CN106434513A - Corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid - Google Patents

Abstract

The invention discloses a recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid. The construction method is as follows: (1) Knocking out the gene ldhA encoding lactate dehydrogenase and the gene pta for generating acetic acid in Corynebacterium glutamicum ATCC 13032 ‑ackA, pqo and cat to obtain bacterial strain CG4; insert the sod promoter in front of the phosphoenolpyruvate carboxylase coding gene ppc in CG4 to obtain bacterial strain CG5; knock out phosphoenolpyruvate carboxykinase in CG5 Encoding gene pck, to obtain bacterial strain CG6; (2) transfer the plasmid of overexpressing 5-aminolevulinic acid synthase gene in CG6 to obtain recombinant strain L; (3) transfer overexpressing 5-aminolevulinic acid synthase gene in L Acid-transporting protein particles, the recombinant strain of the invention can promote the transport of 5-aminolevulinic acid to the extracellular space of Corynebacterium glutamicum, and compared with the control strain, the output of 5-aminolevulinic acid has increased by 112.3%.

Description

Recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid

technical field

The invention belongs to the field of bioengineering technology and application, in particular to a recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid and its construction and application.

Background technique

5-Aminolevulinic acid (5-ALA), molecular weight 131.13, melting point 118°C. It is a non-protein amino acid. Because 5-aminolevulinic acid has the characteristics of less side effects and good permeability, it has been widely used in the diagnosis of skin cancer, bladder cancer, digestive tract cancer, lung cancer and photodynamic therapy (PDT).

5-Aminolevulinic acid is the precursor of pyrrole compounds in organisms and has a wide range of applications. In agriculture, since 5-aminolevulinic acid is easily degraded in the environment, it is harmless to mammals and can selectively kill pests, so it can be widely used as a photodynamic insecticide. In addition, 5-aminolevulinic acid has also received widespread attention in terms of improving the frost resistance and salt tolerance of crops and regulating plant growth. In the field of medicine, 5-aminolevulinic acid, as a second-generation photosensitizer, can not only be used for the treatment of local or systemic skin cancer, but also for the diagnosis of bladder cancer, digestive tract cancer and lung cancer.

According to retrieval, there is no report on overexpressing the 5-aminolevulinic acid transporter gene of Corynebacterium glutamicum to increase the production of 5-ALA.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid.

The second object of the present invention is to provide a method for constructing a recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid.

The third object of the present invention is to provide an application of a recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid.

Technical scheme of the present invention is summarized as follows:

The construction method of the Corynebacterium glutamicum recombinant strain producing 5-aminolevulinic acid comprises the steps:

(1) In Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032, the lactate dehydrogenase encoding gene ldhA and the acetic acid production genes pta-ackA, pqo and cat were knocked out, and the resulting strain was named CG4; the phosphoric acid in the CG4 strain Enol pyruvate carboxylase encoding gene ppc was inserted into the sod promoter to obtain bacterial strain CG5; phosphoenol pyruvate carboxykinase encoding gene pck was knocked out in the CG5 strain to obtain bacterial strain CG6;

(2) Transfer the constructed plasmid pXA overexpressing the 5-aminolevulinic acid synthase gene into the strain CG6 to obtain the recombinant strain L;

(3) Transform the constructed overexpression 5-aminolevulinic acid transport protein pEP-Ncgl2065 into the recombinant strain L to obtain the recombinant strain L2 of Corynebacterium glutamicum producing 5-aminolevulinic acid.

The 5-aminolevulinic acid-producing Corynebacterium glutamicum recombinant strain L2 constructed by the above method.

The use of the above-mentioned Corynebacterium glutamicum recombinant strain L2 producing 5-aminolevulinic acid through fermentation to produce 5-aminolevulinic acid.

The recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid constructed by the present invention can promote the transportation of 5-aminolevulinic acid to the extracellular space of Corynebacterium glutamicum, thereby significantly increasing the output of 5-aminolevulinic acid . Compared with the control strain, the production of 5-aminolevulinic acid was increased by 112.3%.

Description of drawings

Fig. 1 is the enzyme digestion verification map of plasmid pXA.

Fig. 2 is a PCR verification map of gene Ncgl2065 overexpression.

Fig. 3 is the fermentation result of recombinant strains L, L1, L2 in shake flasks.

detailed description

Mechanism: Introducing the Ncgl2065 gene overexpression plasmid (pEP-Ncgl2065) into the recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid can promote the extracellular transport of 5-aminolevulinic acid to Corynebacterium glutamicum, thereby The production of 5-aminolevulinic acid is significantly improved.

The present invention will be further described below in conjunction with specific examples. The following examples are intended to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

Used in the present invention:

The original strain Corynebacterium glutamicum (C.glutamicum) ATCC 13032 was purchased from ATCC (American Type Culture Collection, http://www.atcc.org/ ) in 2013.10;

The original plasmids pK18mobsacB, pXMJ19, pEC-XK99E, pEP2 were purchased from BioVector NTCC (http://www.biovector.net/).

5-aminolevulinic acid standard was purchased from sigma company (http://www.sigmaaldrich.com/sigma-aldrich). Molecular biology reagents such as restriction endonucleases, dephosphorylases, and DNA ligases were purchased from Thermo (http://www.thermoscientificbio.com/fermentas), and other biochemical reagents were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. purchase ( http://www.sangon.com/ ).

Example 1: Construction of knockout plasmid pD-sacB and construction of overexpression 5-aminolevulinic acid synthase gene plasmid pXA

Construction of pD-sacB plasmid:

Firstly, the linear fragment of pK18mobsacB cut by HindIII was used as a template, and the sacB gene was amplified with the following primers sacB-1/sacB-2. The sacB gene fragment was ligated with the plasmid pEC-XK99E after the MunI/EcoRV double digestion and the EcoRI/SmalI double digestion to obtain the plasmid pEC-XK99E-sacB. Use the following primers trcsacB-1/trcsacB-2 to amplify the trcsacB fragment containing the trc promoter using the pEC-XK99E-sacB plasmid as a template.

Use the following primers pD-1/pD-2, and use the pK18mobsacB plasmid as a template to amplify the pD fragment containing kanamycin resistance and E. The pD fragments were ligated to obtain plasmid pD-sacB.

Construction of pXA plasmid:

The 5-aminolevulinic acid synthase gene hemA of Rhodobacter sphaeroides was codon-optimized according to the codon preference of Corynebacterium glutamicum, and the optimized hemA gene was synthesized by total gene synthesis ( As shown in SEQ ID NO.39), utilize primer hemA-1/hemA-2 to amplify the optimized hemA fragment (SEQ ID NO.39), the obtained hemA fragment is digested with PstI/XbaI double enzymes and undergoes the same double The digested shuttle plasmid pXMJ19 was ligated to obtain the pXA plasmid, and the enzyme digestion verification map of the plasmid pXA is shown in Figure 1.

Example 2: Knockout of the lactate dehydrogenase encoding gene ldhA and knockout of the acetate production pathway genes pta-ackA, pqo and cat

Knockout of the gene ldhA encoding lactate dehydrogenase:

Using the genome of Corynebacterium glutamicum (C. glutamicum) ATCC 13032 as a template, using ldh-1/ldh-2 as primers to amplify the upstream fragment of gene ldhA, and ldh-3/ldh-4 as primers to amplify the downstream of gene ldhA fragment. After the two fragments were recovered by gel cutting, the fusion product of the two fragments was amplified using the equimolar proportion of the fragments as a template and ldh-1/ldh-4 as primers. The fused fragment was digested with EcoRI/HindIII and ligated with pD-sacB after the same double digestion to obtain plasmid pD-ldhA.

Transfer the pD-ldhA plasmid into C.glutamicum ATCC 13032, and use kanamycin to screen the positive clones with successful recombination. The selected transformants were inoculated in 5mL BHIS liquid medium, cultivated overnight at 30°C and 220rpm, and the bacteria The solution was diluted 1000 times and spread on the BHIS-Sucrose solid plate. The colony grown on the plate was not resistant to the BHIS solid plate and the BHIS solid plate containing 25 μg/mL kanamycin. The colony that grew on the non-antibiotic plate but not on the kanamycin plate was selected and inoculated into 5 mL of BHIS liquid medium, the genome was extracted, and PCR verification was performed using primers ldh-1/ldh-4 to obtain the ldhA gene knockout strain CG1.

Knockout of the pta-ackA operon

The genome of C. glutamicum ATCC13032 was used as a template, the upstream fragment of operon pta-ackA was amplified with ackA-1/ackA-2 as primers, and the downstream fragment of operon pta-ackA was amplified with ackA-3/ackA-4 as primers. After the two fragments were recovered by gel cutting, the fusion product of the two fragments was amplified using the equimolar proportion of the fragments as a template and ackA-1/ackA-4 as primers. The fused fragment was double digested with SalI/XbaI and ligated with the pD-sacB plasmid after the same double digestion to obtain the pta-ackA operon knockout plasmid pD-pta.

Transfer the pD-pta plasmid into the strain CG1, use kanamycin to screen the positive clones with successful recombination, inoculate the selected transformants in 5mL BHIS liquid medium, cultivate overnight at 30°C, 220rpm, and dilute the bacterial solution by 1000 Double coated on BHIS-Sucrose solid plate. The colony grown on the plate was not resistant to the BHIS solid plate and the BHIS solid plate containing 25 μg/mL kanamycin. Select the colony that grows on the non-antibiotic plate but does not grow on the kanamycin plate and inoculate it into 5mL BHIS liquid medium, extract the genome, use primers ackA-1/ackA-4 for PCR verification, and obtain pta-ackA on the basis of CG1 The operon knockout strain CG2.

Knockout of the pqo gene

C. glutamicum ATCC13032 genome was used as template, pqo-1/pqo-2 was used as primer to amplify the upstream fragment of pqo gene, and pqo-3/pqo-4 was used as primer to amplify the downstream fragment of pqo. After the two fragments were recovered by gel cutting, the fusion product of the two fragments was amplified using the equimolar proportion of the fragments as a template and pqo-1/pqo-4 as primers. The fused fragment was digested with XbaI/PstI and ligated with pD-sacB after the same double digestion to obtain the pqo gene knockout plasmid pD-pqo.

Transfer the pD-pqo plasmid into the strain CG2, use kanamycin to screen the positive clones with successful recombination, inoculate the selected transformants in 5mL BHIS liquid medium, cultivate overnight at 30°C, 220rpm, and dilute the bacterial solution to 1000 Double coated on BHIS-Sucrose solid plate. The colony grown on the plate was not resistant to the BHIS solid plate and the BHIS solid plate containing 25 μg/mL kanamycin. Select the colony that grows on the non-antibiotic plate but does not grow on the kanamycin plate and inoculate it into 5mL BHIS liquid medium, extract the genome, use primers pqo-1/pqo-4 for PCR verification, and obtain the pqo gene on the basis of CG2 Knockout strain CG3.

knockout of the cat gene

The C. glutamicum ATCC13032 genome was used as a template, the upstream fragment of cat gene was amplified with cat-1/cat-2 primers, and the downstream fragment of cat gene was amplified with cat-3/cat-4 primers. After the two fragments were recovered by gel cutting, the fusion product of the two fragments was amplified using the equimolar proportion of the fragments as a template and cat-1/cat-4 as primers. The fused fragment was digested with XbaI/SalI and ligated with pD-sacB after the same double digestion to obtain the cat gene knockout plasmid pD-cat.

Transfer the pD-cat plasmid into the strain CG3, use kanamycin to screen the positive clones with successful recombination, inoculate the selected transformants in 5mL BHIS liquid medium, cultivate overnight at 30°C, 220rpm, and dilute the bacterial solution by 1000 Double coated on BHIS-Sucrose solid plate. The colony grown on the plate was not resistant to the BHIS solid plate and the BHIS solid plate containing 25 μg/mL kanamycin. Select the colony that grows on the non-antibiotic plate but does not grow on the kanamycin plate and inoculate it into 5mL BHIS liquid medium, extract the genome, use primers cat-1/cat-4 for PCR verification, and obtain the cat gene on the basis of CG3 The knockout strain CG4.

The composition of the BHIS medium is (g/L): 37% of bovine brain heart extract powder, 91% of sorbitol, and the balance is water.

The composition of BHIS solid medium is (g/L): bovine brain heart extract powder 37, sorbitol 91, agar 2% (W/V), and the balance is water.

BHIS-Sucrose solid medium (g/L): bovine brain heart extract powder 37, sorbitol 91, agar 2% (W/V), sugarcane

Sugar 10% (W/V), the balance is water.

Example 3: Insertion of sod promoter and phosphoenolpyruvate carboxykinase encoding gene pck in front of ppc gene. Insertion of sod promoter in front of ppc gene

Using C. glutamicum ATCC13032 genome as a template and ppc-1/ppc-2 as primers to amplify the upstream fragment of gene ppc. sod-1/sod-2 are used to amplify the promoter of the sod gene. ppc-3/ppc-4 is used to amplify the downstream fragment of the ppc gene. After the three fragments are cut and recovered, the equimolar proportion of the fragment is used as a template, and ppc-1/ppc-4 is used as a primer to amplify the obtained Fusion product of three fragments. The fused fragment was digested with XbaI/HindIII and ligated with the plasmid vector pD-sacB after the same double digestion. The plasmid pD-ppc was obtained.

The constructed plasmid pD-ppc was transformed into the strain CG4 by electroporation, the positive clones with successful recombination were screened with kanamycin, and the selected transformants were inoculated in 5mL BHIS liquid medium, cultured overnight at 30°C and 220rpm , Dilute the bacterial solution 1000 times and spread it on the BHIS-Sucrose solid plate. The colony grown on the plate was not resistant to the BHIS solid plate and the BHIS solid plate containing 25 μg/mL kanamycin. Inoculate the colony that grew on the non-antibiotic plate but not on the kanamycin plate into 5 mL of BHIS liquid medium, extract the genome, and send it for sequencing. On the basis of CG4, the strain CG5 with the sod promoter inserted in front of the ppc gene was obtained.

Knockout of the pck gene

Using the C. glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the following primers. pck-1/pck-2 was used to amplify the upstream fragment of gene pck. pck-3/pck-4 was used to amplify the downstream fragment of gene pck. The upstream fragment was digested with EcoRI/XbaI and then ligated with the pD-sacB plasmid after the same double digestion to obtain plasmid pD-pck(F), and the constructed plasmid pD-pck(F) was subjected to PstI/HindIII double enzyme After cutting, it was ligated with the downstream fragment digested with PstI/HindIII to obtain the pD-pck plasmid.

Transfer the plasmid into the bacterial strain CG5, use kanamycin to screen the positive clones with successful recombination, inoculate the selected transformants in 5mL BHIS liquid medium, cultivate overnight at 30°C, 220rpm, dilute the bacterial solution 1000 times and spread Cloth on BHIS-Sucrose solid plate. The colony grown on the plate was not resistant to the BHIS solid plate and the BHIS solid plate containing 25 μg/mL kanamycin. Inoculate the colony that grows on the non-antibiotic plate but does not grow on the kanamycin plate into 5mL BHIS liquid medium, extract the genome, use primer pck-1/pck-4 for PCR verification, and obtain the pck gene on the basis of CG5 The knockout strain CG6.

The pXA plasmid was transferred into CG6 and screened with chloramphenicol to obtain the 5-aminolevulinic acid producing strain L carrying the pXA plasmid.

Example 4: Construction of overexpression plasmid pEP-tuf and construction of overexpression transport protein plasmid

Construction of pEP-tuf

Using the C. glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the following primers. TUF-1/TUF-2 were used to amplify genes encoded by the TUF promoter. The amplified fragment was digested with EcoRI/SalI and ligated with the pEP2 plasmid after the same digestion. The overexpression plasmid pEP-tuf was obtained.

Transform the plasmid into strain L, use kanamycin and chloramphenicol to screen the positive clones that have successfully recombined, and inoculate the colonies that can grow on the double-antibody plate into 5mL BHIS liquid medium at 30°C, use primers Tuf-1/tuf-2 was verified by bacterial liquid PCR, and strain L1 was obtained.

Construction of pEP-Ncgl2065

Using the C. glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the following primers. Ncgl2065-1/Ncgl2065-2 were used to amplify the gene Ncgl2065. The amplified fragment was digested with XbaI/SalI and ligated with the pEP-tuf plasmid after the same digestion. The Ncgl2065 overexpression plasmid pEP-Ncgl2065 was obtained.

Transfer the pEP-Ncgl2065 plasmid into the strain L, use kanamycin and chloramphenicol to screen the positive clones with successful recombination, and inoculate the colonies that can grow on the double-antibody plate into 5mL BHIS liquid medium at 30°C. Using primers Ncgl2065-1/Ncgl2065-2 to carry out bacterial liquid PCR verification, the strain L2 was obtained, as shown in FIG. 2 .

Table 1 Primer sequences used for strain construction (artificial synthesis)

Embodiment 5: Utilize the bacterial strain constructed to carry out the shake flask fermentation of 5-aminolevulinic acid

Strains L, L1 (control), L2 were fermented in M2 medium.

The inoculation method of strain L is as follows: first activate the strain on the BHIS solid plate, cultivate it at 30°C until the colony is visible, then pick a single colony and inoculate it into a test tube containing BHIS liquid medium with a final concentration of 10 μg/mL chloramphenicol. After about 15 hours, transfer to the M1 medium containing the corresponding antibiotics, and when the culture reaches OD ₆₀₀ =10, inoculate the seeds into the M2 medium for fermentation, and add chloramphenicol with a final concentration of 10 μg/mL to the medium and 0.5mM isopropylthiogalactopyranoside (IPTG), fermented at 30°C, pH 7.0, 220rpm, for 30h.

The inoculation method of strains L1 and L2 is as follows: first activate the strains on BHIS solid plates, cultivate them at 30°C until the colonies are visible, and inoculate a single colony until the final concentration of chloramphenicol is 8 μg/mL and the final concentration is 20 μg/mL. Cultivate in the test tube of _BHIS liquid medium with mL kanamycin for about 15 hours, and then transfer to the M1 medium containing the corresponding antibiotics respectively. And in the culture medium, add final concentration of 8 μg/mL chloramphenicol and final concentration of 20 μg/mL kanamycin and 0.5 mM isopropylthiogalactopyranoside (IPTG), at 30 ° C, pH 7.0, 220rpm, ferment for 30h.

The fermentation results are shown in Figure 3.

The composition of M1 medium is (g/L): glucose 10, yeast extract 10, tryptone 10, NaCl 2.5, and the balance is water.

The composition of M2 medium is (g/L): glucose 10, yeast extract 7.5, ammonium sulfate 10, potassium dihydrogen phosphate 1, dipotassium hydrogen phosphate 1, magnesium sulfate heptahydrate 0.1, calcium chloride 0.02, 3-(N -morpholine) propanesulfonic acid 21, glycine 7.5, and the balance is water.

SEQUENCE LISTING

<110> Tianjin University

<120> Recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid

<130>

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<170> PatentIn version 3.3

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Claims (3)

1. the construction method of the Corynebacterium glutamicum recombinant bacterial strain of production 5-ALA, is characterized in that including following step Suddenly：

(1) lactic acid dehydrogenase volume is knocked out in Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 Code gene ldhA and acetic acid generate gene pta-ackA, pqo and cat, and the Strain Designation for obtaining is CG4；Phosphorus in CG4 bacterial strain Sour acid enol type pyruvate carboxylase encoding gene ppc is previously inserted into sod promoter, obtains bacterial strain CG5；Knock out in CG5 bacterial strain PCK encoding gene pck, obtains bacterial strain CG6；

(2) the plasmid pXA of the overexpression 5-Aminolevulinate synthase gene for building is proceeded in bacterial strain CG6, is recombinated Bacterial strain L；

(3) the overexpression 5-ALA transport protein plasmid pEP-Ncgl2065 for building is proceeded in recombinant bacterial strain L, Obtain producing the Corynebacterium glutamicum recombinant bacterial strain L2 of 5-ALA.

2. the Corynebacterium glutamicum recombinant bacterial strain L2 of the production 5-ALA that the method described in claim 1 builds.

3. claim 2 production 5-ALA Corynebacterium glutamicum recombinant bacterial strain L2 purposes.