Corynebacterium glutamicum for producing L-citrulline and L-arginine and modification method and application thereof

文档序号：1388831 发布日期：2020-08-18 浏览：36次中文

阅读说明：本技术 生产l-瓜氨酸和l-精氨酸的谷氨酸棒杆菌及其改造方法和应用 (Corynebacterium glutamicum for producing L-citrulline and L-arginine and modification method and application thereof ) 是由满在伟郭静刘典典戴嘉辰许颖琦姚智仁于 2020-06-04 设计创作，主要内容包括：本发明公开了一种生产L-瓜氨酸和L-精氨酸的谷氨酸棒杆菌及其改造方法和应用,通过对谷氨酸棒杆菌基因组进行改造,在argR基因中插入大肠杆菌的argB<Sub>Ec</Sub>基因获得重组菌株。应用该改进的菌株进行发酵在发酵的过程中能够同时生成L-瓜氨酸和L-精氨酸。(The invention discloses a corynebacterium glutamicum for producing L-citrulline and L-arginine, a modification method and application thereof Ec The gene is obtained into a recombinant strain. The improved strain is applied to fermentation, and L-citrulline and L-arginine can be simultaneously generated in the fermentation process.)

1. A method for producing L-citrulline and L-arginine by transforming corynebacterium glutamicum is characterized in that: the genome of the corynebacterium glutamicum is modified,insertion of argB of E.coli into argR Gene_EcA gene.

2. The method for producing L-citrulline and L-arginine by transforming Corynebacterium glutamicum as claimed in claim 1, wherein: PCR amplification of argR gene upstream fragment argR_FAnd argR gene downstream fragment argR_RAnd argB_EcGene acquisition argR_F-argB_Ec-argR_RA gene fragment.

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

(1) with argR_FGene and argB_EcThe gene mixture was used as a template with primers argRF1 and argB_EcObtaining argR by PCR amplification of R overlap extension_F-argB_EcA gene fragment;

(2) with argR_F-argB_EcGene and argR_RThe argR is obtained by using a gene mixture as a template and performing PCR amplification by overlapping extension of primers argRF1 and argR 2_F-argB_Ec-argR_RA gene fragment.

4. The method for producing L-citrulline and L-arginine by transforming Corynebacterium glutamicum as claimed in claim 3, wherein: the sequence of the primer argRF1 is shown in SEQ ID NO. 3.

5. The method for producing L-citrulline and L-arginine by transforming Corynebacterium glutamicum as claimed in claim 3, wherein: the sequence of the primer argrR2 is shown in SEQ ID NO. 6.

6. The method for producing L-citrulline and L-arginine by transforming Corynebacterium glutamicum as claimed in claim 3, wherein: the primer argB_EcThe sequence of R is shown in SEQ ID NO. 8.

7. The method of claim 1A method for producing L-citrulline and L-arginine by transforming corynebacterium glutamicum is characterized in that: coli BL21 strain, argB_EcThe nucleotide sequence of the gene is shown in SEQ ID NO. 2.

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

9. The use of the Corynebacterium glutamicum of claim 8, in the simultaneous production of L-citrulline and L-arginine.

Technical Field

The invention belongs to the field of gene modification, and particularly relates to corynebacterium glutamicum for producing L-citrulline and L-arginine, a modification method and application thereof.

Background

L-citrulline is an important non-protein amino acid, participates in urea circulation to maintain blood ammonia balance, and has physiological functions of resisting oxidation, stabilizing blood pressure and the like. Therefore, the L-citrulline has very wide application prospect in the industries of medical drugs, foods and cosmetics. At present, the research on the synthesis of L-citrulline mainly focuses on an enzymatic conversion method and a fermentation method. The enzymatic conversion method is to generate L-citrulline by taking L-arginine as a substrate and utilizing arginine deiminase to catalyze. The price of the substrate L-arginine is high, so the enzyme conversion method has the problem of high substrate cost. The direct microbial fermentation method for producing L-citrulline can use cheap substrates such as glucose and has the advantages of simple process, low cost, small environmental pollution and the like, so that attention is paid to the method in recent years.

L-arginine is a semi-essential amino acid in human and animal bodies, and is also involved in the urea cycle to maintain blood ammonia balance. Is a synthetic precursor of various bioactive substances, has various unique physiological and pharmacological effects, and has wide application in the aspects of food, medicine, animal feed and the like.

Corynebacterium glutamicum (Corynebacterium glutamicum) is a facultative anaerobic gram-positive safe strain. With the development of research, especially metabolic engineering, glutamicum is widely applied to the fermentation production of various amino acids, organic acids, even biofuels, bio-based chemicals and the like. L-ornithine, L-citrulline and L-arginine in glutamicum exist in the same anabolic pathway, which can be called as L-arginine anabolic pathway. The final product of the metabolic pathway is L-arginine, and L-citrulline is an intermediate product of the metabolic reaction for synthesizing L-arginine from L-ornithine.

Glutaminum ATCC 13032L-arginine anabolic pathway-related genes exist on the chromosome as gene cluster argCJBDFRGH, divided into two operons, argCJBDFR and argGH, respectively. The genes encoding all enzymes in the L-citrulline major synthetic pathway are concentrated on the argCJBDFR operon. Wherein, the N-acetylglutamate kinase coded by the argB gene is a key rate-limiting enzyme for synthesizing L-citrulline and L-arginine, and meanwhile, the activity of the N-acetylglutamate kinase is feedback-inhibited by L-arginine, and the catalytic activity of the N-acetylglutamate kinase can be seriously inhibited by very low-concentration L-arginine. The repressor ArgR encoded by the argR gene can inhibit the transcription of argCJBDFR and argGH operons, thereby inhibiting the entire L-citrulline and L-arginine anabolism in cells(ii) a pathway. Therefore, L-citrulline and L-arginine anabolic pathways in C.glutamcum ATCC13032 cells are precisely metabolically controlled and do not excessively synthesize L-citrulline or L-arginine. And C, the fermentation liquid does not accumulate L-citrulline or L-arginine in the fermentation culture process of the glutamicumATCC13032. The excessive synthesis of L-citrulline and L-arginine can be realized only by releasing the metabolic regulation of cells on the anabolic pathway of L-citrulline and L-arginine. The activity of N-acetylglutamate kinase derived from Escherichia coli (Escherichia coli) is not inhibited by feedback inhibition of L-arginine, and the coding gene is named argB_Ec。

Disclosure of Invention

Aiming at the problems, the invention provides corynebacterium glutamicum for producing L-citrulline and L-arginine, and a modification method and application thereof.

The technical purpose is achieved, the technical effect is achieved, and the invention is realized through the following technical scheme:

a method for producing L-citrulline and L-arginine by transforming Corynebacterium glutamicum comprises transforming Corynebacterium glutamicum genome, and inserting argB of Escherichia coli into argR gene_EcA gene.

As a further improvement of the invention, PCR is adopted to amplify argR gene upstream segment argR_FAnd argR gene downstream fragment argR_RAnd argB_EcGene acquisition argR_F-argB_Ec-argR_RA gene fragment.

As a further improvement of the invention, the method comprises the following steps:

(1) with argR_FGene and argB_EcThe gene mixture was used as a template with primers argRF1 and argB_EcR overlap extension PCR amplification to obtain argRF-argB_EcA gene fragment;

As a further improvement of the invention, the sequence of the primer argRF1 is shown in SEQ ID NO. 3.

As a further improvement of the invention, the sequence of the primer argrR2 is shown in SEQ ID NO. 6.

As a further improvement of the invention, said primer argB_EcThe sequence of R is shown in SEQ ID NO. 8.

As a further improvement of the invention, E.coli BL21 strain is selected as the Escherichia coli, and argB is selected as the Escherichia coli_EcThe nucleotide sequence of the gene is shown in SEQ ID NO. 2.

The invention also provides a corynebacterium glutamicum for producing L-citrulline and L-arginine, and a strain obtained by the method for producing L-citrulline and L-arginine by modifying the corynebacterium glutamicum.

The invention also provides a method for simultaneously producing L-citrulline and L-arginine by using the corynebacterium glutamicum.

The invention has the beneficial effects that: the invention inserts argB in the middle of argR gene in C.glutamicum ATCC13032 strain genome through one-step genome modification_EcGene, corresponding to the deletion of argR gene of C.glutamcum ATCC13032 strain, and argB derived from E.coli_EcA gene. Through one-step genome modification, L-citrulline and L-arginine synthetic operon repressor ArgR are inactivated in a C.glutamicmATCC13032 strain, and N-acetylglutamate kinase which is derived from E.coli and is resistant to feedback inhibition of L-arginine is expressed at the same time. Glutamicum ATCC13032 strain relieved the metabolic regulation of the anabolic pathway of L-citrulline and L-arginine by the cell. The modified strain can simultaneously produce L-citrulline and L-arginine in the fermentation process.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

1. Material selection

Glutamicum mode strain ATCC13032 of corynebacterium glutamicum used, obtained by purchase. And the nucleotide sequence of the glutamicum ATCC13032 argR gene is shown in SEQ NO. 1.

Coli BL21, E.coli BL21 argB_EcThe gene nucleotide sequence is shown as SEQ NO. 2.

2. Primer design

Amplifying an argR gene upstream fragment argR by using a PCR amplification technology and using a C.glutamicum ATCC13032 genome as a template_FAnd the downstream fragment argR_RColi BL21 genome as module for amplification of argB_EcA gene. The designed sequence table 1 is as follows:

table 1: primer sequences

3. Gene recombination

(1) With argR_FGene and argB_EcThe gene mixture was used as a template with primers argRF1 and argB_EcObtaining argR by PCR amplification of R overlap extension_F-argB_EcA gene fragment.

With argR_F-argB_EcGene and argR_RThe argR is obtained by using a gene mixture as a template and performing PCR amplification by overlapping extension of primers argRF1 and argR 2_F-argB_Ec-argR_RA gene fragment.

(2) argR to be obtained_F-argB_Ec-argR_RThe gene fragment was ligated with the C.glutamicum genome integration plasmid pK18mobsacB linearized vector and transferred to E.coli JM109, positive transformants were picked, and the plasmid pK18-argR was constructed_Ec。

(3) Mixing pK18-argR_EcPlasmid electric shock transformation C.glutamicum ATCC13032, after 1800V, 5ms electric shock, spread on LBG solid medium plate containing kanamycin, 30 degrees C culture for 36h, the first homologous recombination transformant grows out. The transformant strain was cultured in liquid LBG medium. Then, secondary screening was performed in a sucrose-containing medium. The secondary screening strain is identified by PCR, and the identified correct strain is named Cg-argR:: argB_Ec。

4. Result verification

Glutaminum ATCC13032 strain (control group) without gene modification and Cg-argR_Ec(experimental group) fermentation was performed simultaneously, and the change of fermentation components in the fermentation broth was observed.

Wherein, in the fermentation process, the components of the fermentation medium are as follows: 80g/L of glucose, 8g/L of yeast powder, 40g/L of ammonium sulfate, 1.5g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.02g/L of manganese sulfate, 0.02g/L of ferrous sulfate and 20g/L of calcium carbonate. The fermentation temperature is 30 ℃, and the rotating speed of a shaking table is 220 r/min.

The fermentation composition in the fermentation broth was varied with the fermentation time as shown in tables 2 and 3.

Table 2: variation of L-citrulline with fermentation time

	12h	24h	36h
				Control group	ND	ND	ND
Experimental group	0.32g/L	1.9g/L	3.6g/L

Note: ND means not detectable.

Table 3: variation of L-arginine with fermentation time

	12h	24h	36h
				Control group	ND	ND	ND
Experimental group	0.14g/L	0.47g/L	1.1g/L

Note: ND means not detectable.

From this, it can be seen that the strain Cg-argR:: argB_EcArgB is inserted in middle of argR gene in genome_EcGene, corresponding to the deletion of argR gene of C.glutamcum ATCC13032 strain, and argB derived from E.coli_EcA gene. Through one-step genome transformation, L-citrulline and L-arginine are inactivated in a C.glutamicum ATCC13032 strain to synthesize an operon repressor protein ArgR, and N-acetylglutamate kinase which is derived from E.coli and is resistant to feedback inhibition of L-arginine is expressed at the same time. The fermentation production of L-citrulline and L-arginine by the C.glutamicum ATCC13032 strain is realized through one-step genome transformation.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

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