CN106047916A - Corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain - Google Patents

Abstract

The invention discloses a Corynebacterium glutamicum strain producing 5-aminolevulinic acid and its construction and application. The construction method is as follows: (1) Knocking out the lactate dehydrogenase encoding gene ldhA and the acetic acid generation gene in Corynebacterium glutamicum pta‑ackA, pqo and cat, the resulting strain was named CB4; a strong sod promoter was inserted in front of the phosphoenolpyruvate carboxylase encoding gene ppc in the CB4 strain to obtain strain CB5; knockout in the CB5 strain Gene pck, obtain bacterial strain CB6; (2) transfer plasmid pXA and plasmid pEP2 ^{tuf ‑} rhtA into CB6 bacterial strain. The bacterial strain constructed by the present invention can produce 2.78g/L 5-aminolevulinic acid in a medium with 10g/L glucose as a carbon source, which improves the output and yield of 5-aminolevulinic acid for continuous feeding of subsequent fermentation tanks. rate laid the foundation.

Description

Corynebacterium glutamicum strain producing 5-aminolevulinic acid and its construction and application

technical field

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

Background technique

5-Aminolevulinic acid has a molecular weight of 131.13 and a melting point of 118°C. It is a non-protein amino acid. 5-aminolevulinic acid has the characteristics of less side effects and good permeability, and has been widely used in the diagnosis of skin cancer, bladder cancer, digestive tract cancer, lung cancer and photodynamic therapy (PDT). At the same time, 5-aminolevulinic acid also has very important applications in agriculture such as plant growth regulators, herbicides and green pesticides.

In nature, there are two main pathways for the biosynthesis of 5-aminolevulinic acid: the C4 pathway and the C5 pathway. The C4 pathway is the condensation of succinyl CoA and glycine under the action of 5-aminolevulinic acid synthase to generate 5-aminolevulinic acid. This pathway exists in animals, fungi and non-sulfur photosynthetic bacteria. The C5 pathway is catalyzed by tRNA to generate 5-aminolevulinic acid through a three-step reaction. This pathway widely exists in plants, algae and bacteria, including Escherichia coli and Corynebacterium glutamicum.

At present, there are only a few reports about the production of 5-aminolevulinic acid by Corynebacterium glutamicum using the C5 pathway, but the pathway is relatively complicated, and the yield and yield of 5-aminolevulinic acid are very low.

According to retrieval, there is no report about Corynebacterium glutamicum using C4 pathway to produce 5-aminolevulinic acid.

Contents of the invention

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

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

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

Technical scheme of the present invention is summarized as follows:

A method for constructing a Corynebacterium glutamicum strain producing 5-aminolevulinic acid, comprising the steps of:

(1) In Corynebacterium glutamicum (C.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 CB4; in the CB4 strain A strong sod promoter was inserted in front of the phosphoenolpyruvate carboxylase encoding gene ppc to obtain strain CB5; the phosphoenolpyruvate carboxykinase encoding gene pck was knocked out in the CB5 strain to obtain strain CB6;

(2) Transfer the constructed plasmid pXA overexpressing the 5-aminolevulinic acid synthase gene and the plasmid pEP2 ^tuf -rhtA overexpressing the RhtA transport protein into the CB6 strain to obtain the gluten for producing 5-aminolevulinic acid Corynebacterium acidic acid strain AL2.

Corynebacterium glutamicum strain AL2 producing 5-aminolevulinic acid constructed by the above method.

The use of the Corynebacterium glutamicum strain AL2 producing 5-aminolevulinic acid through fermentation to produce 5-aminolevulinic acid. Beneficial effect

The present invention constructs a strain of Corynebacterium glutamicum that utilizes the C4 pathway to produce 5-aminolevulinic acid, which can produce 2.78g/L 5-aminolevulinic acid in a medium with 10g/L glucose as a carbon source acid, which laid the foundation for the continuous feeding of subsequent fermenters to increase the production and yield of 5-aminolevulinic acid.

Description of drawings

Figure 1 is the gene manipulation target.

Fig. 2A is the restriction map of plasmid pXA, and Fig. 2B is the restriction map of plasmid pEP2 ^tuf- rhtA.

detailed description

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 TypeCulture Collection, http://www.atcc.org/);

E. coli MG1655 was purchased from CGSC (Coli Genetic Stock Center, http://cgsc.biology.yale.edu/).

The original plasmids pK18mobsacB, pEC-XK99E, pXMJ19 and 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

First, the linear fragment of pK18mobsacB cut by HindIII was used as a template to amplify the sacB gene with the following primers sacB-1 (SEQ ID NO.1)/sacB-2 (SEQ ID NO.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. The following primers trcsacB-1 (SEQ ID NO.3)/trcsacB-2 (SEQ ID NO.4) were used to amplify the trcsacB fragment containing the trc promoter using the pEC-XK99E-sacB plasmid as a template.

Use the following primers pD-1 (SEQ ID NO.5)/pD-2 (SEQ ID NO.6), use the pK18mobsacB plasmid as a template to amplify the pD fragment containing kanamycin resistance and the Escherichia coli replicon, and finally The fragment trcsacB digested by AatII was ligated with the pD fragment digested by the same restriction enzyme to obtain plasmid pD-sacB.

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 Corynebacterium glutamicum (C.glutamicum) ATCC 13032 genome as a template, using ldh-1 (SEQ ID NO.7)/ldh-2 (SEQ ID NO.8) as primers to amplify the upstream fragment of gene ldhA, ldh- 3(SEQ ID NO.9)/ldh-4(SEQ ID NO.10) are primers for amplifying the downstream fragment of gene ldhA. 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.

Transform the pD-ldhA plasmid into Corynebacterium glutamicum (C.glutamicum) ATCC 13032, use kanamycin to screen the positive clones with successful recombination, and inoculate the selected transformants in 5mL BHIS liquid medium, at 30°C , overnight culture at 220rpm, and the bacterial solution was diluted to a certain number of 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 CB1.

Knockout of the pta-ackA operon

Using the C.glutamicum ATCC13032 genome as a template, using ackA-1 (SEQ ID NO.11)/ackA-2 (SEQ ID NO.12) as primers to amplify the upstream fragment of operon pta-ackA, ackA-3 (SEQ ID NO.13)/ackA-4 (SEQ ID NO.14) is the primer to amplify the downstream fragment of operon pta-ackA. 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 CB1, 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 a certain The multiples were coated on BHIS-Sucrose solid plates. 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 operon knockout Strain CB2.

Knockout of the pqo gene

Using the C. glutamicum ATCC13032 genome as a template, using pqo-1 (SEQ ID NO.15)/pqo-2 (SEQ ID NO.16) as primers to amplify the upstream fragment of the pqo gene, pqo-3 (SEQ ID NO.17) /pqo-4 (SEQ ID NO.18) is the 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 CB2, 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 a certain The multiples were coated on BHIS-Sucrose solid plates. 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 5 mL of BHIS liquid medium, extract the genome, and use primers pqo-1/pqo-4 for PCR verification to obtain the pqo gene knockout strain CB3 .

knockout of the cat gene

Using the C. glutamicum ATCC13032 genome as a template, using cat-1 (SEQ ID NO.19)/cat-2 (SEQ ID NO.20) as primers to amplify the upstream fragment of the cat gene, cat-3 (SEQ ID NO.21) /cat-4 (SEQ ID NO.22) is a primer to amplify the downstream fragment of cat. 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 CB3, 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 a certain The multiples were coated on BHIS-Sucrose solid plates. 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, and use primers cat-1/cat-4 for PCR verification to obtain the cat gene knockout strain CB4 .

Among them, the composition of BHIS medium is (g/L): bovine brain heart extract powder 37, sorbitol 91.

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

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

Sugar 10% (W/V).

Example 3: Insertion of a strong sod promoter in front of the ppc gene and knockout of the pck gene, the gene encoding phosphoenolpyruvate carboxykinase

A strong sod promoter was inserted in front of the ppc gene

C. glutamicum ATCC13032 genome was used as a template, and ppc-1 (SEQ ID NO. 23)/ppc-2 (SEQ ID NO. 24) was used as primers to amplify the upstream fragment of gene ppc. sod-1 (SEQ ID NO.25)/sod-2 (SEQ ID NO.26) was used to amplify the promoter of sod gene. ppc-3 (SEQ ID NO.27)/ppc-4 (SEQ ID NO.28) is used to amplify the downstream fragments of the ppc gene. After the three fragments are gel-cut and recovered, the equimolar ratio fragments are used as templates. Using ppc-1/ppc-4 as primers, the fusion product of the three fragments was amplified. 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 was transferred into the strain CB4 by electroporation, and the positive clones with successful recombination were screened with kanamycin, and the selected transformants were inoculated in 5mL BHIS liquid medium, cultivated overnight at 30°C and 220rpm, and the bacteria The solution was diluted by a certain multiple 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. 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 to obtain the strain CB5 with the sod promoter inserted in front of the ppc gene.

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 (SEQ ID NO. 29)/pck-2 (SEQ ID NO. 30) is used to amplify the upstream fragment of gene pck. pck-3 (SEQ ID NO. 31)/pck-4 (SEQ ID NO. 32) is 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 strain CB5, use kanamycin to screen the positive clones with successful recombination, inoculate the selected transformant in 5mL BHIS liquid medium, cultivate overnight at 30°C, 220rpm, and dilute the bacterial solution by a certain number of times Spread on BHIS-Sucrose solid plates. 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 anti-antibiotic plate but did not grow on the kanamycin plate into 5 mL of BHIS liquid medium, extract the genome, and perform PCR verification using primers pck-1/pck-4 to obtain the knockout strain CB6 of the pck gene.

Embodiment 4: Construction of plasmid pXA and pEP2 ^tuf- rhtA

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 gene was synthesized by total gene synthesis ( As shown in SEQ ID NO.39), utilize primer hemA-1 (SEQ ID NO.33)/hemA-2 (SEQ ID NO.34) to amplify the optimized hemA fragment, and pass the obtained hemA fragment through PstI/XbaI After the double digestion, it was ligated with the shuttle plasmid pXMJ19 after the same double digestion to obtain the pXA plasmid. Figure 2A is the restriction map of the plasmid pXA.

Using the genome of C. glutamicum ATCC 13032 as a template, use tuf-1 (SEQ ID NO.35)/tuf-2 (SEQ ID NO.36) as primers to amplify the tuf promoter, digest the tuf fragment with EcoRI/SalI and combine with The pEP2 plasmids digested with the same restriction enzymes were ligated to obtain a low-copy plasmid pEP2 ^tuf with a tuf promoter. Using the E.coli MG1655 genome as a template, rhtA-1 (SEQ ID NO.37)/rhtA-2 (SEQ ID NO.38) was used as primers to amplify the rhtA fragment, and the fragment was digested with XbaI/BamHI and processed The pEP2 ^tuf plasmid digested with the same restriction enzyme was ligated to obtain the pEP2 ^tuf- rhtA plasmid, and Figure 2B is a verification map of the plasmid pEP2 ^tuf- rhtA digestion.

The constructed pXA plasmid was introduced into strain CB6, and screened by chloramphenicol to obtain strain AL1 with pXA plasmid. The constructed pEP2 ^tuf -rhtA plasmid was introduced into AL1 by electroporation and screened by chloramphenicol and kanamycin to obtain the final 5-aminolevulinic acid producing strain AL2.

The gene manipulation involved in the present invention is shown in Fig. 1, "X" in Fig. 1 represents knockout, and underline represents overexpression.

Table 1 The sequences of primers used for strain construction

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

The constructed strain AL2 was fermented in shake flasks.

The specific fermentation method is as follows

The inoculation method 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 5 mL of kanamycin sulfate with a final concentration of 25 μg/mL and a final concentration of 10 μg/mL After being cultured in the BHIS liquid medium of chloramphenicol for 15 hours, it was transferred to the M1 medium, and the seeds were inoculated into the M2 medium when the culture reached the middle and late logarithmic stages. Isopropylthio-β-D-galactoside was added to the medium at a final concentration of 0.5 mM as an inducer. After the fermentation, the output of 5-ALA detected was 2.78g/L.

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

The composition of M2 medium is (g/L): glucose 10, yeast extract 10, tryptone 10, NaCl 2.5, 3-(N-morpholine) propanesulfonic acid 21, glycine 7.5.

Claims (3)

1. produce a construction method for the Corynebacterium glutamicum strain of 5-ALA, it is characterized in that including walking as follows Rapid:

(1) in Corynebacterium glutamicum (C.glutamicum) ATCC 13032, lactic dehydrogenase enzyme coding gene ldhA and second are knocked out Acid generates gene pta-ackA, pqo and cat, and the Strain Designation obtained is CB4；Phosphoenolpyruvic acid in CB4 bacterial strain Carboxylase encoding gene ppc is previously inserted into strong sod promoter, obtains bacterial strain CB5；Phosphoenolpyruvate is knocked out in CB5 bacterial strain Pyruvate carboxykinase encoding gene pck, obtains bacterial strain CB6；

(2) by the plasmid pXA of process LAN 5-Aminolevulinate synthase gene built and process LAN RhtA transport protein Plasmid pEP2^tuf-rhtA is transferred in CB6 bacterial strain, it is thus achieved that produce the Corynebacterium glutamicum strain AL2 of 5-ALA.

2. the Corynebacterium glutamicum strain AL2 producing 5-ALA that the method described in claim 1 builds.

3. the purposes of the Corynebacterium glutamicum strain AL2 producing 5-ALA of claim 2.