CN115851657A - Acetyl glutamate kinase for promoting arginine synthesis - Google Patents

Abstract

Compared with the wild type acetylglutamate kinase SEQ ID NO. 2, the acetylglutamate kinase mutant SEQ ID NO. 11 screened by the genetic engineering technology has the advantage that the L-arginine synthesis promoting capability is remarkably improved, and the method can be used for the transformation of L-arginine producing bacteria.

Description

Acetyl glutamate kinase for promoting arginine synthesis

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to an acetylglutamate kinase mutant and application thereof in improving the ability of microorganisms to synthesize L-arginine.

Background

L-arginine is a conditionally essential amino acid in humans and has important physiological functions. In vivo, it is involved in regulating the balance of the immune system, enhancing the immune function of the body, and also in ornithine circulation, converting the ammonia in vivo into non-toxic urea to be discharged out of the body, thereby reducing the blood ammonia concentration (Guo Changjiang, xuqishou. Arginine has an in vitro regulating effect on the immune cell function. Amino acids journal, 1991 (1): 4-5.). In addition, arginine is also involved in the synthesis of nitric oxide, phosphoric acid, creatine, and the like in vivo. The L-arginine has important economic value and is widely applied in the fields of medicine, cosmetics, food, animal husbandry and the like at present. Particularly in the pharmaceutical industry, intravenous injection of L-arginine can promote the detoxification of liver, has remarkable curative effect on hepatic coma caused by ammonia poisoning, and arginine supplementation can effectively promote the synthesis of collagen at wounds and accelerate the healing of damaged tissues (Sax HC. Arginine coatings around healing and immunity functions in the absence of collagen leather. JPEN J Parenter Enterprise Nutr,1994,18 (6): 559-560.).

Currently, protein hydrolysis or microbial fermentation is the primary method for producing arginine. The protein hydrolysis method is to heat cheap animal hair, gelatin and the like serving as raw materials under an acidic condition to release an amino acid mixture, and finally obtain an L-arginine product through steps of ion exchange resin, activated carbon decoloration and the like. The method can obtain L-arginine finished products, but has a plurality of defects: one is that the yield is low, and the purity of the product is difficult to reach the drug approval standard; secondly, the process is complex, and a large amount of wastewater containing salt and organic reagents is generated, thereby causing environmental pollution. Therefore, the protein hydrolysis method is limited in large-scale application. The microbial fermentation method is a technical means of utilizing genetic engineering, mutation breeding, fermentation engineering and the like, synthesizes arginine based on microorganisms, and has the advantages of low production cost, high product purity, mild production conditions and the like. Corynebacterium glutamicum and Corynebacterium crenatum are frequently used as the basal cell synthesis. In 2014, C.glutamicum ATCC 21831 was modified by Park SH et al using Metabolic engineering technology to synthesize L-arginine by fermentation, and the fermentation level of L-arginine in 5L and 1500L bioreactors reached 92.5g/L and 81.2g/L, respectively (Park SH, kim HU, kim TY, metabolism engineering of Corynebacterium glutamicum for L-arginine production. Nat. Commin., 2014, 5. However, the use of C.glutamicum has the disadvantages of low production intensity, long fermentation period, and easily fluctuating production, and is not an optimal production strain, and acetyl glutamate kinase involved in the L-arginine synthesis pathway in C.glutamicum is subject to feedback inhibition by the product L-arginine (Schendzielorz G, dipong M, gr ü nberger A, et al. Taking control over: use of production sensing in single cells to remove fluoro control at enzymes in biosthesis pathways. Synth.biol.,2014,3 (1): 21-29).

Disclosure of Invention

In order to overcome the defect of feedback inhibition of a product L-arginine during the production of L-arginine by a Corynebacterium glutamicum fermentation method and expand the range of a host of engineering bacteria, the inventor deeply researches the biosynthesis way of the L-arginine, makes a breakthrough on the modification of the acetoglutamate kinase SEQ ID NO. 2, and obtains a mutant with the enzyme activity improved by a plurality of times by using a random mutation and high-throughput screening method. Accordingly, the present invention provides the following technical solutions.

An acetylglutamate kinase which is a polypeptide selected from the group consisting of:

(1) A polypeptide having the amino acid sequence of SEQ ID NO. 11;

(2) A polypeptide having 90% or more homology with SEQ ID NO. 11, preferably 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, more preferably 99% or more homology with SEQ ID NO. 11, and having an improved enzymatic activity as compared with SEQ ID NO. 11.

MNDLIKDLGSEVGANVLAEALPWLQHFRDKIVVVKYGGSAMVDHDLKAAFAADMVFLRTVGAKPVVVHGGGPQISEMLNRVGLQGEFKGGFRVTTPEVMDIVRMVLFGQVGRDLVGLINSHGPYAVGTSGEDAGLFTAQKRMVNIDGVPTDIGLVGDIINVDASSLMDIIEAGRIPVVSTIAPGEDGQIYSINADTAAGALAAAIDAERLLVLTNVEGLYNDWPDKSSLVSKIKATELEAILPGLDSGMIPKMESCLNAVRGGVSAAHVIDGRIAHSVLLELLTMGGIGTMVLPDVFDRENYPEGTVFRKDDKDGEL(SEQ ID NO:11)。

In a second aspect, the present invention provides a polynucleotide which is a gene encoding the above-mentioned polypeptide.

Preferably, the gene encoding SEQ ID NO. 11 is a polynucleotide represented by SEQ ID NO. 10 or a polynucleotide having 90% or more, preferably 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, more preferably 99% or more homology with SEQ ID NO. 10.

The third aspect of the present invention provides the use of the above-mentioned polypeptide, or the above-mentioned polynucleotide, for promoting the production of L-arginine by a microorganism.

Specifically, the above-mentioned microorganism can produce L-arginine by fermentation. The microorganism is L-arginine producing bacteria selected from Corynebacterium glutamicum, corynebacterium crenatum, and Escherichia coli.

A fourth aspect of the present invention provides a microorganism having an ability to synthesize L-arginine, which expresses the above-mentioned gene.

The microbial host bacteria are selected from Corynebacterium glutamicum, corynebacterium crenatum and Escherichia coli, for example the host bacteria can be Corynebacterium glutamicum ATCC 21831 or Escherichia coli W3110.

In one embodiment, the microorganism is an engineered bacterium, i.e., the native acetylglutamate kinase coding gene in the genome of the microorganism host is replaced by the above-mentioned gene; or the genome of the microbial host bacterium is integrated with the gene.

The integration of the above-mentioned coding gene can be achieved by transforming a plasmid containing the gene into the microbial host bacterium, or by directly cloning the gene into the genome of the host bacterium.

Wherein the cloning of the above genes into the genome of the microbial host bacteria and the knock-out of a certain gene are performed by a gene editing technique.

The gene editing technology can adopt homologous double exchange, a CRISPR-Cas9 system, a CRISPR-Cpf1 system, a CRISPR-Cas related transposition system INTEGRATE system or a CAST system.

The INTEGRATE system refers to a gene editing tool (Insertion of RNA-assisted targeting transposable element for guiding RNA to assist in targeting) developed by Sam Sternberg research group; the CAST system is a gene editing tool (CRISPR-associated transposase) developed by the tensor research group.

When the host bacterium is Escherichia coli W3110, the L-arginine producing bacterium (engineering bacterium) can be constructed by the following steps:

A. knocking out repressor protein gene argR in a genome to obtain a strain ARG-1 (E.coliW3110 delta argR);

B. knocking out an endogenous acetylglutamate kinase gene argB in the genome of the strain obtained in the step A to obtain a strain ARG-2 (E.coli W3110 delta argR delta argB);

C. integrating the exogenous gene into the strain genome obtained in the step B to obtain a strain ARG-3 (E.coli W3110 delta argR delta argB/pARGB) for expressing the acetylglutamate kinase, which is an L-arginine engineering bacterium.

Optionally, in order to facilitate screening of arginine expression level capable of visually displaying the engineering bacteria, the construction of the engineering bacteria may further include the following steps: and (3) constructing an arginine-RFP-sacB biosensing system, and transferring a plasmid pCARS for expressing the arginine-RFP-sacB biosensing system into the strain obtained in the step (C) to obtain a strain ARG-5 (E.coli W3110 delta argR delta argB/pARGB/pCARS).

The microorganism having the above-mentioned acetylglutamate kinase gene integrated into the genome has a potential for use in the production of L-arginine.

The acetyl glutamate kinase SEQ ID NO. 11 screened by the invention is a mutant of wild acetyl glutamate kinase SEQ ID NO. 2, compared with wild enzyme, the mutant can improve the L-arginine synthesis level of the strain by about 3 times, and has huge application potential.

Drawings

FIG. 1 is a plasmid map of plasmid pARGB constructed in the present invention for expressing an acetylglutamate kinase gene derived from Corynebacterium glutamicum ATCC 13032.

FIG. 2 is a plasmid map of plasmid pCARS constructed in accordance with the present invention for expression of the arginine-RFP-sacB biosensing system.

Detailed Description

Modification of L-arginine tolerance by mutation of the wild-type acetylglutamate kinase which is feedback-inhibited by the product L-arginine in C.glutamicum for the production of L-arginine may be a way to increase L-arginine production.

Therefore, wild-type acetyl glutamate kinase with the amino acid sequence of SEQ ID NO:2 (GenBank: CP 025533.1) from Corynebacterium glutamicum ATCC13032 is taken as a modified object, and random mutation and high-throughput screening are carried out in Escherichia coli which is most commonly used in the field of genetic engineering so as to obtain mutant enzyme with higher enzyme activity or resistance to feedback inhibition of L-arginine.

As used herein, the terms "wild type", "wild enzyme" and "wild-type enzyme" are intended to have the same meaning and refer to the wild-type acetylglutamate kinase SEQ ID NO:2:

MNDLIKDLGSEVRANVLAEALPWLQHFRDKIVVVKYGGNAMVDDDLKAAFAADMVFLRTVGAKPVVVHGGGPQISEMLNRVGLQGEFKGGFRVTTPEVMDIVRMVLFGQVGRDLVGLINSHGPYAVGTSGEDAGLFTAQKRMVNIDGVPTDIGLVGDIINVDASSLMDIIEAGRIPVVSTIAPGEDGQIYNINADTAAGALAAAIGAERLLVLTNVEGLYTDWPDKSSLVSKIKATELEAILPGLDSGMIPKMESCLNAVRGGVSAAHVIDGRIAHSVLLELLTMGGIGTMVLPDVFDRENYPEGTVFRKDDKDGEL(SEQ ID NO:2)。

similarly, the terms "acetylglutamate kinase mutant", "mutant acetylglutamate kinase" and "mutant enzyme" have the same meaning, and refer to the mutant of acetylglutamate kinase, SEQ ID NO:11, in which the 13 th arginine is mutated to glycine (R13G), the 39 th asparagine is mutated to serine (N39S), the 44 th aspartic acid is mutated to histidine (D44H), the 191 th asparagine is mutated to serine (N191S), the 206 th glycine is mutated to aspartic acid (G206D), and the 221 th threonine is mutated to asparagine (T221N).

Sometimes for convenience of expression, the wild enzyme SEQ ID NO 2 and its mutant SEQ ID NO 11 and the like may be collectively referred to herein as "acetylglutamate kinase".

After the acetyl glutamate kinase mutant is over-expressed in Escherichia coli W3110 producing L-arginine, the L-arginine producing capacity of the strain is improved by 2.7 times.

It is expected that the acetyl glutamate kinase mutant can also be used for other L-arginine producing strains including Corynebacterium glutamicum and Corynebacterium crenatum to improve the L-arginine biosynthesis level of engineering bacteria.

It is easily understood that in order to express exogenous acetylglutamate kinase in different host bacteria, the gene for expression of the enzyme may be codon-optimized. Codon optimization is one technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms often show a special preference for one of several codons encoding the same amino acid due to mutation tendencies and natural selection. For example, in rapidly growing microorganisms such as E.coli, the optimized codons reflect the composition of their respective pools of genomic tRNA's. Thus, in a fast growing microorganism, low frequency codons for an amino acid can be replaced with codons for the same amino acid but with a high frequency. Thus, expression of optimized DNA sequences is improved in fast growing microorganisms.

For example, for expression of a mutant of acetylglutamate kinase, SEQ ID NO. 11, in E.coli, the codon-optimized encoding gene may be SEQ ID NO. 10, but is not limited thereto.

In order to facilitate the high-throughput screening of mutant enzymes, the invention also designs a detection system for coupling detection of the acetylglutamate kinase, namely an arginine-RFP-sacB biosensing system, so as to intuitively display the arginine expression level of the strain, thereby improving the screening efficiency of the forward mutant. When the biosensing system is applied to different microorganism species, preferential codon optimization should be carried out.

Examples

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

In the examples, the addition, content and concentration of various substances are mentioned, wherein the percentages refer to mass percentages unless otherwise indicated.

Materials and methods

The whole gene synthesis, primer synthesis and sequencing in the examples were performed by Jinzhi Biotechnology, inc., suzhou.

The molecular biological experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, culture medium preparation, and the like, and are mainly performed with reference to "molecular cloning experimental manual" (third edition), sambrook, d.w. rasel (american), translation of huang peitang et al, scientific press, beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.

The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.

LB medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2. (20 g/L agar powder was additionally added to LB solid medium.)

AE medium components: glucose 20g/L, peptone 4g/L, yeast extract 2g/L, mgSO ₄ ·7H ₂ O 2.5g/L，FeCl ₃ 17.2mg/L，MnSO ₄ ·4H ₂ O 5mg/L，ZnSO ₄ ·7H ₂ O 0.2mg/L，CuSO ₄ ·5H ₂ O0.15 mg/L, vitamin B1 mg/L, vitamin B5 mg/L, vitamin B12 mg/L, pH 7.0-7.1.

Chromatographic conditions for L-arginine:

example 1: knockout of repressor Gene argR

1.1 construction of the targeting fragment ARGR-3

The ARGR-1 fragment was amplified by using E.coli W3110 genome (GenBank: AP 009048.1) as a template and the ARGR-51/ARGR-31 primer pair, the ARGR-2 fragment was amplified by using the ARGR-52/ARGR-32 primer pair, both fragments were recovered, the ARGR-1 and ARGR-2 fragments were used as templates, the ARGR-51/ARGR-32 primer pair was amplified to obtain the ARGR-3 targeting fragment, and the fragments were recovered by cutting.

And (3) PCR reaction system: 10ng of template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNAA polymerase.

And (3) PCR reaction conditions: 95 ℃ for 3min; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

The primer sequences are as follows:

ARGR-51：5’-TATACCCTACCGTTCAGCCAA-3’，

ARGR-31：5’-CATAAGTCACCCGATATGGTG-3’，

ARGR-52：5’-CCATATCGGGTGACTTATGTAATCTCTGCCCCGTCGTTTC-3’，

ARGR-32：5’-CTGTTTCAGTTGATACTAAGT-3’。

2.2 construction of pTargetF-argR

Circular amplification was performed using the Plasmid pTargetF (Addgene Plasmid: # 62226) as a template and argRSpc/gTEMdn as primers to obtain a pTarget-argR Plasmid of about 2.2 kb. The primer sequences are as follows:

argRspc：

5’-TAATACTAGTTTACTGCCTGCCAGCTGAACGTTTTAGAGCTAGAAATAGC-3’.

gTEMdn：5’-CTCAAAAAAAGCACCGACTCGG-3’。

the PCR reaction system comprises: 10ng of template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNA polymerase.

The PCR reaction conditions were: 3min at 95 ℃; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

mu.L of the PCR product was amplified, and 1. Mu.L of DpnI restriction enzyme (Thermo Co.) was added directly to digest the plasmid template and transform E.coli DH5a competent E.coli.

2.3 knockout of E.coli W3110 endogenous argR Gene

Plasmid pCas (Addgene Plasmid: # 62225) was electroporated into E.coli W3110 competent cells, positive clones were picked up, transferred to a tube containing 5mL of LB (containing 50. Mu.g/mL kanamycin sulfate), and cultured overnight at 220rm at 30 ℃. The following day, the mixture was inoculated at a volume concentration of 1% into a 250mL shake flask containing 50mL of LB liquid medium (containing 10mM of L-arabinose), and incubated at 30 ℃ and 220rpm until OD _600nm 0.4-0.6, at 4 deg.C and 4000rpmThe thalli is collected in the heart for 10min, and the electrotransformation competent cells are prepared.

Mixing 100ng of pTargetF-argR plasmid and 500ng of ARGR-3 targeting fragment with 100 μ L of E.coli W3110/pCas electroporation competent cells, transferring into a precooled 2mm electroporation cuvette, performing electroporation transformation with an electroporation apparatus, immediately adding precooled 1mL of LB medium and transferring into a 1.5mL centrifuge tube after electroporation is finished, recovering at 30 ℃, and coating after 1.5h at 180rpm

LB plates containing 50. Mu.g/mL kanamycin sulfate and 50. Mu.g/mL spectinomycin hydrochloride were cultured in an inverted state at 30 ℃ for 24 hours. Colony PCR was verified using ARGR-01 and ARGR-02 as primers, and about 0.6kb was positive, yielding E.coli W3110 Δ argR/pCas9/pTargetF-argR.

ARGR-01：5’-GTAATCAGGAACCTAACTTAC-3’，

ARGR-02：5’-CTTTGTTTCGTGACCCATATC-3’。

Helper plasmid elimination: a single colony of E.coli W3110. Delta. ArgR/pCas9/pTargetF-argR was picked, inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin sulfate and 1mM IPTG, and incubated overnight at 30 ℃ and 220 rpm. The next day overnight colonies were plated on solid LB medium containing 50. Mu.g/mL kanamycin sulfate and incubated at 30 ℃ for 24h, indicating successful elimination of pTargetF-argR plasmid if a single colony was sensitive to 50. Mu.g/mL spectinomycin hydrochloride. Single colonies that successfully eliminated the pTargetF-argR plasmid were picked, incubated at 42 ℃ for 6h at 220rpm, and then plated on LB plates without antibiotic addition and incubated at 42 ℃ for 12h. If a single colony is sensitive to 50. Mu.g/mL kanamycin sulfate, successful elimination of the pCas plasmid is indicated, and finally the strain ARG-1 (E.coli W3110. Delta. ArgR) is obtained.

Example 2: knock-out endogenous acetylglutamate kinase gene argB of Escherichia coli

2.1 construction of the targeting fragment ARGB-3

Taking an Escherichia coli W3110 genome (GenBank: AP 009048.1) as a template, and designing the following primer pair ARGB-51/ARGB-31 for amplification to obtain an ARGB-1 fragment:

forward primer ARGB-51:5 'GTGACCAGGGCTTTGGACCAG-containing 3',

reverse primer ARGB-31:5 'GCACTGTTTAACGGTATGCCG-doped 3'.

The following primer pair ARGB-52/ARGB-32 is designed for amplification to obtain an ARGB-2 fragment:

forward primer ARGB-52:

5’-GGCATACCGTTAAACAGTGCATTCACCAGTGCGCTAAACAG-3’，

reverse primer ARGB-32:5 'GTTGGGCTGCCATTTTGCGAT-3'.

The ARGB-1 and ARGB-2 fragments are simultaneously taken as templates, and the ARGB-51/ARGB-32 is amplified by a primer pair to obtain an ARGB-3 targeting fragment.

The PCR reaction system comprises: 10ng of template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNA polymerase.

The PCR reaction conditions are as follows: 3min at 95 ℃; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

2.2 construction of pTargetF-argB

The pTarget-argB Plasmid was obtained by circular amplification of argBspc/gTEMdn primer pair designed using Plasmid pTargetF (Addge Plasmid: # 62226) as a template:

forward primer argBspc:

5’-TAATACTAGTGACCTAACTCTTCATCAAGCGTTTTAGAGCTAGAAATAGC-3’,

reverse primer gtemmdn: 5 'CTCAAAAAAAGCACCGATCGG-doped 3'.

PCR amplification was performed using the plasmid pTargetF as a template to obtain pTargetF-argB plasmid of about 2.2 kb.

The 50 μ L PCR reaction included: 10ng of template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNA polymerase.

The PCR reaction conditions are as follows: 3min at 95 ℃; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

After 50. Mu.L of the amplification, 1. Mu.L of DpnI restriction enzyme (Thermo Co.) was directly added to the PCR solution to digest the plasmid template, thereby transforming E.coli DH5a.

2.3 knockout of argB Gene

Plasmid pCas (Addgene Plasmid: # 62225) was electroporated into strain ARG-1 competent cells, and positive cells were pickedThe sex clones were transferred to a tube containing 5mL LB (containing 50. Mu.g/mL kanamycin sulfate) and cultured overnight at 30 ℃ and 220 rm. The following day, the cells were inoculated at a volume concentration of 1% into a 250mL flask containing 50mL of LB liquid medium (containing 10mM of L-arabinose) and incubated at 30 ℃ and 220rpm to OD _600nm 0.4-0.6, centrifuging at 4 deg.C and 4000rpm for 10min to collect thallus, and preparing electroporation competent cells.

Mixing 100ng of pTargetF-argB plasmid and 500ng of ARGB-3 targeting fragment with 100 μ L of E.coli W3110/pCas electroporation competent cells, transferring into a precooled 2mm electroporation cuvette, performing electroporation transformation with an electroporation apparatus, immediately adding precooled 1mL of LB medium and transferring into a 1.5mL centrifuge tube after electroporation is finished, recovering at 30 ℃, and coating after 1.5h at 180rpm

LB plates containing 50. Mu.g/mL kanamycin sulfate and 50. Mu.g/mL spectinomycin hydrochloride were cultured in an inverted state at 30 ℃ for 24 hours. Colony PCR was verified using ARGB-01 and ARGB-02 as primers, and about 0.6kb was positive and 1.2kb was negative, to obtain E.coli ARG-1. Delta. ArgB/pCas9/pTargetF-argB.

ARGB-01：5’-GCGAACATCTTCCAGCAACAC-3’，

ARGB-02：5’-TGCGGCTGTATGACAAAGGCG-3’。

Helper plasmid elimination: single colonies of ARG-1. Delta. ArgB/pCas9/pTargetF-argB were picked, inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin sulfate and 1mM IPTG, and incubated overnight at 220rpm at 30 ℃. The next day overnight colonies were plated on solid LB medium containing 50. Mu.g/mL kanamycin sulfate and incubated at 30 ℃ for 24h, indicating successful elimination of the pTargetF-argB plasmid if a single colony was sensitive to 50. Mu.g/mL spectinomycin hydrochloride. Single colonies that successfully eliminated the pTargetF-argB plasmid were picked, incubated at 42 ℃ for 6h at 220rpm, and then plated on LB plates without antibiotic addition and incubated at 42 ℃ for 12h. If a single colony is sensitive to 50. Mu.g/mL kanamycin sulfate, successful elimination of the pCas plasmid is indicated, and ARG-2 (E.coli W3110. Delta. ArgR. Delta. ArgB) is finally obtained.

Example 3: construction of Strain overexpressing C.Glutamicum derived acetylglutamate kinase Gene argB

The following primer pair ARGB-54/ARGB-34 is designed for amplification to obtain an ARGB-4 fragment:

forward primer ARGB-54:

5’-TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCTCGAGTAAGGAGGATATTTAGATGAATGACTTGATCAAAGATTTAG-3’，

reverse primer ARGB-34:

5’-TTACAGTTCCCCATCCTTGTC-3’。

PCR amplification was performed using Corynebacterium glutamicum ATCC13032 genome (GenBank: CP 025533.1) as a template.

The 50 μ L PCR reaction included: 100ng of genomic template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNA polymerase.

The PCR reaction conditions are as follows: 3min at 95 ℃; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

mu.L of the amplified PCR product was recovered by cutting gel to obtain an ARGB-4 fragment of about 1.0 kb. The sequence of the C.glutamicum argB gene contained in the ARGB-4 fragment is shown as SEQ ID NO. 1, and the corresponding protein sequence is shown as SEQ ID NO. 2.

The following primer pair TRC-5/TRC-3 is designed to be amplified to obtain a TRC framework:

forward primer TRC-5:

5’-ACAAGGATGGGGAACTGTAAAGAGTAGGGAACTGCCAGGCAT-3’，

reverse primer TRC-3:

5’-AGGACTGAGCTAGCTGTCAACAGCTCATTTCAGAATATTTGC-3’。

PCR amplification was performed using the plasmid pTrc99A as a template.

The 50 μ L PCR reaction included: 10ng of template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNA polymerase.

The PCR reaction conditions are as follows: 3min at 95 ℃; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

After 50. Mu.L of amplification, 1. Mu.L of DpnI restriction enzyme (Thermo Co.) was directly added to the PCR solution to digest the plasmid template, and the gel was cut and recovered to obtain a TRC backbone of about 3.9 kb.

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TRC backbone and ARGB-4 fragment were assembled in vitro using the Seamless Cloning and Assembly Kit to obtain plasmid pARGB, the map of which is shown in FIG. 1.

The plasmid pARGB was transformed into the strain ARG-2 to obtain the strain ARG-3 (E.coli W3110. DELTA. ArgR. DELTA. ArgB/pARGB)

Example 4: shaking flask horizontal fermentation synthesis of L-arginine

The deposited glycerol strain was streaked onto LB plates (strains W3110, ARG-1, and ARG-2 used no antibiotics, and strain ARG-3 added 100. Mu.g/mL ampicillin), and a single colony was picked up and dropped into 5mL of liquid LB medium, and incubated at 37 ℃ and 220rpm for 10 to 14 hours to be used as a seed solution. The L-arginine synthesis level was determined by inoculating 1mL of the seed solution into a 500mL shake flask containing 100mL of LAE medium, incubating at 220rpm and 30 ℃ for 48 hours, and sampling, and the results are shown in Table 1.

TABLE 1L-arginine Synthesis levels

Bacterial strains	L-arginine level of synthesis (g/L)
		W3110	0.07
ARG-1	0.94
		ARG-2	0
ARG-3	1.07

As can be seen from Table 1, after the argR gene is knocked out by the starting strain W3110, the L-arginine synthesis level is greatly improved by 12 times; however, after the endogenous acetylglutamate kinase gene argB is continuously knocked out, the strain loses the L-arginine production capacity because the L-arginine synthesis path is interrupted; when the acetoglutamate kinase gene argB derived from corynebacterium glutamicum is integrated, the strain recovers an L-arginine synthesis path, so that the L-arginine production capacity is recovered, and a small increase of the L-arginine production capacity probably indicates that the enzyme activity of exogenous acetoglutamate kinase SEQ ID NO. 2 is slightly higher than that of the endogenous acetoglutamate kinase of the strain W3110.

Example 5: construction of arginine-RFP-sacB biosensing System

The following primer pair ARGR-54/ARGR-34 was designed to amplify to obtain the segment ARGR-4 containing the gene argR, and the constitutive promoter pJ23119 was introduced:

forward primer ARGR-54:

5’-TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCTCGAGTAAGGAGGATATTTAGATGTCCCTTGGCTCAACCCC-3’，

reverse primer ARGR-34:

5’-GGATGATTTGGACCTTGGTTAAGTGGTGCGCCCGCTGA-3’。

PCR amplification was performed using Corynebacterium glutamicum ATCC13032 genome (GenBank: CP 025533.1) as a template.

The 50 μ L PCR reaction included: 100ng of genomic template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNA polymerase.

The PCR reaction conditions are as follows: 3min at 95 ℃; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

After 50. Mu.L of amplification, PCR, electrophoresis and gel recovery, an ARGR-4 fragment of about 0.5kb was obtained.

The argR gene nucleic acid sequence contained in the ARGR-4 segment is shown as SEQ ID NO. 3, and the corresponding protein sequence is shown as SEQ ID NO. 4.

The protein sequence (GenBank: AAM 54544.1) of Red Fluorescent Protein (RFP) is shown as SEQ ID NO. 6, and the obtained nucleic acid sequence is shown as SEQ ID NO. 5 by optimizing according to the codon preference of escherichia coli.

The protein sequence (GenBank: WP-187954058.1) of the levosucrase (sacB) is shown in SEQ ID NO. 8, and the nucleic acid sequence obtained by optimization according to the codon bias of Escherichia coli is shown in SEQ ID NO. 7.

The argC promoter-red fluorescent protein-levansucrase nucleic acid sequence is shown in SEQ ID NO 9, and is synthesized by Jinzhi Biotechnology Limited, suzhou and cloned to EcoRI-HindIII sites of a pUC57 vector to obtain pUC57-RS.

Designing the following primer pair RS-51/RS-31 amplification fragment RS-1:

forward primer RS-51:

5’-CCAAGGTCCAAATCATCCACTAAAAATTCATGCTTTTACCCAC-3’，

reverse primer RS-31:

5’-TTATTTGTTCACGGTCAGCTGAC-3’。

PCR amplification was performed using the plasmid pUC57-RS as a template.

The 50 μ L PCR reaction included: 10ng of template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNA polymerase.

The PCR reaction conditions are as follows: 3min at 95 ℃; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

After 50. Mu.L of PCR amplification, 1. Mu.L of DpnI restriction enzyme (Thermo Co.) was directly added to digest the plasmid template, followed by electrophoresis and gel recovery to obtain an RS-1 fragment of about 2.3 kb.

The following primer pair CM-51/CM-31 was designed

Forward primer CM-51:

5’-AGCTGACCGTGAACAAATAATGCCATTCATCCGCTTATTa-3’，

reverse primer CM-31:

5’-AGGACTGAGCTAGCTGTCAAGAGCCTTCAACCCAGTCAG-3’。

PCR amplification was performed using plasmid pACYCDuet-1 as a template.

The 50 μ L PCR reaction included: 10ng of template, 10pmol of primer set, 1 XKODplus buffer,0.2mM dNTP,1.5mM MgSO ₄ 1 unit of KOD-plus DNA polymerase.

The PCR reaction conditions are as follows: 3min at 95 ℃; 30 cycles of 98 ℃ 10s,57 ℃ 30s,68 ℃ 2 min/kb; 10min at 68 ℃.

After 50. Mu.L of amplification, 1. Mu.L of DpnI restriction enzyme (Thermo Co.) was directly added to digest the plasmid template, followed by electrophoresis and gel recovery to obtain a CM-1 skeleton of about 2.1 kb.

By Beijing-Authentic Biotechnology Ltd

The scaffold CM-1, fragment RS-1 and fragment ARGR-1 were assembled in vitro using the Seamless Cloning and Assembly Kit to obtain plasmid pCARS, the plasmid map is shown in FIG. 2.

The plasmid pCARS was transformed into the strain ARG-2 to obtain a strain ARG-4 (E.coli W3110. DELTA. ArgR. DELTA. ArgB/pCARS).

Plasmid pCARS was transformed into strain ARG-3 to obtain ARG-5 (E.coli W3110. DELTA. ArgR. DELTA. ArgB/pARGB/pCARS).

Example 6: construction of Glutamicam-derived acetylglutamate kinase argB mutant

And (3) constructing a random mutant library by using an error-prone PCR (polymerase chain reaction) technology by taking the plasmid pARGB as a template.

The following primer pair ARGB-55/ARGR-35 is designed:

forward primer ARGB-55:5 'ATGAATGACTTGATCAAAGATTTAG-3',

reverse primer ARGB-34:5 'TTACAGTTCCCCCATCTCTTGTTC-3'.

PCR amplification was performed using the plasmid pARGB as a template to obtain a mutant DNA sequence of about 1.0 kb.

The 50. Mu.L error-prone PCR reaction system comprises: 10ng of plasmid (pARGB) template, 50pmol of a pair of primers ARGB-55 and ARGB-34,1 XTaq buffer,0.2mM dGTP,0.2mM dATP,1mM dCTP,1mM dTTP,7mM MgCl ₂ ，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl ₂ 2.5 units of Taq enzyme (Takara).

The PCR reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ 30s,55 ℃ 30s,72 ℃ 2 min/kbp; 10min at 72 ℃.

And cutting and recovering PCR products. Megaprimer PCR was performed using KOD-plus DNA polymerase using plasmid pARGB as a template and about 1.0kb of the recovered product (randomly mutated fragment) as a large primer: 5min at 94 ℃; 10s at 98 ℃, 30s at 60 ℃, 2min/kb at 68 ℃ and 25 cycles; 10min at 68 ℃.

The plasmid template was digested with DpnI restriction enzyme (Thermo Co., ltd.), and E.coli ARG-4 was electroporated to a yield of more than 10 ⁴ (iv) a library of randomly mutated acetylglutamate kinase argB from individual clones C.

Example 7: construction of acetylglutamate kinase mutant library

The C.glutamicum-derived argB random mutant library obtained in example 6 was subjected to high-throughput screening, and a single colony was picked up in a 96-well deep-well plate (containing 300. Mu.L of liquid LB-Amp-Cm medium per well), incubated at 37 ℃ and 300rpm for 18 hours, and then 50. Mu.L of the bacterial suspension was taken out from each well in a 96-well deep-well plate (containing 450. Mu.L of liquid AE-Amp-Cm medium per well), and incubated at 30 ℃ and 400rpm for 24 hours. Subsequently, sucrose stock solution was added to reach a final concentration of 10% per well, and incubation was continued for 12h. Selecting the clone with better growth and lighter red, centrifuging for 10min at 4 ℃ and 4000rpm, and taking the supernatant to detect the content of the L-arginine by using HPLC.

When the concentration of L-arginine in the cell is higher, repressor (argR) is activated, transcription of an argC promoter is inhibited, expression of levosucrase (sacB) and Red Fluorescent Protein (RFP) is further inhibited, and the cell grows better and has light red. When the intracellular L-arginine concentration was low, the L-sucrase (sacB) and Red Fluorescent Protein (RFP) were normally expressed, and the cells were deep red and died in the presence of 10% sucrose. Therefore, the content of the L-arginine is preliminarily judged according to the growth condition and the color of the thalli.

And selecting strains with obviously improved activity for genome sequencing, determining amino acid mutation sites through gene comparison, taking the strains with the highest activity improvement as starting strains for establishing a library of the next round of random mutant library, and repeating the establishment of the random mutant library and high-throughput screening. The screening results are shown in Table 2

TABLE 2 high throughput screening results of random mutation pools from 1 st to 3 rd round

Remarking: "-" indicates no product was detected; "+" indicates that the product content is more than 0% and less than or equal to 50% relative to the respective starting strain; "+ +" indicates a product content of greater than 50% and less than or equal to 100% relative to the respective starting strain; "+++" indicates a product content of more than 100% with respect to the respective starting strain.

After 3 rounds of screening, the strain ARG-13 has the highest L-arginine production capacity, which indicates that the enzyme activity of the acetylglutamate kinase is the highest. Through gene sequencing, the nucleotide sequence of the acetylglutamate kinase is determined to be SEQ ID NO. 10, the corresponding amino acid sequence is determined to be SEQ ID NO. 11, and 6-site amino acid mutation is generated relative to the original enzyme SEQ ID NO. 2.

The strain ARG-13 was transferred to LB liquid medium containing Amp resistance and incubated overnight at 220rpm at 37 ℃. After plasmids are extracted by a plasmid miniextraction kit, the plasmids are transformed into a strain ARG-2 and are coated on an LB-Amp plate. A single clone was picked for validation and the clone resistant to Amp and sensitive to Cm was ARG-14 (E.coli W3110. Delta. ArgR. Delta. ArgB/pARGB) ^{R13G，N39S，D44H，N191S，G206D，T221N} )。

Example 8: synthesis of L-arginine by shake flask horizontal fermentation of strain ARG-14

With reference to the method of example 4, the preserved glycerol strain was streaked onto LB plates (100. Mu.g/mL ampicillin was added), and a single colony was picked up into 5mL of liquid LB medium and incubated at 37 ℃ and 220rpm for 10 to 14 hours to be used as a seed solution. 1mL of the seed solution was inoculated into a 500mL shake flask containing 100mL of LAE medium, and incubated at 220rpm and 30 ℃ for 48 hours to sample the L-arginine synthesis level to 2.91g/L.

Experimental results show that the acetyl glutamate kinase mutant SEQ ID NO. 11 obtained by screening effectively promotes the level of microorganism synthesis of L-arginine, can be used for gene modification of L-arginine producing bacteria, and has development and application prospects.

Claims (10)

1. An acetylglutamate kinase which is a polypeptide selected from the group consisting of:

(1) A polypeptide having the amino acid sequence of SEQ ID NO. 11;

(2) The polypeptide has more than 90 percent of homology with SEQ ID NO. 11 and has higher enzyme activity compared with the polypeptide of SEQ ID NO. 11.

2. A gene encoding the acetylglutamate kinase of claim 1.

3. The gene as claimed in claim 2, wherein the gene encoding SEQ ID NO. 11 is a polynucleotide represented by SEQ ID NO. 10 or a polynucleotide having a homology of 90% or more with SEQ ID NO. 10.

4. Use of the acetylglutamate kinase of claim 1 or the gene of any one of claims 2 to 3 for promoting the production of L-arginine by a microorganism.

5. The use according to claim 4, wherein the microorganism is an L-arginine producing bacterium selected from the group consisting of Corynebacterium glutamicum, corynebacterium crenatum, and Escherichia coli.

6. A microorganism having an ability to synthesize L-arginine, which expresses the gene of claim 2 or 3.

7. The microorganism of claim 6, wherein the microbial host bacteria are selected from the group consisting of Corynebacterium glutamicum, corynebacterium crenatum, and Escherichia coli.

8. The microorganism according to claim 7, wherein the gene encoding acetylglutamate kinase present in the genome of the host microorganism is replaced by a gene according to claim 2 or 3; or the microbial host genome incorporates the gene of claim 2 or 3.

9. The microorganism of claim 7, wherein the integration of the gene is achieved by transformation of a plasmid comprising the gene into the host microorganism or by direct cloning of the gene into the genome of the host microorganism.

10. Use of the microorganism according to claim 6 for the production of L-arginine.

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