CN111647617A - Corynebacterium glutamicum for producing L-citrulline and modification method and application thereof - Google Patents

Abstract

The invention discloses a L-citrulline-producing Corynebacterium glutamicum, a modification method and application thereof. By modifying the genome of Corynebacterium glutamicum, the downstream fragment of argR gene and the upstream fragment of argG gene are knocked out, And insert the argB _Ec gene of Escherichia coli into the middle of the knockout fragment to obtain a recombinant strain. The use of the improved strain for fermentation can enhance the L-citrulline synthesis pathway during the fermentation process, and at the same time, block the L-citrulline to L-arginine conversion pathway, thereby obtaining high-purity, high-yield L-citrulline. ‑Citrulline.

Description

Corynebacterium glutamicum for producing L-citrulline and its transformation method and application

technical field

The invention belongs to the field of genetic modification, and in particular relates to a L-citrulline-producing Corynebacterium glutamicum and a modification method and application thereof.

Background technique

L-citrulline is an important non-protein amino acid that participates in the urea cycle to maintain blood ammonia balance, and has physiological functions such as antioxidant and blood pressure stabilization. Therefore, L-citrulline has very broad application prospects in the medical drug, food and cosmetic industries. At present, L-citrulline synthesis research mainly focuses on enzymatic conversion and fermentation. The enzymatic conversion method uses L-arginine as a substrate to catalyze the production of L-citrulline by arginine deiminase. The substrate L-arginine is expensive, so the enzymatic conversion method has the problem of high substrate cost. The direct fermentation of microorganisms to produce L-citrulline can use cheap substrates such as glucose, and has the advantages of simple process, low cost, and low environmental pollution, so it has gradually received attention in recent years.

Corynebacterium glutamicum is a facultative anaerobic Gram-positive safe strain. With the deepening of research, especially the development of metabolic engineering, C.glutamicum has been widely used in the fermentation production of various amino acids, organic acids and even biofuels and biobased chemicals. C.glutamicum includes a metabolic pathway integrating L-ornithine, L-citrulline and L-arginine synthesis, which can be called the L-arginine anabolic pathway. The final product of this metabolic pathway is L-arginine, and L-citrulline is the intermediate product of the metabolic reaction of L-ornithine to L-arginine.

The genes related to the L-arginine anabolic pathway in C. glutamicum ATCC13032 exist on the chromosome in the form of the gene cluster argCJBDFRGH, which is divided into two operons, argCJBDFR and argGH. The genes encoding all enzymes in the main L-citrulline synthesis pathway are concentrated on the argCJBDFR operon. Among them, the N-acetylglutamate kinase encoded by the argB gene is the key rate-limiting enzyme in the synthesis of L-citrulline and L-arginine. At the same time, its activity is feedback-inhibited by L-arginine. L-arginine can severely inhibit the catalytic activity of N-acetylglutamate kinase. The repressor protein ArgR encoded by the argR gene can inhibit the transcription of the argCJBDFR and argGH operons, thereby inhibiting the entire L-citrulline and L-arginine anabolic pathway in cells. Therefore, the L-citrulline and L-arginine anabolic pathways in C.glutamicum ATCC13032 cells are under precise metabolic regulation, and L-citrulline or L-arginine will not be excessively synthesized. C. glutamicum ATCC13032 did not accumulate L-citrulline or L-arginine in the fermentation broth during the fermentation process. Excessive synthesis of L-citrulline and L-arginine can only be achieved by releasing the metabolic regulation of cells on L-citrulline and L-arginine anabolic pathways. At the same time, if we want to achieve L-citrulline fermentative production of C. glutamicum ATCC13032 without accumulating L-arginine, we need to block the conversion pathway of L-citrulline to L-arginine. The argininosuccinate synthase encoded by the argG gene catalyzes the synthesis of argininosuccinate from L-citrulline, and inactivation of argininosuccinate synthase can block the continued conversion of L-citrulline to L-arginine. The argG and argR genes are adjacent on the C. glutamicum genome. The N-acetylglutamate kinase activity derived from Escherichia coli is not subject to feedback inhibition by L-arginine, and its encoding gene is named argB _Ec .

SUMMARY OF THE INVENTION

In view of the above problems, the present invention proposes a L-citrulline-producing Corynebacterium glutamicum and its transformation method and application.

To achieve the above-mentioned technical purpose and achieve the above-mentioned technical effect, the present invention is realized through the following technical solutions:

A method for transforming Corynebacterium glutamicum to produce L-citrulline, characterized in that: modifying the genome of Corynebacterium glutamicum, knocking out the downstream fragment of argR gene and the upstream fragment of argG gene, and in the knockout The argB _Ec gene of E. coli is inserted in the middle of the fragment.

As a further improvement of the present invention, the argR _F -argB _Ec -argG _R gene fragment is obtained by amplifying the upstream fragment argR _F of the argR gene, the argB _Ec gene and the downstream fragment argG _R of the argG gene by PCR.

As a further improvement of the present invention, comprise the following steps:

(1) Using the mixture of _argRF gene and argB _Ec gene as a template, the argRF- _{argB Ec} gene fragment was obtained by using primers argRF and argB _Ec R to overlap and extend PCR amplification;

(2) Using the mixture of argRF- _{argB Ec} gene and argG _R gene as a template, the argRF- _{argB Ec} -argG _R gene fragment was obtained by PCR amplification of primers argRF and argGR.

As a further improvement of the present invention, the primer argRF sequence is shown in SEQ ID NO.4.

As a further improvement of the present invention, the primer argGR sequence is shown in SEQ ID NO.7.

As a further improvement of the present invention, the primer argB _Ec R sequence is shown in SEQ ID NO.9.

As a further improvement of the present invention, the E. coli BL21 strain is selected as the E. coli strain, and the nucleotide sequence of the argB _Ec gene is shown in SEQ ID NO.3.

The present invention also provides a L-citrulline-producing Corynebacterium glutamicum, a strain obtained according to the above-mentioned method for transforming Corynebacterium glutamicum to produce L-citrulline.

The present invention also provides a method for producing L-citrulline by using the Corynebacterium glutamicum.

Beneficial effects of the present invention: The present invention inserts the argB _Ec gene between the argR and argG genes in the genome of the C.glutamicum ATCC13032 strain through one-step genome modification. Equivalent to C. glutamicum ATCC13032 strain argR and argG gene knockout simultaneously introduced the argB _Ec gene derived from E. coli. Through one-step genome modification, the L-citrulline synthesis operon repressor ArgR and argininosuccinate synthase were inactivated in C.glutamicum ATCC13032 strain, and the anti-L-arginine feedback from E. coli was expressed Inhibited N-acetylglutamate kinase. In C.glutamicum ATCC13032 strain, the metabolic regulation of cells on the L-citrulline synthesis and metabolism pathway was relieved, the L-citrulline synthesis pathway was enhanced, and at the same time, the conversion of L-citrulline to L-arginine was blocked. transformation pathway. Corynebacterium glutamicum capable of producing high-purity, high-yield L-citrulline was obtained.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

1. Material selection

The C. glutamicum type strain ATCC13032 of Corynebacterium glutamicum used was obtained by purchase. The C.glutamicum ATCC13032 argR gene sequence is shown in SEQ NO.1, and the C.glutamicum ATCC13032 argG gene sequence is shown in SEQ NO.2.

The selected strain of E. coli is E. coli BL21, and the E. coli BL21 argB _Ec gene sequence is shown in SEQ NO.3.

2. Primer Design

PCR amplification technology was used to amplify the upstream fragment argR _F of argR gene and the downstream fragment argG _R of argG gene using C.glutamicum ATCC13032 genome as a template, and amplify argB _Ec gene using E.coli BL21 genome as a module. Sequence Listing 1 of the design is as follows:

Table 1: Primer sequences

3. Genetic recombination

(1) Using the mixture of _argRF gene and argB _Ec gene as a template, the argRF- _{argB Ec} gene fragment was obtained by overlapping extension PCR with primers argRF and argB _Ec R.

Using the mixture of argRF- _{argB Ec} gene and argG _R gene as a template, the argRF- _{argB Ec} -argG _R gene fragment was obtained by overlapping extension PCR with primers argRF and argGR.

(2) The obtained argR _F -argB _Ec -argG _R gene fragment was ligated with the C.glutamicum genome integration plasmid pK18mobsacB linearized vector, and transferred into E.coli JM109, and the positive transformants were picked to construct the plasmid pK18-argRG:: argB _Ec .

(3) The pK18-argRG::argB _Ec plasmid was electroporated into C.glutamicum ATCC13032, and after electroporation at 1800V for 5ms, it was spread on the LBG solid medium plate containing kanamycin, and cultured at 30°C for 36h, the first homologous Recombinant transformants grow. Transformant strains were cultured in liquid LBG medium. Then, a secondary screen was performed in sucrose-containing medium. The secondary screening strain was identified by PCR, and the correct identified strain was named Cg-argRG::argB _Ec .

4. Result verification

The unmodified C.glutamicum ATCC13032 strain (control group) and Cg-argRG::argB _Ec (experimental group) were simultaneously fermented, and the changes of fermentation components in the fermentation broth were observed.

Among them, in the fermentation process, the fermentation medium components are: glucose 80g/L, yeast powder 8g/L, ammonium sulfate 40g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.02g/L L, ferrous sulfate 0.02g/L, calcium carbonate 20g/L. The fermentation temperature was 30 °C, and the shaking speed was 220 r/min.

Changes of fermentation components in the fermentation broth with fermentation time are shown in Tables 2 and 3.

Table 2: Variation of L-citrulline with fermentation time

12h 24h 36h control group ND ND ND test group 0.53g/L 2.6g/L 5.7g/L

Note: ND means not detected.

Table 3: Variation of L-arginine with fermentation time

12h 24h 36h control group ND ND ND test group ND ND ND

Note: ND means not detected.

It can be seen from this that the strain Cg-argRG::argB _Ec obtained by the genetic modification method adopted in the present invention has the argB _Ec gene inserted between the argR and argG genes in its genome, and meanwhile, the argR gene lacks the downstream part of the fragment, and the argG gene Missing upstream segments. Equivalent to C. glutamicum ATCC13032 strain argR and argG gene knockout simultaneously introduced the argB _Ec gene derived from E. coli. Through one-step genome modification, the L-citrulline synthesis operon repressor ArgR and argininosuccinate synthase were inactivated in C.glutamicum ATCC13032 strain, and the anti-L-arginine feedback from E. coli was expressed Inhibited N-acetylglutamate kinase. Through one-step genome modification, the L-citrulline synthesis pathway was enhanced in C.glutamicum ATCC13032 strain, while the L-citrulline to L-arginine conversion pathway was blocked.

The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and the descriptions in the above-mentioned embodiments and the description are only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

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Claims (9)

1. A method for producing L-citrulline by transforming corynebacterium glutamicum is characterized in that: modifying the genome of Corynebacterium glutamicum, knocking out a downstream fragment of argR gene and an upstream fragment of argG gene, and inserting argB of Escherichia coli into the knocked-out fragments_EcA gene.

2. The method for producing L-citrulline by transforming Corynebacterium glutamicum as claimed in claim 1, wherein: PCR amplification of argR gene upstream fragment argR_F、argB_EcGene and argG Gene downstream fragment argG_RObtaining argR_F-argB_Ec-argG_RA gene fragment.

3. The method for producing L-citrulline by modifying Corynebacterium glutamicum as claimed in claim 2, comprising the following steps:

(1) with argR_FGene and argB_EcThe gene mixture is used as a template, and primers argRF and argB are used_EcObtaining argR by PCR amplification of R overlap extension_F-argB_EcA gene fragment;

(2) with argR_F-argB_EcGene and argG_RThe gene mixture is used as a template, and the argR is obtained by PCR amplification by overlapping extension of primers arggRF and argGR_F-argB_Ec-argG_RA gene fragment.

4. The method for producing L-citrulline by transforming Corynebacterium glutamicum as claimed in claim 3, wherein: the sequence of the primer argRF is shown in SEQ ID NO. 4.

5. The method for producing L-citrulline by transforming Corynebacterium glutamicum as claimed in claim 3, wherein: the sequence of the primer argGR is shown as SEQ ID NO. 7.

6. The method for producing L-citrulline by transforming Corynebacterium glutamicum as claimed in claim 3, wherein: the primer argB_EcThe sequence of R is shown in SEQ ID NO. 9.

7. The method for producing L-citrulline by transforming Corynebacterium glutamicum as claimed in claim 1, wherein: coli BL21 strain, argB_EcThe nucleotide sequence of the gene is shown in SEQ ID NO. 3.

8. A corynebacterium glutamicum producing L-citrulline, comprising: the strain obtained by the method for producing L-citrulline by modifying corynebacterium glutamicum according to any one of claims 1 to 7.

9. L-citrulline is produced using Corynebacterium glutamicum of claim 8.