realized. The synthesized thiolase and the fatty acid synthetase in the forward path are co-expressed to enhance the reduction reaction in the beta-oxidation reverse cycle and accelerate the synthesis of fatty acid. The yield of fatty acid is influenced by various factors, wherein the selection of key enzyme and the synergistic expression of various fatty acid metabolism related genes play an important role in improving the synthesis efficiency of fatty acid and regulating the length of a carbon chain synthesized by fatty acid.

Disclosure of Invention

The invention aims to: in order to solve the defects of the prior art, the invention provides the bifunctional thiolase gene MvaE.

The technical problem to be solved by the invention is to provide the bifunctional thiolase.

The invention also aims to solve the technical problem of providing an expression cassette, a recombinant vector, a recombinant bacterium or a cell, which contains the bifunctional thiolase gene MvaE.

The invention also aims to solve the technical problem of providing the application of the bifunctional thiolase gene MvaE, the bifunctional thiolase, the expression cassette, the recombinant vector, the recombinant bacterium or the cell in fatty acid synthesis.

The invention finally solves the technical problem of providing a method for producing fatty acid.

The technical scheme is as follows: in order to solve the technical problems, the invention provides a bifunctional thiolase gene MvaE, wherein the nucleotide sequence of the bifunctional thiolase gene MvaE is as follows:

i) The amino acid sequence of SEQ ID NO:1 or the nucleotide sequence shown in SEQ ID NO: 2; or

ii) SEQ ID NO:1 or SEQ ID NO:2 is substituted, deleted and/or added with one or more nucleotides and has the same function; or

iii) A nucleotide sequence having 90% or more homology with the nucleotide sequence of i) and ii) and having the same function.

The invention also comprises a bifunctional thiolase, the amino acid sequence of which is shown in SEQ ID NO:3, respectively.

The invention also comprises an expression cassette, a recombinant vector, a recombinant bacterium or a cell, which contains the bifunctional thiolase gene MvaE.

Wherein the recombinant vector is obtained by introducing the bifunctional thiolase gene MvaE into a vector plasmid pSTV 28.

The recombinant strain is a recombinant strain containing expressible MvaE, wherein the recombinant strain is obtained by introducing the bifunctional thiolase gene MvaE into host bacteria, and screening and identifying the host bacteria and then retaining a positive strain.

Wherein, the host bacteria include but are not limited to Escherichia coli MG1655.

The invention also comprises the application of the bifunctional thiolase gene MvaE, the bifunctional thiolase, the expression cassette, the recombinant vector, the recombinant bacterium or the cell in fatty acid synthesis.

The invention also discloses a method for producing fatty acid, which is obtained by inoculating the recombinant bacterium into a fermentation culture medium for fermentation culture.

Wherein the fermentation temperature is 25-38 ℃; the initial pH value of the fermentation system is 6.5-7.5; the carbon source in the fermentation medium is one or more of glycerol, glucose and starch; the nitrogen source in the fermentation medium is one or more of yeast powder, peptone, ammonia water, ammonium salt and urea.

Wherein the fermentation medium comprises glycerol, peptone, yeast powder and sodium chloride, and is dissolved in MOPS buffer solution with pH of 7.0-7.5.

The recombinant strain can over-express the difunctional thiolase gene MvaE,thereby remarkably improving C in host bacteria _14：0 ，C _16：0 And C _18：0 Fatty acid content.

Has the advantages that: compared with the prior art, the invention has the following advantages: the over-expressed artificially designed bifunctional thiolase gene MvaE can utilize rich culture medium containing glucose, glycerol or other carbon sources as raw materials under the condition of facultative anaerobism, and can remarkably improve C content in host bacteria _14：0 ，C _16：0 And C _18：0 The fatty acid content, the increase range of which varies from 27% to 213% depending on the host strain.

Drawings

FIG. 1 is a graph of the effect of MvaE on the colony morphology of E.coli MG 1655;

FIG. 2 is an Anthony capsular staining experiment;

FIG. 3 shows Sudan black staining experiment.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples using Escherichia coli as a host bacterium, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: artificial design and synthesis of bifunctional thiolase gene MvaE based on codon preference of escherichia coli

According to SEQ ID NO:1 the MvaE gene codon is optimized to facilitate its expression in E.coli. The optimized nucleic acid sequence is shown as SEQ NO:2, respectively. The gene sequence after codon optimization is synthesized by the company Limited in the Biotechnology engineering (Shanghai), and the 5 'end and the 3' end of the synthesized gene sequence are respectively provided with BamH I restriction enzyme cutting sites and Hind III restriction enzyme cutting sites.

Example 2: construction of expression vector pSTmvAE for expression of MvaE Gene in Large intestine rods

The peptide has the sequence shown in SEQ NO:2 on an expression vector, such as the pSTV28 vector. The specific implementation steps are as follows:

the synthetic DNA fragment of example 1 and the vector plasmid pSTV28 were double digested with BamH I and Hind III, respectively; and (3) recovering the enzyme-cut DNA fragment and the large vector fragment by agarose electrophoresis, and connecting the enzyme-cut gene fragment to a corresponding enzyme-cut product of pSTV28 at 16 ℃ sequentially by using DNA ligase to form pSTmvAE. Each ligation product was used to transform E.coli MG1655, which was plated on a petri dish containing 30. Mu.g/mL of Cmp (chloramphenicol) and incubated at 37 ℃ for 10-12h. And selecting a single colony on the next day, and extracting plasmids by using a Shanghai biological medium particle small quantity extraction kit. And carrying out double enzyme digestion verification on the obtained plasmid and sequencing the obtained recombinant plasmid. The results show that the pSTmvAE plasmid was successfully constructed.

Example 3 construction of recombinant Escherichia coli overexpressing bifunctional thiolase Gene MvaE

The pSTV28 and pSTmvae plasmids in example 2 were respectively transformed into Escherichia coli MG1655 to respectively obtain engineered Escherichia coli MG: pSTV28 and pSMG: pSTmvae.

Example 4: fermentation culture of recombinant Escherichia coli

The engineered strain MG obtained in example 3: pSTV28 and pSMG: positive single colonies of pSTmvae were inoculated into 5mL LB tubes containing 30. Mu.g/mL chloramphenicol for 8 hours at 37 ℃ and 50mL of fermentation medium containing 30. Mu.g/mL chloramphenicol at an inoculum size of 1% (v/v).

Fermentation medium formula (1L): 2g glycerol, 10g peptone, 10g yeast powder and 5g sodium chloride were dissolved in 800ml 10mM MOPS buffer, pH7.0-7.5 and finally made up to 1000ml with 10mM MOPS.

And (3) inoculating the engineering bacteria MG of the escherichia coli in a shaker at 30 ℃ and 250 rpm: pSTV28 and pSMG: fermentation of pSTmvae culture to OD ₆₀₀ Isopropyl beta-D-thiogalactoside (IPTG) was added at 0.4-0.6 to a final concentration of 0.1mM as an inducer and incubated under these conditions for 24 hours for sampling for subsequent cell concentration and fatty acid component detection (see example 5 for the results of specific fatty acid component detection).

Example 5: effect of overexpression of MvaE on colony morphology of Escherichia coli MG1655 and enrichment of neutral oil

The colony morphology of the escherichia coli recombinant expression strain of the bifunctional thiolase MvaE is remarkably changed compared with that of a control group which does not express the enzyme, and the colony morphology is in an oil drop shape or a viscous liquid shape (figure 1A) to show a sample application result; fig. 1B shows the scribing result.

An Anthony capsular staining experiment was performed on the MvaE recombinant expression strain and the control group (fig. 2). The specific experimental steps are as follows: a negative dyeing method: (1) tabletting: taking a clean glass slide, adding a drop of distilled water, taking a small amount of thalli, putting the thalli into the drop of distilled water, uniformly mixing and coating; and (2) drying: drying the smear in air or blowing dry with cold air; (3) dyeing: adding red dye solution on the coated surface for dyeing for 2-3min; (4) washing with water: washing with water to remove red dye liquor; (5) drying: drying the dyed piece by air or blowing by electric blowing cold air; (6) melanin coating: adding a small drop of melanin on the left side of the dyeing coating surface, lightly contacting the melanin with a slide glass with a smooth edge to disperse the melanin along the edge of the slide glass, dragging the melanin to the right to form a thin layer on the dyeing coating surface, and quickly drying the melanin in the air; (7) microscopic examination: observing with a low power lens and then with a high power lens.

The results show that no capsule formation was found, thus excluding the possibility that oiling or sliming of the colonies was caused by capsule formation.

And measuring the content of neutral lipid in the saccharomyces cerevisiae by taking the sudan black B as a coloring agent. The yeast cells after induction are prepared in 70% ethanol for 10min by using 0.1% Sudan black B solution, and then are washed for more than 3 times by using the 70% ethanol to prepare a sample slide for microscopic detection. Results of sudan black B staining, a liposoluble dye, showed that the intracellular concentration of sudan black in the MvaE recombinant expression strain was significantly greater than that in the control group (fig. 3).

Example 6: c of E.coli MG1655 by overexpression of MvaE _14：0 、C _16：0 And C _18：0 Gas chromatography detection of fatty acid content

The extraction and esterification method of the fatty acid of the escherichia coli comprises the following steps:

(1) Taking 4mL of the liquid fermentation liquid obtained by the fermentation method of the embodiment 4, centrifuging at 8000 rpm for 15min, removing the supernatant, adding 1mL of deionized water for resuspension, adding 100 μ L of acetic acid for acidification, (using a 15mL plastic tube);

(2) 3mL of chloroform-methanol (1: 1 by volume) was lysed and mixed well with shaking every 10 minutes at room temperature for 2 hours.

Standing for 2 hours at room temperature;

(3) 0.5mL of the lower organic phase was placed in a fresh 1.5mL centrifuge tube with a lid and vacuum dried (longer time, overnight);

(4) The dried organic phase was resuspended in 300. Mu.L of 5% sulfuric acid/methanol (5% sulfuric acid in methanol; vol/vol) solution. The containers were used as 1.5ml centrifuge tubes with lids. Carrying out warm bath at 60 ℃ for 4h (60 ℃ oven);

(5) Adding 300 mu L of 0.9% (wt/vol) NaCl solution into the reaction mixed solution, shaking and uniformly mixing;

(6) Extracting fatty acid methyl ester with 600 μ L of dichloromethane (shaking and mixing every 10 minutes, room temperature 1 hr);

(7) Transferring the lower dichloromethane layer of 300-400 mu L to a gas chromatography glass bottle for gas chromatography detection.

The operation method of the gas chromatograph comprises the following steps: (1) And (3) temperature programming, setting the initial temperature to be 40 ℃, the retention time to be 3min, heating to 120 ℃ at the speed of 10 ℃/min, heating to 230 ℃ at the speed of 20 ℃/min, and the retention time to be 2min. (2) Then setting the temperature of a sample inlet to be 250 ℃, the temperature of an FID detector to be 280 ℃, the sample injection volume to be 0.2 mu L and the split ratio to be 25: 1, after setting, putting the gas chromatographic columns in the gas chromatograph in sequence, and clicking start to start operation.

The gas chromatography detection result shows that C _14：0 ，C _16：0 And C _18：0 Total fatty acid content was 78% higher than that of the empty plasmid control group (Table 1), further confirming that MvaE overexpresses C in E.coli _14：0 ，C _16：0 And C _18：0 The fatty acid content is increased to various degrees.

TABLE 1 gas chromatographic determination of the intracellular content of the three major fatty acids of E.coli ^a

a. Each strain was cultured in LB liquid medium without induction by an inducer (using leaky expression) for 24 hours.

b. A fatty acid.

c. Strain MG: pSTmvae to MG: increase in the fatty acid content of pSTV 28.

d. The increase rate of the fatty acid content; (Δ FA/MG: pSTV 28) 100.

e. Here the total amount of fatty acids is represented by C _14：0 ，C _16：0 And C _18：0 Sum of fatty acid content.

Sequence listing

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

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Ala Ala Gln Phe Leu Ala Val Gly Lys Asn Leu Gly Phe Asp Ser Glu

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Gly Gly Gly Ser Met Ser Pro Asp Glu Arg Leu Gly Ser Leu Leu Gln

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Leu Ser Gln Gly Gly His Leu Ala Asn Gly Phe Lys Thr Val Asn Gln

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Arg Asp Leu Gln Tyr Arg Ala Phe Asp Glu Ser Arg Val Ser Val Asp

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Lys Ile Leu Phe Ser Ile Leu Ser Asn Tyr Ala Thr Glu Ser Val Val

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

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Gly Arg Glu Ile Ala Glu Lys Ile Val Leu Ala Ser Arg Tyr Ala Ser

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Leu Asp Pro Tyr Arg Ala Val Thr His Asn Lys Gly Ile Met Asn Gly

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Ile Glu Ala Val Val Leu Ala Thr Gly Asn Asp Thr Arg Ala Val Ser

660 665 670

Ala Ser Cys His Ala Phe Ala Val Lys Glu Asp Arg Tyr Gln Gly Leu

675 680 685

Thr Ser Trp Asn Leu Asp Gly Glu Gln Leu Ile Gly Glu Ile Ser Val

690 695 700

Pro Leu Ala Leu Ala Thr Val Gly Gly Ala Thr Lys Val Leu Pro Lys

705 710 715 720

Ser Gln Ala Ala Ala Asp Leu Leu Ala Val Thr Asp Ala Lys Glu Leu

725 730 735

Ser Arg Val Val Ala Ala Val Gly Leu Ala Gln Asn Leu Ala Ala Leu

740 745 750

Arg Ala Leu Val Ser Glu Gly Ile Gln Lys Gly His Met Ala Leu Gln

755 760 765

Ala His Pro Ile Ala Met Thr Val Gly Ala Thr Gly Lys Glu Val Glu

770 775 780

Ala Val Ala Gln Gln Leu Lys Arg Gln Lys Thr Met Asn Gln Asp Arg

785 790 795 800

Ala Leu Ala Ile Leu Asn Asp Leu Arg Lys Gln

805 810

Claims

1. Difunctional thiolase geneMvaE，Characterized in that the bifunctional thiolase geneMvaEThe nucleotide sequence of (A) is the nucleotide sequence shown in SEQ ID NO. 1 or the nucleotide sequence shown in SEQ ID NO. 2.

2. The bifunctional thiolase is characterized in that the amino acid sequence of the bifunctional thiolase is shown as SEQ ID NO 3.

3. An expression cassette, recombinant vector or recombinant bacterium comprising the bifunctional thiolase gene of claim 1MvaE。

4. The recombinant vector according to claim 3, wherein the recombinant vector is a bifunctional thiolase geneMvaEThe vector plasmid pSTV28 was introduced.

5. The recombinant bacterium according to claim 3, wherein the recombinant bacterium is a bifunctional thiolase geneMvaEIntroducing into host bacteria, screening and identifying, and retaining positive strain as expressible strainMvaEThe recombinant strain of (1).

6. The recombinant bacterium according to claim 5, wherein the host bacterium is Escherichia coli MG1655.

7. The bifunctional thiolase gene as claimed in claim 1MvaEThe bifunctional thiolase of claim 2, the expression cassette, the recombinant vector, or the recombinant bacterium of claim 3, for use in fatty acid synthesis.

8. A method for producing fatty acids, comprising inoculating the recombinant bacterium according to claim 5 or 6 to a fermentation medium and carrying out fermentation culture.

9. The method for producing fatty acids according to claim 8, wherein the fermentation temperature is 25-38 ℃; the initial pH value of the fermentation system is 6.5-7.5; the carbon source in the fermentation medium is one or more of glycerol, glucose and starch; the nitrogen source in the fermentation medium is one or more of yeast powder, peptone, ammonia water, ammonium salt and urea.