CN110951664B - Recombinant corynebacterium glutamicum and application thereof in production of 2-pyrrolidone - Google Patents
Recombinant corynebacterium glutamicum and application thereof in production of 2-pyrrolidone Download PDFInfo
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Abstract
The invention discloses a recombinant corynebacterium glutamicum and application thereof in production of 2-pyrrolidone, and belongs to the technical field of biology. The invention provides a recombinant Corynebacterium glutamicum Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act capable of highly producing 2-pyrrolidone, and the recombinant Corynebacterium glutamicum Corynebacterium/pXMJ 19/delta argB-gad-N2-act is inoculated into a fermentation medium for fermentation for 72 hours, so that the yield of 2-pyrrolidone in fermentation liquor can reach 8 +/-0.3 g/L.
Description
Technical Field
The invention relates to a recombinant corynebacterium glutamicum and application thereof in production of 2-pyrrolidone, and belongs to the technical field of biology.
Background
2-Pyrrolidone (2-Pyrrolidone), also known as Pyrrolidone, azavalerone, butyrolactam, can be used as a precursor for a variety of compounds such as nylon, polyvinylpyrrolidone (PVP), and vinylpyrrolidone, and has many industrially important applications.
Currently, 2-pyrrolidone is industrially produced by a chemical method. The chemical method is mainly characterized in that 1, 4-butanediol is dehydrogenated to obtain gamma-butyrolactone, and then the aqueous solution of the gamma-butyrolactone reacts with ammonia to obtain the 2-pyrrolidone. However, the chemical method for producing 2-pyrrolidone has the defects of high energy consumption, complex process, harsh reaction conditions, serious pollution, low safety and the like, and is not suitable for the environment which limits the development of high energy consumption industries and gradually implements the policy of energy conservation and emission reduction in China.
Attempts have also been made to produce 2-pyrrolidone biologically. The biological method is mainly to obtain the 2-pyrrolidone by the mild fermentation of an ORF27 gene in heterologous expression source Streptomyces aizunensis and L-glutamic acid as a substrate. Compared with a chemical method, the biological method has the advantages of safety, green, sustainable development and the like. However, the existing biological methods still have certain defects, for example, Zhang, J. (2016) and others construct a recombinant Escherichia coli capable of producing 2-pyrrolidone, and the recombinant Escherichia coli is fermented for 31h, so that the content of 2-pyrrolidone in the fermentation broth can only reach 1.1g/L, and the yield is too low (see references "Zhang, J., Kao, E., Wang, G., Baidoo, E.E.K., Chen, M., and Keasling, J.D (2016) metabolism engineering of Escherichia coli for the biosynthesis of 2-pyrollidone, MetEng Commun 3, 1-7.").
The above-mentioned defects make the existing biological method unable to really realize the large-scale industrial production of 2-pyrrolidone, and therefore, it is urgently needed to find a microorganism capable of producing 2-pyrrolidone with high yield to overcome the above-mentioned defects.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to provide the recombinant corynebacterium glutamicum capable of producing 2-pyrrolidone in a high yield.
[ solution ]
In order to solve the technical problems, the invention provides a recombinant corynebacterium glutamicum which takes corynebacterium glutamicum as a host, knocks out a gene coding N-acetyl glutamate kinase, and expresses a gene coding glutamate decarboxylase and a gene coding beta-alanine CoA transferase.
In one embodiment of the invention, the upstream of the gene coding for beta-alanine CoA transferase is connected with a regulatory gene with a nucleotide sequence shown as SEQ ID No. 1.
In one embodiment of the invention, the nucleotide sequence of the gene encoding N-acetylglutamate kinase is shown in SEQ ID No. 2.
In one embodiment of the invention, the gene encoding glutamate decarboxylase is derived from Escherichia coli (Escherichia coli).
In one embodiment of the invention, the nucleotide sequence of the gene encoding glutamate decarboxylase is shown as SEQ ID No. 3.
In one embodiment of the invention, the gene encoding beta-alanine CoA transferase is derived from clostridium propionicum (clostridium propionicum).
In one embodiment of the invention, the nucleotide sequence of the gene encoding beta-alanine CoA transferase is shown in SEQ ID No. 4.
In one embodiment of the present invention, the recombinant corynebacterium glutamicum uses corynebacterium glutamicum as a host, knockouts a gene encoding N-acetylglutamate kinase, and uses a pET-28a (+) plasmid, a pXMJ19 plasmid, a pET-24b (+) plasmid, and/or a pDXW-10 plasmid as a vector to express a gene encoding glutamate decarboxylase and a gene encoding beta-alanine CoA transferase.
In one embodiment of the present invention, the recombinant corynebacterium glutamicum uses corynebacterium glutamicum as a host, knockouts a gene encoding N-acetylglutamate kinase, and uses pXMJ19 plasmid as a vector to express a gene encoding glutamate decarboxylase and a gene encoding beta-alanine CoA transferase.
In one embodiment of the invention, the gene encoding glutamate decarboxylase is located upstream of the gene encoding beta-alanine CoA transferase.
In one embodiment of the invention, the regulatory gene is located downstream of the gene encoding glutamate decarboxylase and upstream of the gene encoding beta-alanine CoA transferase.
In one embodiment of the invention, the Corynebacterium glutamicum is Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032.
The invention also provides a method for producing 2-pyrrolidone, which comprises the steps of inoculating the recombinant corynebacterium glutamicum into a culture medium containing glucose, fermenting at the temperature of 28-35 ℃, the rotating speed of 100-250 rpm and the pH of 6-8 to obtain fermentation liquor containing 2-pyrrolidone, and extracting the fermentation liquor containing 2-pyrrolidone to obtain the 2-pyrrolidone.
In one embodiment of the present invention, the concentration of glucose in the culture medium is 30 to 130 g/L.
The invention also provides a section of regulatory gene, and the nucleotide sequence of the regulatory gene is shown in SEQ ID No. 1.
The invention also provides a recombinant plasmid which carries the regulatory gene and the target gene.
In one embodiment of the present invention, the recombinant plasmid is pET-28a (+) plasmid, pXMJ19 plasmid, pET-24b (+) plasmid or pDXW-10 plasmid as a vector.
In one embodiment of the present invention, the recombinant plasmid is pXMJ19 plasmid as a vector.
In one embodiment of the invention, the gene of interest is a gene encoding glutamate decarboxylase and/or a gene encoding beta-alanine CoA transferase.
In one embodiment of the present invention, the genes of interest are a gene encoding glutamate decarboxylase and a gene encoding beta-alanine CoA transferase.
In one embodiment of the invention, the regulatory gene is located downstream of the gene encoding glutamate decarboxylase and upstream of the gene encoding beta-alanine CoA transferase.
The invention also provides a host cell, which carries the regulatory gene and the target gene, or carries the recombinant plasmid.
In one embodiment of the invention, the gene of interest is a gene encoding glutamate decarboxylase and/or a gene encoding beta-alanine CoA transferase.
In one embodiment of the present invention, the genes of interest are a gene encoding glutamate decarboxylase and a gene encoding beta-alanine CoA transferase.
In one embodiment of the invention, the regulatory gene is located downstream of the gene encoding glutamate decarboxylase and upstream of the gene encoding beta-alanine CoA transferase.
In one embodiment of the invention, the host cell is a bacterial, fungal or animal cell.
In one embodiment of the invention, the host cell is Corynebacterium glutamicum (Corynebacterium glutamicum)
In one embodiment of the invention, the host cell is Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032.
The invention also provides the application of the recombinant corynebacterium glutamicum, the method, the regulatory gene, the recombinant plasmid or the host cell in the production of 2-pyrrolidone.
[ advantageous effects ]
(1) The invention provides a recombinant Corynebacterium glutamicum Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act capable of highly producing 2-pyrrolidone, and the recombinant Corynebacterium glutamicum Corynebacterium/pXMJ 19/delta argB-gad-N2-act is inoculated into a fermentation medium for fermentation for 72 hours, so that the yield of 2-pyrrolidone in fermentation liquor can reach 8 +/-0.3 g/L.
(2) The invention provides a recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act capable of highly producing 2-pyrrolidone, and when the recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act is used for producing 2-pyrrolidone by fermentation, cheap glucose can be used as a substrate, and the cost is low.
(3) The invention provides a regulatory gene capable of improving the yield of recombinant bacteria 2-pyrrolidone, which is characterized in that recombinant Corynebacterium glutamicum Glutamicum/pXMJ 19/delta argB-gad-N2-act containing the regulatory gene is inoculated into a fermentation culture medium for fermentation for 72 hours, so that the yield of 2-pyrrolidone in fermentation liquor can reach 8 +/-0.3 g/L, and is improved by 45.2% compared with the recombinant Corynebacterium glutamicum Glutamicum/pXMJ 19/delta argB-gad-act containing no regulatory gene.
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FIG. 1: PCR verification results of different Corynebacterium glutamicum; wherein, M: DL2000 Marker, 1: wild-type corynebacterium glutamicum, 2: recombinant Corynebacterium glutamicum Glutamicum/delta argB.
FIG. 2: fluorescence intensity/OD in fermentation broth obtained by fermentation of different corynebacterium glutamicum600The ratio of (A) to (B); wherein, CK: recombinant Corynebacterium glutamicum/pXMJ19/gfp, NCS 2: recombinant Corynebacterium glutamicum/pXMJ19/gfp-N2, NCS 28: recombinant Corynebacterium glutamicum/pXMJ 19/gfp-N28, NCS 38: recombinant Corynebacterium glutamicum Glutamicum/pXMJ 19/gfp-N38.
FIG. 3: the contents of glucose, L-glutamic acid, gamma-aminobutyric acid and 2-pyrrolidone in fermentation liquor obtained by fermenting recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act at different times and the OD of the fermentation liquor obtained by fermenting recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act at different times600The value is obtained.
Detailed Description
Coli (Escherichia coli) BL21 referred to in the examples below was purchased from North Nay organisms; the plasmids Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 and pXMJ19 mentioned in the examples were obtained from Bio-wind corporation; the pK18mobsacB plasmids referred to in the examples below were purchased from Youbao organisms; tween-80, isoniazid, gamma-aminobutyric acid (standard), 2-pyrrolidone (standard), sodium hexane sulfonate, corn steep liquor and FeSO4 & 7H2O referred to in the following examples were purchased from Shanghai Allantin Biotech Co., Ltd.
The media involved in the following examples are as follows:
LB liquid medium: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract.
LB solid medium: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract and 15g/L of agar.
Brain heart immersion liquid culture medium: brain heart infusion 37.5g/L (purchased from Haibo Bio Inc.).
Brain heart soaking solid medium: 37.5g/L brain heart infusion and 15g/L agar.
Competent medium: 37.5g/L brain-heart infusion, 801g/L tween-801 g/L, 3g/L glycine and 0.4g/L isoniazid.
Seed culture medium: glucose 25g/L, K2HPO4·3H201.5 g/L、MgSO40.6 g/L, 30g/L corn steep liquor and 2.5g/L, FeSO g urea4·7H2O 0.005g/L、MnSO4·4H2O0.005 g/L and pH 7.0.
Fermentation medium: glucose 140g/L, K2HPO4·3H201g/L、MgSO40.6 g/L, 5g/L corn steep liquor and 7g/L, FeSO g urea4·7H2O 0.002g/L、MnSO4·4H2O0.002 g/L and pH 7.0.
non-anti-LBG medium: 5g/L of glucose, 5g/L of yeast extract, 10g/L of sodium chloride and 10g/L of tryptone.
The detection methods referred to in the following examples are as follows:
and (3) determination of the content of glucose and L-glutamic acid: adopting a Bio-SBA bioanalyzer to analyze, absorbing 25 mu L of glucose or L-glutamic acid standard solution for calibration, after the calibration is finished, taking 1mL of fermentation liquor for dilution by 100 times, absorbing 25 mu L of diluted fermentation liquor for determination, and recording data.
And (3) determining the content of gamma-aminobutyric acid and arginine: analysis was performed by OPAFMOC pre-column derivatization HPLC method, where pretreatment of the sample: protein in the fermentation liquor needs to be removed, the fermentation liquor is subjected to membrane filtration to remove particle impurities, and the detection results are accurate when the concentrations of the gamma-aminobutyric acid standard substance and the amino acid in the fermentation liquor to be detected are controlled within 0.5 g/L; chromatographic conditions are as follows: c18 chromatographic column, the column temperature is controlled at 40 ℃, the flow rate is 1.0mL/min, and the detection is carried out under an ultraviolet detector with the wavelength of 338 nm; preparation of OPA derivative: weighing 10mg OPA (o-phthalaldehyde), adding 0.5mL methanol for dissolving, adding 2mL 0.05M boric acid buffer solution and 50 mu L beta-mercaptoethanol for uniformly mixing, storing at low temperature in a dark place, and optimally using in situ; derivatization process (either pre-column derivatization or on-line derivatization using an amino wheel liquid chromatograph, with on-line derivatization used in the examples below): 200 mu L of OPA derivative agent and 400 mu L of sample are mixed evenly, and then 400 mu L of 0.1M potassium dihydrogen phosphate solution is added to stabilize the system; mobile phase: phase A: weighing 8.0g of crystalline sodium acetate in a 1000mL beaker, adding 1000mL of water, stirring until all the crystalline water is dissolved, then adding 225 μ L of triethylamine, stirring, dropwise adding 5% acetic acid, adjusting the pH value to 7.20 +/-0.05, adding 5mL of tetrahydrofuran, and mixing for later use; phase B: weighing 6.0g of crystalline sodium acetate in a 1L beaker, adding 200mL of water, stirring until all crystals are dissolved, dropwise adding 5% acetic acid, adjusting the pH to 7.20 +/-0.05, adding 400mL of acetonitrile and 400mL of methanol into the solution, and mixing for later use; mobile phase elution procedure (gradient elution): 0-27.5 min, A92% -40%, and the flow rate is 1 mL/min; 27.5-31.5 min, A40% -0%, and the flow rate is 1-1.5 mL/min; 31.5-32 min, A0% -0%, and the flow rate is 1.5 mL/min; 32-34 min, A0% -0%, and the flow rate is 1.5-1 mL/min; 34-35.5 min, A0% -92%, and the flow rate is 1.0 mL/min; 35.5-45 min, A92% and the flow rate is 1.0 mL/min.
Determination of 2-pyrrolidone content: HPLC analysis, wherein chromatographic conditions: a C18 chromatography column; mobile phase: potassium dihydrogen phosphate solution (potassium dihydrogen phosphate 10.0g and sodium hexanesulfonate 1.1g, dissolved in water and diluted to 1000mL, pH adjusted to 2.1 with phosphoric acid) -methanol (90: 10); flow rate: 1.0 mL/min; ultraviolet detection wavelength: 210 nm; the column temperature is 30 ℃; sample introduction amount: 10 μ L.
Example 1: construction of recombinant Corynebacterium glutamicum capable of producing 2-pyrrolidone
1. Deletion of Gene argB encoding N-acetylglutamate kinase
Designing knock-out primers argB-L-F, argB-L-R, argB-R-F and argB-R-R according to a gene argB of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032, which codes for N-acetyl glutamate kinase; using the genome of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 as a template and argBCarrying out PCR amplification by taking L-F, argB-L-R as a primer to obtain an amplification product 1; carrying out PCR amplification by taking the genome of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 as a template and argB-R-F, argB-R-R as a primer to obtain an amplification product 2; connecting the amplification products 1 and 2 by overlapping extension PCR by using argB-L-F, argB-R-R as a primer to obtain an argB knockout fragment; connecting the argB knockout fragment with pK18mobsacB plasmid after restriction enzyme digestion by restriction enzymes EcoR I and HindIII to obtain a connection product; transforming the ligation product into Escherichia coli (Escherichia coli) BL21 to obtain a transformation product; the transformation product was plated on LB solid medium (containing 50. mu.g.mL)-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 120-180 rpm, extracting plasmids for enzyme digestion verification and sequencing verification, and obtaining successfully transformed recombinant Escherichia coli/pK18mobsacB-argB and knockout plasmid pK18mobsacB-argB after verification is correct;
transforming Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 by electric shock on the knock-out plasmid pK18mobsacB-argB to obtain a transformation product; the transformation product was spread on brain heart infusion solid medium (containing 50. mu.g.mL)-1Kanamycin) is inversely cultured in a constant-temperature incubator at 30 ℃ for 36 hours to obtain a first-level transformant; selecting a first-stage transformant, inoculating the first-stage transformant to a non-resistance LBG culture medium, and performing inverted culture in a constant temperature incubator at 30 ℃ for 24 hours to obtain a second-stage transformant; inoculating the second-level transformant to a non-resistance LBG culture medium, and performing inverted culture in a constant temperature incubator at 30 ℃ for 24 hours to obtain a third-level transformant; the identification of the restored wild type/gene deletion type of the tertiary transformant is carried out (the identification method of the restored wild type/gene deletion type can be specifically referred to as the following documents:
A,TauchA,
W,et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum[J]gene,1994,145 (1: 69); extracting and identifying a correct genome of the tertiary transformant, and carrying out PCR identification by taking a gene argB for coding N-acetyl glutamate kinase as a target gene, wherein the recombinant Corynebacterium glutamicum/pXMJ 19/delta argB (the sequences can be shown in Table 1) can be obtained only if the complete target gene cannot be amplified.
Selecting a single colony of a recombinant Corynebacterium glutamicum Corynebacterium/pXMJ 19/delta argB to inoculate into a brain-heart infusion liquid culture medium by taking a wild Corynebacterium glutamicum ATCC 13032 as a control, and performing shake culture for 24 hours at 30 ℃ and 180r/min to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 5% (v/v), and fermenting for 72h at 30 ℃ and 180r/min to obtain a fermentation liquid; the content of arginine and L-glutamic acid in the fermentation liquor is detected, and the arginine can be hardly detected in the fermentation liquor, and the accumulation amount of the L-glutamic acid is improved to 28.4g/L from 25.6g/L of wild Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032. Therefore, the knockout of the gene argB of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032, which codes for N-acetylglutamate kinase, can enhance the accumulation of L-glutamic acid.
2. Expression of Gene gad encoding glutamate decarboxylase and Gene act encoding beta-alanine CoA transferase
Chemically synthesizing a gene gad (nucleotide sequence shown in SEQ ID No.3 and synthesized by Jinzhi Co., Ltd. Suzhou) encoding glutamate decarboxylase derived from Escherichia coli (Escherichia coli) and a gene act (nucleotide sequence shown in SEQ ID No.4 and synthesized by Jinzhi Co., Ltd. Suzhou) encoding beta-alanine CoA transferase derived from Clostridium propionicum (Clostridium propionicum); designing connection primers GAD-F, GAD-Act-R, Act-GAD-F and Act-R according to a gene GAD encoding glutamate decarboxylase and a gene Act encoding beta-alanine CoA transferase; GAD-F, GAD-Act-R is used as a primer, and the gene GAD for coding glutamate decarboxylase is subjected to PCR amplification to obtain an amplification product 1; uses Act-GAD-F and Act-R as primers to carry out PCR amplification on the gene Act for coding beta-alanine CoA transferase,obtaining an amplification product 2; using GAD-F, Act-R as a primer, connecting the amplification product 1 with the amplification product 2 through overlap extension PCR to obtain a target gene ad-act formed by connecting a gene GAD coding glutamate decarboxylase and a gene act coding beta-alanine CoA transferase; connecting the target gene ad-act and the pXMJ19 plasmid after being digested by restriction enzymes EcoR I and HindIII to obtain a connection product; transforming the ligation product into Escherichia coli (Escherichia coli) BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 10. mu.g.mL)-1Chloramphenicol), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 120-180 rpm, extracting plasmids, performing enzyme digestion verification and sequencing verification, and obtaining successfully transformed recombinant Escherichia coli/pXMJ 19/gad-act and recombinant plasmid pXMJ 19/gad-act after verification is correct;
transforming the recombinant plasmid pXMJ 19/gad-act into recombinant Corynebacterium glutamicum/pXMJ 19/delta argB by electric shock to obtain a transformation product; the transformation product was spread on a brain heart infusion solid medium (containing 10. mu.g.mL)-1Chloramphenicol), and performing inverted culture in a constant temperature incubator at 30 ℃ for 36h to obtain a transformant; streaking the transformant on a brain-heart infusion solid culture medium, and carrying out inverted culture in a constant-temperature incubator at 30 ℃ for 36h to obtain a single colony; PCR verification is carried out on a single colony (the PCR verification result is shown in figure 1), and the recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-act is obtained after the verification is correct (the sequences can be seen in the table 1).
Selecting a single colony of recombinant Corynebacterium glutamicum Corynebacterium/pXMJ 19/delta argB-gad-act by taking wild type Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032) as a control, inoculating the single colony into a brain-heart immersion liquid culture medium, and performing shake culture for 24 hours at 30 ℃ and 180r/min to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 5% (v/v), and fermenting for 72h at 30 ℃ and 180r/min to obtain a fermentation liquid; the content of the gamma-aminobutyric acid and the 2-pyrrolidone in the fermentation liquor is detected, and the accumulation amount of the gamma-aminobutyric acid in the fermentation liquor reaches 593 +/-6 mg/L, and the accumulation amount of the 2-pyrrolidone in the fermentation liquor reaches 124 +/-8 mg/L, which are respectively improved by 20.0 percent and 10.5 percent compared with the wild type. It can be seen that knocking out the gene argB coding for N-acetyl glutamate kinase of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 and expressing the gene gad coding for glutamate decarboxylase and the gene act coding for beta-alanine CoA transferase in Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 can enhance the accumulation of gamma-aminobutyric acid and 2-pyrrolidone.
TABLE 1 nucleotide sequences of primers and genes
Example 2: construction of recombinant Corynebacterium glutamicum capable of producing 2-pyrrolidone at high yield
1. Obtaining regulatory genes
The N-terminal coding sequence of green fluorescent protein was designated "AAAGGANNNNNNNN (regulatory gene NCS)"; chemically synthesizing a gene encoding green fluorescent protein whose N-terminal coding sequence is designated "AAAGGANNNNNNNN"; designing connection primers Ngfp-F and Ngfp-R according to a gene encoding green fluorescent protein with an N-terminal coding sequence of 'AAAGGANNNNNNNN'; taking Ngfp-F, Ngfp-R as a primer, and carrying out PCR amplification on a gene of green fluorescent protein with a coded N-terminal coding sequence designed to be AAAGGANNNNNNNN to obtain an amplification product; connecting the amplified product and the pXMJ19 plasmid after enzyme digestion by restriction enzymes EcoR I and HindIII to obtain a connection product; transforming the ligation product into Escherichia coli (Escherichia coli) BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting transformants, inoculating the transformants into an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ under the condition of 120-180 rpm, extracting plasmids, performing enzyme digestion verification and sequencing testVerifying that the recombinant Escherichia coli/pXMJ 19/Ngfp library and the recombinant plasmid pXMJ 19/Ngfp library which are successfully transformed and contain different regulatory genes NCS are obtained;
transforming Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 by electric shock of recombinant plasmid pXMJ 19/Ngfp containing different regulatory genes NCS to obtain a transformation product; the transformation product was spread on a brain heart infusion solid medium (containing 10. mu.g.mL)-1Chl and 0.2mmol/L IPTG) in a constant temperature incubator at 30 ℃ for 24h, and after 24h, selecting a conversion product capable of emitting green fluorescence under ultraviolet rays by a transmission protective cover ultraviolet analyzer; selecting a single colony of a conversion product capable of emitting green fluorescence to inoculate into a 96-well plate containing 1mL of brain heart infusion liquid culture medium (containing 0.2mmol/L IPTG) by taking recombinant Corynebacterium glutamicum Glutamicum/pXMJ19/gfp as a control, culturing for 24h in a 30 ℃ constant temperature incubator, and detecting the fluorescence intensity and OD (optical density) of fermentation liquor in the 96-well plate by a multifunctional microplate reader after 24h600Value, selection of fluorescence intensity and OD600The three transformation products are named as recombinant Corynebacterium glutamicum Glutamicum/pXMJ19/gfp-N2, recombinant Corynebacterium glutamicum Glutamicum/pXMJ 19/gfp-N28 and recombinant Corynebacterium glutamicum Glutamicum/pXMJ19/gfp-N38 respectively (the detection result is shown in figure 2); extracting recombinant Corynebacterium glutamicum/pXMJ19/gfp-N2, recombinant plasmid pXMJ 19/Ngfp contained in recombinant Corynebacterium glutamicum glutamcum/pXMJ 19/gfp-N38, sequencing to find that the NCSs of the regulatory genes contained in the recombinant Corynebacterium glutamicum/pXMJ19/gfp-N38, recombinant Corynebacterium glutamicum/pXMJ 19/gfp-N28 and recombinant Corynebacterium glutamicum/pXMJ 19/gfp-N567 are respectively NCS2 with the nucleotide sequence shown in SEQ ID No.1, NCS 3516 with the nucleotide sequence shown in SEQ ID No.16 and NCS 3617 with the nucleotide sequence shown in SEQ ID No. 17.
2. Regulating gene expression
Chemical synthesis ofA control gene NCS 2; designing connection primers GAD-NCS2-Act-R and Act-NCS2-GAD-F according to the regulatory gene NCS 2; performing PCR amplification on a regulatory gene NCS2 by using GAD-NCS2-Act-R and Act-NCS2-GAD-F as primers to obtain an amplification product; using GAD-F, Act-R as a primer, connecting the amplification product with the gene GAD coding for glutamate decarboxylase and the gene act coding for beta-alanine CoA transferase obtained in example 1 by overlap extension PCR to obtain a target gene ad-N2-act formed by connecting a regulatory gene NCS2, the gene GAD coding for glutamate decarboxylase and the gene act coding for beta-alanine CoA transferase (the regulatory gene NCS2 is positioned at the downstream of the gene GAD coding for glutamate decarboxylase and at the upstream of the gene act coding for beta-alanine CoA transferase); connecting the target gene ad-N2-act and the pXMJ19 plasmid after enzyme digestion by restriction enzymes EcoR I and HindIII to obtain a connection product; transforming the ligation product into Escherichia coli (Escherichia coli) BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 120-180 rpm, extracting plasmids, performing enzyme digestion verification and sequencing verification, and obtaining the successfully transformed recombinant Escherichia coli/pXMJ 19/gad-N2-act and recombinant plasmid pXMJ 19/gad-N2-act after verification is correct;
the recombinant plasmid pXMJ 19/gad-N2-act is transformed into recombinant Corynebacterium glutamicum/pXMJ 19/delta argB by electric shock to obtain a transformation product; the transformation product was spread on a brain heart infusion solid medium (containing 10. mu.g.mL)-1Chloramphenicol), and performing inverted culture in a constant temperature incubator at 30 ℃ for 36h to obtain a transformant; streaking the transformant on a brain-heart infusion solid culture medium, and carrying out inverted culture in a constant-temperature incubator at 30 ℃ for 36h to obtain a single colony; PCR verification is carried out on the single colony, and the recombinant Corynebacterium glutamicum is obtained after the verification is correct, namely the recombinant Corynebacterium glutamicum Glutamicum/pXMJ 19/delta argB-gad-N2-act is obtained (the sequences can be shown in the table 2).
Taking wild Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 as a control, selecting a single colony of recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act, inoculating the single colony in a brain-heart infusion liquid culture medium, and performing shake culture at 30 ℃ and 180r/min for 24 hours to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 5% (v/v), and fermenting for 72h at 30 ℃ and 180r/min to obtain a fermentation liquid; the content of gamma-aminobutyric acid and 2-pyrrolidone in the fermentation liquor is detected, and the accumulation amount of the gamma-aminobutyric acid in the fermentation liquor is 30 +/-3 mg/L, the accumulation amount of the heavier Corynebacterium glutamicum Glutamicum/pXMJ 19/delta argB-gad-act is reduced by 18.7 times, the accumulation amount of the 2-pyrrolidone in the fermentation liquor reaches 180 +/-7 mg/L, and the accumulation amount of the heavier Corynebacterium glutamicum Glutamicum/pXMJ 19/delta argB-gad-act is improved by 45.2%. Therefore, the expression regulation gene NCS2 in the recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-act can improve and weaken the accumulation of gamma-aminobutyric acid and enhance the accumulation of 2-pyrrolidone.
TABLE 2 nucleotide sequences of primers and genes
Example 3: application of recombinant corynebacterium glutamicum capable of producing 2-pyrrolidone at high yield
Picking a single colony of the recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act obtained in the example 2, inoculating the single colony into a seed culture medium (containing 10 mu g/mL chloramphenicol), and performing shake culture at 30 ℃ and 180r/min for 24 hours to obtain a seed solution; inoculating the seed solution into 200mL of fermentation medium with the inoculation amount of 10% (v/v), and performing shake cultivation for 18h at 30 ℃ and 180r/min to obtain a culture solution; transferring all the culture solution into a 5L fermentation tank, fermenting for 8h under the conditions of 30 ℃ and 180r/min, after 8h, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.2mM into a fermentation medium, and continuing to perform induction fermentation for 84h under the conditions of 30 ℃ and 180r/min to obtain fermentation liquor; in the whole fermentation process, the ventilation amount is controlled to be maintained at 40%, the rotating speed is controlled to be maintained at 600-800 r/min, the pH is controlled to be maintained at 7.0, and the concentration of glucose in a fermentation medium is controlled to be maintained at 50g/L by feeding a glucose solution with the concentration of 80 g/L.
During the fermentation period, sampling the fermentation liquor at intervals, and detecting the contents of glucose, L-glutamic acid, gamma-aminobutyric acid and 2-pyrrolidone in the fermentation liquor and the OD of the fermentation liquor600Values (see FIG. 3 for test results).
As can be seen from FIG. 3, the content of 2-pyrrolidone in the fermentation broth reaches the highest value, up to 8. + -. 0.3g/L, when the fermentation is carried out for 72 hours. Therefore, the recombinant Corynebacterium glutamicum/pXMJ 19/delta argB-gad-N2-act is used as the chassis cell, and cheap glucose is used as a substrate to efficiently synthesize the 2-pyrrolidone.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of south of the Yangtze river
<120> recombinant corynebacterium glutamicum and application thereof in production of 2-pyrrolidone
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<170> PatentIn version 3.3
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Claims (7)
1. A recombinant Corynebacterium glutamicum, which is characterized in that the recombinant Corynebacterium glutamicum takes Corynebacterium glutamicum as a host, knockouts a gene encoding N-acetyl glutamate kinase, and expresses a gene encoding glutamate decarboxylase and a gene encoding beta-alanine CoA transferase; a regulating gene with a nucleotide sequence shown as SEQ ID No.1 is connected between the downstream of the gene coding the glutamate decarboxylase and the upstream of the gene coding the beta-alanine CoA transferase.
2. The recombinant corynebacterium glutamicum of claim 1, wherein the gene encoding N-acetylglutamate kinase has the nucleotide sequence shown in SEQ ID No. 2.
3. The recombinant corynebacterium glutamicum of claim 2, wherein the gene encoding glutamate decarboxylase has the nucleotide sequence shown in SEQ ID No. 3.
4. The recombinant corynebacterium glutamicum of claim 3, wherein the gene encoding β -alanine CoA transferase has a nucleotide sequence set forth in SEQ ID No. 4.
5. The recombinant Corynebacterium glutamicum of any of claims 1 to 4, wherein the Corynebacterium glutamicum is used as a host, the gene encoding N-acetylglutamate kinase is knocked out, and the gene encoding glutamate decarboxylase and the gene encoding β -alanine CoA transferase are expressed from a vector selected from the group consisting of pET-28a (+) plasmid, pXMJ19 plasmid, pET-24b (+) plasmid, and pDXW-10 plasmid.
6. A method for producing 2-pyrrolidone, comprising inoculating the recombinant Corynebacterium glutamicum of any one of claims 1 to 5 to a glucose-containing medium, fermenting at 28 to 35 ℃ at 100 to 250rpm and pH 6 to 8 to obtain a fermentation broth containing 2-pyrrolidone, and extracting the fermentation broth containing 2-pyrrolidone to obtain 2-pyrrolidone.
7. Use of the recombinant corynebacterium glutamicum of any of claims 1 to 5 or the method of claim 6, for producing 2-pyrrolidone.
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