An acetylglutamate kinase that promotes arginine synthesis

Abstract

The present invention screened out an acetylglutamate kinase mutant SEQ ID NO: 11 through genetic engineering technology. Compared with the wild-type acetylglutamate kinase SEQ ID NO: 2, the ability of the mutant to promote L-arginine synthesis is significantly improved, and the mutant can be used for the transformation of L-arginine-producing bacteria.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to an acetylglutamate kinase mutant and its use in improving the ability of microorganisms to synthesize L-arginine. Background Art

[0002] L-arginine is a conditionally essential amino acid for the human body and has important physiological functions. It participates in regulating the balance of the immune system in the body, enhancing the body's immune function, and also participates in the ornithine cycle, converting ammonia in the body into non-toxic urea and excreting it from the body, thereby reducing blood ammonia concentration (Guo Changjiang, Xu Qishou. The regulatory effect of arginine on immune cell function in vitro. Amino Acid Journal, 1991 (1): 4-5.). In addition, arginine is also involved in the synthesis of substances such as nitric oxide, phosphoric acid, and creatine in the body. L-arginine has important economic value and is currently widely used in the fields of medicine, cosmetics, food, and animal husbandry. Especially in the pharmaceutical industry, intravenous injection of L-arginine can promote liver detoxification, and has a significant effect on hepatic coma caused by ammonia poisoning. Supplementing arginine can effectively promote collagen synthesis at the wound and accelerate the healing of damaged tissues (Sax HC. Arginine stimulates swound healing and immune function in elderly human beings. JPEN J Parenter Enteral Nutr, 1994, 18 (6): 559-560.).

[0003] At present, protein hydrolysis or microbial fermentation is the main method for producing arginine. Protein hydrolysis is to use cheap animal hair and gelatin as raw materials, heat them under acidic conditions, release amino acid mixtures, and finally obtain L-arginine products through ion exchange resin, activated carbon decolorization and other steps. This method can obtain L-type arginine products, but there are many shortcomings: one is that the yield is low and the product purity is difficult to meet the drug licensing standards; the second is that the process is complicated and produces a large amount of wastewater containing salt and organic reagents, causing environmental pollution. Therefore, protein hydrolysis is limited in large-scale applications. Microbial fermentation is a method of synthesizing arginine based on microorganisms themselves using technical means such as genetic engineering, mutagenesis breeding and fermentation engineering, with the advantages of low production cost, high product purity and mild production conditions. Corynebacterium glutamicum and Corynebacterium blunt tooth are often used as chassis cells for synthesis. In 2014, Park SH et al. used metabolic engineering technology to transform Corynebacterium glutamicum ATCC 21831 to ferment and synthesize L-arginine. The fermentation levels of L-arginine in 5L and 1500L bioreactors reached 92.5g / L and 81.2g / L, respectively (Park SH, Kim HU, Kim TY, Metabolic engineering of Corynebacterium glutamicum for L-arginine production. Nat. Commun., 2014, 5: 4618). However, the use of Corynebacterium glutamicum has the disadvantages of low production intensity, long fermentation cycle, and easy production fluctuations, and is not the optimal production strain. In addition, the acetylglutamate kinase involved in the L-arginine synthesis pathway in Corynebacterium glutamicum is subject to feedback inhibition by the product L-arginine (Schendzielorz G, Dippong M, Grünberger A, et al. Taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways. ACS Synth. Biol., 2014, 3(1): 21-29). Summary of the invention

[0004] In order to overcome the defect of feedback inhibition of the product L-arginine encountered when the fermentation method of Corynebacterium glutamicum produces L-arginine, the scope of the engineering bacteria host is expanded, the inventor has conducted in-depth research on the L-arginine biosynthetic pathway, and has made a breakthrough in the transformation of acetylglutamate kinase SEQ ID NO:2, and obtained a mutant with enzyme activity increased several times by the method of random mutation and high-throughput screening, and the acetylglutamate kinase mutant can effectively improve the ability of strains to synthesize L-arginine, and is suitable for the most commonly used model bacteria Escherichia coli in the field of genetic engineering to realize L-arginine synthesis. Accordingly, the present invention provides the following technical scheme.

[0005] An acetylglutamate kinase, which is a polypeptide selected from the group consisting of:

[0006] (1) a polypeptide having an amino acid sequence of SEQ ID NO: 11;

[0007] (2) A polypeptide having 90% or more homology, preferably 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, and more preferably 99% or more homology to SEQ ID NO: 11, and having improved enzyme activity compared to SEQ ID NO: 11.

[0008] MNDLIKDLGSEVGANVLAEALPWLQHFRDKIVVVKYGGSAMVDHDLKAAFAADMVFLRTVGAKPVVVHGGGPQISEMLNRVGLQGEFKGGFRVTTPEVMDIVRMVLFGQVGRDLVGLINSHGPYAVGTSGEDAGLFTAQKRMVNIDGVPTDIGLVGDIIN VDASSLMDIIEAGRIPVVSTIAPGEDGQIYSINADTAAGALAAAIDAERLLVLTNVEGLYNDWPDKSSLVSKIKATELEAILPGLDSGMIPKMESCLNAVRGGVSAAHVIDGRIAHSVLLELLTMGGIGTMVLPDVFDRENYPEGTVFRKDDKDGEL(SEQ ID NO:11).

[0009] The second aspect of the present invention provides a polynucleotide, which is a gene encoding the above polypeptide.

[0010] Preferably, the gene encoding SEQ ID NO:11 is a polynucleotide shown in SEQ ID NO:10, or a polynucleotide having more than 90%, preferably more than 95%, preferably more than 96%, preferably more than 97%, preferably more than 98%, and more preferably more than 99% homology with SEQ ID NO:10.

[0011] The third aspect of the present invention provides the use of the above-mentioned polypeptide or the above-mentioned polynucleotide in promoting the production of L-arginine by microorganisms.

[0012] Specifically, the microorganism can produce L-arginine by fermentation. The microorganism is an L-arginine producing bacterium selected from Corynebacterium glutamicum, Corynebacterium crenata and Escherichia coli.

[0013] The fourth aspect of the present invention provides a microorganism capable of synthesizing L-arginine, which expresses the above-mentioned gene.

[0014] The microbial host bacteria is selected from Corynebacterium glutamicum, Corynebacterium crenata and Escherichia coli. For example, the host bacteria can be Corynebacterium glutamicum ATCC 21831 or Escherichia coli W3110.

[0015] In one embodiment, the above-mentioned microorganism is an engineered bacterium, that is, the original acetylglutamate kinase encoding gene in the genome of the microorganism host is replaced by the above-mentioned gene; or the above-mentioned gene is integrated into the genome of the microorganism host.

[0016] The integration of the above-mentioned coding gene can be achieved by transforming a plasmid containing the gene into the microbial host bacteria, or directly cloning the gene into the host bacteria genome.

[0017] Among them, the above-mentioned genes are cloned into the genome of the microbial host bacteria, and the knockout of a certain gene is implemented through gene editing technology.

[0018] The above-mentioned gene editing technology can adopt homologous double crossover, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas related transposition system INTEGRATE system or CAST system.

[0019] The above-mentioned INTEGRATE system refers to the gene editing tool (Insertion of transposable elements by guide RNA-assisted targeting) developed by Sam Sternberg's research group; the CAST system refers to the gene editing tool (CRISPR-associated transposase) developed by Zhang Feng's research group.

[0020] When the host bacterium is Escherichia coli W3110, the L-arginine producing bacterium (engineered bacterium) can be constructed by the following steps:

[0021] A. Knock out the repressor protein gene argR in the genome to obtain strain ARG-1 (E. coli W3110ΔargR);

[0022] B. knocking out the endogenous acetylglutamate kinase gene argB in the genome of the strain obtained in step A to obtain strain ARG-2 (E. coli W3110ΔargRΔargB);

[0023] C. Integrate the exogenous gene into the genome of the strain obtained in step B to obtain the strain ARG-3 (E. coli W3110ΔargRΔargB / pARGB) expressing the acetylglutamate kinase, which is an L-arginine engineered bacterium.

[0024] Optionally, in order to facilitate screening and intuitively display the arginine expression level of the engineered bacteria, the construction of the engineered bacteria may also include the following steps: constructing an arginine-RFP-sacB biosensor system, and transferring the plasmid pCARS expressing the arginine-RFP-sacB biosensor system into the strain obtained in step C to obtain strain ARG-5 (E. coli W3110ΔargRΔargB / pARGB / pCARS).

[0025] The microorganism having the exogenous acetylglutamate kinase gene integrated into the genome has the potential to be used in the production of L-arginine.

[0026] The acetylglutamate kinase SEQ ID NO: 11 screened by the present invention is a mutant of the wild-type acetylglutamate kinase SEQ ID NO: 2. Compared with the wild enzyme, the mutant can increase the L-arginine synthesis level of the strain by nearly 3 times, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The invention discloses a plasmid map of a plasmid pARGB constructed by the present invention and used for expressing the acetylglutamate kinase gene derived from Corynebacterium glutamicum ATCC13032.

[0028] Figure 2 The present invention is a plasmid map of the plasmid pCARS for expressing the arginine-RFP-sacB biosensor system. DETAILED DESCRIPTION

[0029] For the wild-type acetylglutamate kinase in Corynebacterium glutamicum used for producing L-arginine, which is feedback inhibited by the product L-arginine, changing its tolerance to L-arginine by mutation may be a way to improve the L-arginine production capacity.

[0030] To this end, the wild-type acetylglutamate kinase with an amino acid sequence of SEQ ID NO: 2 (GenBank: CP025533.1) from Corynebacterium glutamicum ATCC13032 was used as the modification object, and random mutagenesis and high-throughput screening were carried out in Escherichia coli, which is the most commonly used bacterium in the field of genetic engineering, in order to obtain mutant enzymes with higher enzyme activity or resistance to L-arginine feedback inhibition.

[0031] In this article, the terms "wild (type)", "wild enzyme", and "wild-type enzyme" have the same meaning, and all refer to the wild-type acetylglutamate kinase SEQ ID NO: 2:

[0032] MNDLIKDLGSEVRANVLAEALPWLQHFRDKIVVVKYGGNAMVDDDLKAAFAADMVFLRTVGAKPVVVHGGGPQISEMLNRVGLQGEFKGGFRVTTPEVMDIVRMVLFGQVGRDLVGLINSHGPYAVGTSGEDAGLFTAQKRMVNIDGVPTDIGLVGDIIN VDASSLMDIIEAGRIPVVSTIAPGEDGQIYNINADTAAGALAAAIGAERLLVLTNVEGLYTDWPDKSSLVSKIKATELEAILPGLDSGMIPKMESCLNAVRGGVSAAHVIDGRIAHSVLLELLTMGGIGTMVLPDVFDRENYPEGTVFRKDDKDGEL(SEQ ID NO:2).

[0033] Similarly, the terms "acetylglutamate kinase mutant", "mutant acetylglutamate kinase", "mutated acetylglutamate kinase" and "mutant enzyme" have the same meaning and refer to the mutant of acetylglutamate kinase SEQ ID NO:11, which is a mutant of the wild enzyme in which the arginine at position 13 is mutated to glycine (R13G), the asparagine at position 39 is mutated to serine (N39S), the aspartic acid at position 44 is mutated to histidine (D44H), the asparagine at position 191 is mutated to serine (N191S), the glycine at position 206 is mutated to aspartic acid (G206D), and the threonine at position 221 is mutated to asparagine (T221N).

[0034] Sometimes, for the sake of convenience, the wild enzyme SEQ ID NO: 2 and its mutant SEQ ID NO: 11 may be collectively referred to as "acetylglutamate kinase" herein.

[0035] After the acetylglutamate kinase mutant was overexpressed in L-arginine-producing Escherichia coli W3110, the strain's ability to produce L-arginine increased by 2.7 times.

[0036] It can be expected that the acetylglutamate kinase mutant can also be used in other L-arginine-producing strains including Corynebacterium glutamicum and Corynebacterium crenata to improve the L-arginine biosynthesis level of the engineered bacteria.

[0037] It is easy to understand that in order to express exogenous acetylglutamate kinase in different host bacteria, the expression gene of this enzyme can be codon optimized. Codon optimization is a kind of technology that can be used for maximizing protein expression in organism by increasing the translation efficiency of gene of interest. Different organisms usually show the special preference for one of some codons of the same amino acid being encoded due to mutation tendency and natural selection. For example, in fast-growing microorganisms such as Escherichia coli, the optimization codon reflects the composition of its respective genome tRNA library. Therefore, in fast-growing microorganisms, the low-frequency codon of amino acid can be used for the same amino acid but the high-frequency codon replacement. Therefore, the expression of the optimized DNA sequence is improved in the fast-growing microorganism.

[0038] For example, in order to express the mutant SEQ ID NO: 11 of acetylglutamate kinase in Escherichia coli, the codon-optimized encoding gene may be SEQ ID NO: 10, but is not limited thereto.

[0039] In order to facilitate high-throughput screening of mutant enzymes, the present invention also designs a coupled detection system for acetylglutamate kinase, namely, the arginine-RFP-sacB biosensor system, so as to intuitively display the arginine expression level of the strain, thereby improving the screening efficiency of positive mutants. When the biosensor system is applied to different microbial species, preferential codon optimization should also be performed.

[0040] Example

[0041] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0042] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0043] Materials and methods

[0044] The whole gene synthesis, primer synthesis and sequencing in the examples were all completed by Suzhou Genewise Biotechnology Co., Ltd.

[0045] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0046] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0047] LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, pH 7.2. (LB solid medium plus 20g / L agar powder.)

[0048] 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 O 0.15mg / L, vitamin B1 1mg / L, vitamin B5 1mg / L, vitamin B12 1mg / L, pH 7.0-7.1.

[0049] Chromatographic conditions for L-arginine:

[0050]

[0051] Example 1: Knockout of the repressor gene argR

[0052] 1.1 Construction of the targeting fragment ARGR-3

[0053] Using the Escherichia coli W3110 genome (GenBank: AP009048.1) as a template, the ARGR-1 fragment was amplified using the primer pair ARGR-51 / ARGR-31, and the ARGR-2 fragment was amplified using the primer pair ARGR-52 / ARGR-32. The two fragments were recovered, and then the ARGR-1 and ARGR-2 fragments were used as templates. The ARGR-3 target fragment was amplified using the primer pair ARGR-51 / ARGR-32 and recovered by gel cutting.

[0054] PCR reaction system: 10 ng template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0055] PCR reaction conditions: 95°C, 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0056] The primer sequences are as follows:

[0057] ARGR-51: 5'-TATACCCTACCGTTCAGCCAA-3',

[0058] ARGR-31: 5'-CATAAGTCACCCGATATGGTG-3',

[0059] ARGR-52: 5'-CCATATCGGGTGACTTATGTAATCTCTGCCCCGTCGTTTC-3',

[0060] ARGR-32: 5'-CTGTTTCAGTTGATACTAAGT-3'.

[0061] 2.2 Construction of pTargetF-argR

[0062] Using plasmid pTargetF (Addgene Plasmid: #62226) as template and argRspc / gTEMdn as primers, circular amplification was performed to obtain a pTarget-argR plasmid of about 2.2 kb. The primer sequences are as follows:

[0063] argRspc:

[0064] 5'-TAATACTAGTTTACTGCCTGCCAGCTGAACGTTTTAGAGCTAGAAATAGC-3'.

[0065] gTEMdn: 5'-CTCAAAAAAAGCACCGACTCGG-3'.

[0066] The PCR reaction system included: 10 ng template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0067] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0068] 50 μL of amplified PCR product was directly added with 1 μL of DpnI restriction endonuclease (Thermo Company) to digest the plasmid template and transformed into E. coli DH5a competent cells.

[0069] 2.3 Knockout of the endogenous argR gene in E. coli W3110

[0070] Plasmid pCas (Addgene Plasmid: #62225) was electroporated into E. coli W3110 competent cells, and positive clones were picked and transferred to a test tube containing 5 mL LB (containing 50 μg / mL kanamycin sulfate) and cultured overnight at 30°C and 220 rpm. The next day, the inoculum was inoculated into a 250 mL shake flask containing 50 mL LB liquid medium (containing 10 mM L-arabinose) at a volume concentration of 1%, and incubated at 30°C and 220 rpm until OD 600nm The concentration of 0.4 to 0.6 was adjusted, and the cells were collected by centrifugation at 4°C and 4000 rpm for 10 min to prepare electroporation competent cells.

[0071] Take 100 ng of pTargetF-argR plasmid, 500 ng of ARGR-3 targeting fragment and 100 μL of E. coli W3110 / pCas electroporation competent cells, transfer them into a pre-cooled 2 mm electroporation cup, and perform electroporation transformation using an electroporator. After the electroporation is completed, immediately add 1 mL of pre-cooled LB medium and transfer to a 1.5 mL centrifuge tube. Resuscitate at 30°C, 180 rpm for 1.5 h before coating

[0072] LB plates containing 50 μg / mL kanamycin sulfate and 50 μg / mL spectinomycin hydrochloride were incubated at 30°C for 24 h. Colony PCR was performed using ARGR-01 and ARGR-02 as primers, and about 0.6 kb was positive, obtaining E. coli W3110ΔargR / pCas9 / pTargetF-argR.

[0073] ARGR-01: 5'-GTAATCAGGAACCTAACTTAC-3',

[0074] ARGR-02: 5'-CTTTGTTTTCGTGACCCATATC-3'.

[0075] Auxiliary plasmid elimination: Pick a single colony of E. coli W3110ΔargR / pCas9 / pTargetF-argR and inoculate it into a liquid LB medium containing 50μg / mL kanamycin sulfate and 1mM IPTG, and incubate it overnight at 30°C and 220rpm. The next day, spread the overnight bacteria on a solid LB medium containing 50μg / mL kanamycin sulfate and incubate it at 30°C for 24h. If the single colony is sensitive to 50μg / mL spectinomycin hydrochloride, it means that the pTargetF-argR plasmid has been successfully eliminated. Pick a single colony that successfully eliminates the pTargetF-argR plasmid, incubate it at 42°C and 220rpm for 6h, then spread the bacterial solution on an LB plate without antibiotics, and incubate it at 42°C for 12h. If the single colony is sensitive to 50μg / mL kanamycin sulfate, it means that the pCas plasmid has been successfully eliminated, and finally the strain ARG-1 (E. coliW3110ΔargR) is obtained.

[0076] Example 2: Knockout of the endogenous acetylglutamate kinase gene argB in Escherichia coli

[0077] 2.1 Construction of the targeting fragment ARGB-3

[0078] Using the E. coli W3110 genome (GenBank: AP009048.1) as a template, the following primer pair ARGB-51 / ARGB-31 was designed to amplify the ARGB-1 fragment:

[0079] Forward primer ARGB-51: 5'-GTGACCAGGGCTTTGGACCAG-3',

[0080] Reverse primer ARGB-31: 5′-GCACTGTTTAACGGTATGCCG-3′.

[0081] The following primer pair ARGB-52 / ARGB-32 was designed to amplify the ARGB-2 fragment:

[0082] Forward primer ARGB-52:

[0083] 5'-GGCATACCGTTAAACAGTGCATTCACCAGTGCGCTAAACAG-3',

[0084] Reverse primer ARGB-32: 5′-GTTGGGCTGCCATTTTGCGAT-3′.

[0085] The ARGB-1 and ARGB-2 fragments were used as templates at the same time, and the primer pair ARGB-51 / ARGB-32 was used to amplify the ARGB-3 targeting fragment.

[0086] The PCR reaction system included: 10 ng template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0087] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0088] 2.2 Construction of pTargetF-argB

[0089] Using plasmid pTargetF (Addgene Plasmid: #62226) as template, the following primer pair was designed for circular amplification of argBspc / gTEMdn to obtain pTarget-argB plasmid:

[0090] Forward primer argBspc:

[0091] 5'-TAATACTAGTGACCTAACTCTTCATCAAGCGTTTTAGAGCTAGAAATAGC-3',

[0092] Reverse primer gTEMdn: 5′-CTCAAAAAAAGCACCGACTCGG-3′.

[0093] Using plasmid pTargetF as a template, PCR amplification was performed to obtain a plasmid pTargetF-argB of about 2.2 kb.

[0094] 50 μL PCR reaction system includes: 10 ng template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0095] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0096] After 50 μL of amplified PCR, 1 μL of DpnI restriction endonuclease (Thermo Company) was directly added to digest the plasmid template and transformed into E. coli DH5a.

[0097] 2.3 Knockout of argB gene

[0098] Plasmid pCas (Addgene Plasmid: #62225) was electroporated into competent cells of strain ARG-1, and positive clones were picked and transferred to a test tube containing 5 mL LB (containing 50 μg / mL kanamycin sulfate) and cultured overnight at 30°C and 220 rpm. The next day, the inoculum was inoculated into a 250 mL shake flask containing 50 mL LB liquid medium (containing 10 mM L-arabinose) at a volume concentration of 1%, and incubated at 30°C and 220 rpm until OD 600nm The concentration of 0.4 to 0.6 was adjusted, and the cells were collected by centrifugation at 4°C and 4000 rpm for 10 min to prepare electroporation competent cells.

[0099] Take 100 ng of pTargetF-argB plasmid, 500 ng of ARGB-3 targeting fragment and 100 μL of E. coli W3110 / pCas electroporation competent cells, transfer them into a pre-cooled 2 mm electroporation cup, and use an electroporator for electroporation. After the electroporation is completed, immediately add 1 mL of pre-cooled LB medium and transfer to a 1.5 mL centrifuge tube. Resuscitate at 30°C, 180 rpm for 1.5 h before coating

[0100] LB plates containing 50 μg / mL kanamycin sulfate and 50 μg / mL spectinomycin hydrochloride were incubated at 30°C for 24 h. Colony PCR was performed using primers ARGB-01 and ARGB-02. About 0.6 kb was positive and 1.2 kb was negative, and E. coli ARG-1ΔargB / pCas9 / pTargetF-argB was obtained.

[0101] ARGB-01: 5'-GCGAACATCTTCCAGCAACAC-3',

[0102] ARGB-02: 5'-TGCGGCTGTATGACAAAGGCG-3'.

[0103] Auxiliary plasmid elimination: Pick a single colony of ARG-1ΔargB / pCas9 / pTargetF-argB and inoculate it into a liquid LB medium containing 50μg / mL kanamycin sulfate and 1mM IPTG, and incubate it overnight at 30°C and 220rpm. The next day, spread the overnight bacteria on a solid LB medium containing 50μg / mL kanamycin sulfate and incubate it at 30°C for 24h. If the single colony is sensitive to 50μg / mL spectinomycin hydrochloride, it means that the pTargetF-argB plasmid has been successfully eliminated. Pick a single colony that successfully eliminates the pTargetF-argB plasmid, incubate it at 42°C and 220rpm for 6h, then spread the bacterial solution on an LB plate without antibiotics, and incubate it at 42°C for 12h. If the single colony is sensitive to 50μg / mL kanamycin sulfate, it means that the pCas plasmid has been successfully eliminated, and ARG-2 (E.coli W3110ΔargRΔargB) is finally obtained.

[0104] Example 3: Construction of a strain overexpressing the C. glutamicum derived acetylglutamate kinase gene argB

[0105] The following primer pair ARGB-54 / ARGB-34 was designed to amplify the ARGB-4 fragment:

[0106] Forward primer ARGB-54:

[0107] 5'-TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCTCGAGTAAGGAGGATATTTAGATGAATGACTTGATCAAAGATTTAG-3',

[0108] Reverse primer ARGB-34:

[0109] 5'-TTACAGTTCCCCATCCTTGTC-3'.

[0110] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC13032 (GenBank: CP025533.1) as a template.

[0111] 50 μL PCR reaction system includes: 100 ng genomic template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0112] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0113] 50 μL of the amplified PCR product was excised and recovered to obtain an ARGB-4 fragment of about 1.0 kb. The C. glutamicum argB gene sequence contained in the ARGB-4 fragment is shown in SEQ ID NO: 1, and the corresponding protein sequence is shown in SEQ ID NO: 2.

[0114] Design the following primer pair to amplify TRC-5 / TRC-3 to obtain the TRC backbone:

[0115] Forward primer TRC-5:

[0116] 5'-ACAAGGATGGGGAACTGTAAAGAGTAGGGAACTGCCAGGCAT-3',

[0117] Reverse primer TRC-3:

[0118] 5'-AGGACTGAGCTAGCTGTCAACAGCTCATTTCAGAATATTTGC-3'.

[0119] PCR amplification was performed using plasmid pTrc99A as a template.

[0120] 50 μL PCR reaction system includes: 10 ng template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0121] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0122] After 50 μL of amplified PCR, 1 μL of DpnI restriction endonuclease (Thermo Company) was directly added to digest the plasmid template, and the gel was cut and recovered to obtain a TRC backbone of about 3.9 kb.

[0123] Beijing Quanshijin Biotechnology Co., Ltd. The TRC backbone and ARGB-4 fragment were assembled in vitro using the Seamless Cloning and Assembly Kit to obtain the plasmid pARGB. The plasmid map is shown in Figure 1 shown.

[0124] Transform plasmid pARGB into strain ARG-2 to obtain strain ARG-3 (E. coli W3110ΔargRΔargB / pARGB)

[0125] Example 4: Synthesis of L-arginine by shake flask fermentation

[0126] The preserved glycerol bacteria were streaked onto LB plates (strains W3110, ARG-1, and ARG-2 were not treated with antibiotics, and strain ARG-3 was supplemented with 100 μg / mL ampicillin), and a single colony was picked into 5 mL of liquid LB medium and incubated at 37°C and 220 rpm for 10 to 14 h as seed solution. 1 mL of seed solution was inoculated into a 500 mL shake flask containing 100 mL of AE medium, and incubated at 220 rpm and 30°C for 48 h. Samples were taken to detect the synthesis level of L-arginine, and the results are shown in Table 1.

[0127] Table 1. L-arginine synthesis level

[0128] strain L-arginine synthesis level (g / L) W3110 0.07 ARG-1 0.94 ARG-2 0 ARG-3 1.07

[0129] As can be seen from Table 1, after the argR gene was knocked out of the starting strain W3110, the L-arginine synthesis level was greatly improved, by 12 times; but after the endogenous acetylglutamate kinase gene argB was further knocked out, the strain lost the ability to produce L-arginine because the L-arginine synthesis pathway was interrupted; when the acetylglutamate kinase gene argB from Corynebacterium glutamicum was integrated, the strain restored the L-arginine synthesis pathway, thereby restoring the L-arginine production capacity. The slight increase in the ability to produce L-arginine may indicate that the enzyme activity of the exogenous acetylglutamate kinase SEQ ID NO: 2 is slightly higher than the endogenous acetylglutamate kinase of the strain W3110.

[0130] Example 5: Construction of Arginine-RFP-sacB Biosensor System

[0131] The following primer pair ARGR-54 / ARGR-34 was designed to amplify the fragment ARGR-4 containing the gene argR and introduce the constitutive promoter of pJ23119:

[0132] Forward Primer ARGR-54:

[0133] 5'-TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCTCGAGTAAGGAGGATATTTAGATGTCCCTTGGCTCAACCC-3',

[0134] Reverse Primer ARGR-34:

[0135] 5'-GGATGATTTGGACCTTGGTTAAGTGGTGCGCCCGCTGA-3'.

[0136] PCR amplification was performed using the genome of Corynebacterium glutamicum ATCC13032 (GenBank: CP025533.1) as a template.

[0137] 50 μL PCR reaction system includes: 100 ng genomic template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0138] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0139] After 50 μL amplification, PCR, electrophoresis and gel recovery, an ARGR-4 fragment of about 0.5 kb was obtained.

[0140] The nucleic acid sequence of the argR gene contained in the ARGR-4 fragment is shown in SEQ ID NO:3, and the corresponding protein sequence is shown in SEQ ID NO:4.

[0141] The protein sequence of red fluorescent protein (RFP) (GenBank: AAM54544.1) is shown in SEQ ID NO:6. The nucleic acid sequence obtained by optimizing according to the codon preference of Escherichia coli is shown in SEQ ID NO:5.

[0142] The protein sequence of L-sucrase (sacB) (GenBank: WP_187954058.1) is shown in SEQ ID NO:8. The nucleic acid sequence obtained by optimization according to the codon preference of Escherichia coli is shown in SEQ ID NO:7.

[0143] The argC promoter-red fluorescent protein-L-sucrase nucleic acid sequence is shown in SEQ ID NO:9, which was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and cloned into the EcoRI-HindIII site of the pUC57 vector to obtain pUC57-RS.

[0144] Design the following primer pair RS-51 / RS-31 to amplify fragment RS-1:

[0145] Forward primer RS-51:

[0146] 5'-CCAAGGTCCAAATCATCCACTAAAAATTCATGCTTTTTACCCAC-3',

[0147] Reverse primer RS-31:

[0148] 5'-TTATTTGTTCACGGTCAGCTGAC-3'.

[0149] PCR amplification was performed using plasmid pUC57-RS as a template.

[0150] 50 μL PCR reaction system includes: 10 ng template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0151] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0152] After 50 μL of amplified PCR, 1 μL of DpnI restriction endonuclease (Thermo Company) was directly added to digest the plasmid template, and electrophoresis was performed. After gel recovery, an RS-1 fragment of about 2.3 kb was obtained.

[0153] Design the following primer pair CM-51 / CM-31

[0154] Forward primer CM-51:

[0155] 5'-AGCTGACCGTGAACAAATAATGCCATTCATCCGCTTATTa-3',

[0156] Reverse Primer CM-31:

[0157] 5'-AGGACTGAGCTAGCTGTCAAGAGCCTTCAACCCAGTCAG-3'.

[0158] PCR amplification was performed using plasmid pACYCDuet-1 as a template.

[0159] 50 μL PCR reaction system includes: 10 ng template, 10 pmol primer pair, 1× KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , 1 unit of KOD-plus DNA polymerase.

[0160] The PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kb, 30 cycles; 68°C for 10 min.

[0161] After 50 μL of amplified PCR, 1 μL of DpnI restriction endonuclease (Thermo Company) was directly added to digest the plasmid template, and electrophoresis was performed. After gel recovery, a CM-1 backbone of about 2.1 kb was obtained.

[0162] Beijing Quanshijin Biotechnology Co., Ltd. The Seamless Cloning and Assembly Kit was used to assemble the backbone CM-1, fragment RS-1 and fragment ARGR-1 in vitro to obtain the plasmid pCARS. The plasmid map is shown in Figure 2 shown.

[0163] The plasmid pCARS was transformed into strain ARG-2 to obtain strain ARG-4 (E. coli W3110ΔargRΔargB / pCARS).

[0164] The plasmid pCARS was transformed into strain ARG-3 to obtain ARG-5 (E. coli W3110ΔargRΔargB / pARGB / pCARS).

[0165] Example 6: Construction of C. glutamicum derived acetylglutamate kinase argB mutant

[0166] The random mutant library was constructed using error-prone PCR technology with plasmid pARGB as template.

[0167] The following primer pair ARGB-55 / ARGR-35 was designed:

[0168] Forward primer ARGB-55: 5'-ATGAATGACTTGATCAAAGATTTAG-3',

[0169] Reverse primer ARGB-34: 5′-TTACAGTTCCCCATCCTTGTC-3′.

[0170] Using plasmid pARGB as a template, PCR amplification was performed to obtain a mutant DNA sequence of approximately 1.0 kb.

[0171] 50 μL error-prone PCR reaction system includes: 10 ng plasmid (pARGB) template, 50 pmol primer pair ARGB-55 and ARGB-34, 1× Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM)MnCl 2 , 2.5 units of Taq enzyme (Takara).

[0172] The PCR reaction conditions were: 95°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min / kbp, 30 cycles; 72°C for 10 min.

[0173] The PCR product was excised and recovered. Using plasmid pARGB as template and the recovered product of about 1.0 kb (random mutation fragment) as large primer, MegaPrimer PCR was performed with KOD-plus DNA polymerase: 94℃5min; 98℃10s, 60℃30s, 68℃2min / kb, 25 cycles; 68℃10min.

[0174] The plasmid template was digested with DpnI restriction endonuclease (Thermo Company) and electroporated into E. coli ARG-4 to obtain more than 10 4 A random mutation library of argB acetylglutamate kinase from C. glutamicum containing 15 clones.

[0175] Example 7: Construction of an acetylglutamate kinase mutant library

[0176] The argB random mutation library from C. glutamicum obtained in Example 6 was subjected to high-throughput screening, and single colonies were picked to a 96-well deep-well plate (each well containing 300 μL of liquid LB-Amp-Cm medium), and incubated at 37°C and 300 rpm for 18 h. Then, 50 μL of bacterial solution was taken from each well to a 96-well deep-well plate (each well containing 450 μL of liquid AE-Amp-Cm medium), and incubated at 30°C and 400 rpm for 24 h. Sucrose mother solution was then added to make the final concentration of each well reach 10%, and the incubation continued for 12 h. The clones with better growth and lighter red were selected, and the supernatant was centrifuged at 4°C and 4000 rpm for 10 min to detect the L-arginine content by HPLC.

[0177] When the intracellular L-arginine concentration is high, the repressor protein (argR) is activated, inhibiting the transcription of the argC promoter, thereby inhibiting the expression of sacB and red fluorescent protein (RFP), and the cells grow well and the red color is lighter. When the intracellular L-arginine concentration is low, sacB and red fluorescent protein (RFP) are expressed normally, the cells are darker red and die in the presence of 10% sucrose. In this way, the L-arginine content can be preliminarily judged based on the growth and color of the bacteria.

[0178] The strains with significantly improved vitality were selected for genome sequencing, and the amino acid mutation sites were determined by gene comparison. The strains with the highest vitality improvement were used as the starting strains for the next round of random mutant library construction, and the random mutation library construction and high-throughput screening were repeated. The screening results are listed in Table 2

[0179] Table 2. Results of high-throughput screening of random mutation libraries from round 1 to round 3

[0180]

[0181] Note: “-” means no product was detected; “+” means the product content is greater than 0% and less than or equal to 50% relative to the respective starting strain; “++” means the product content is greater than 50% and less than or equal to 100% relative to the respective starting strain; “+++” means the product content is greater than 100% relative to the respective starting strain.

[0182] After three rounds of screening, strain ARG-13 had the highest L-arginine production capacity, indicating that its acetylglutamate kinase had the highest enzyme activity. Gene sequencing determined that the nucleic acid sequence of the acetylglutamate kinase was SEQ ID NO: 10, and the corresponding amino acid sequence was SEQ ID NO: 11. Compared with the original enzyme SEQ ID NO: 2, there were 6 amino acid mutations at the sites.

[0183] The strain ARG-13 was transferred to LB liquid medium containing Amp resistance and incubated overnight at 37°C and 220rpm. The plasmid was extracted using a plasmid extraction kit and transformed into the strain ARG-2, and LB-Amp plates were coated. A single clone was picked for verification, and the clone with Amp resistance and Cm sensitivity was ARG-14 (E. coli W3110ΔargRΔargB / pARGB R13G，N39S，D44H，N191S，G206D，T221N ).

[0184] Example 8: Synthesis of L-arginine by shake flask fermentation of strain ARG-14

[0185] Referring to the method of Example 4, the preserved glycerol bacteria were streaked onto LB plates (100 μg / mL ampicillin was added), and a single colony was picked into 5 mL of liquid LB medium, and incubated at 37°C and 220 rpm for 10 to 14 h as seed solution. 1 mL of seed solution was inoculated into a 500 mL shake flask containing 100 mL of AE medium, and incubated at 220 rpm and 30°C for 48 h. The synthesis level of L-arginine was detected and reached 2.91 g / L.

[0186] The experimental results show that the acetylglutamate kinase mutant SEQ ID NO: 11 screened by the present invention effectively promotes the level of L-arginine synthesis by microorganisms, can be used for genetic modification of L-arginine-producing bacteria, and has development and application prospects.

Claims

1. An acetylglutamate kinase, whose amino acid sequence is SEQ ID NO:

11.

2. A gene encoding the acetylglutamate kinase as claimed in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

10.

4. The use of the acetylglutamate kinase according to claim 1 or the gene according to any one of claims 2 to 3 in promoting the production of L-arginine by microorganisms, wherein the microorganism is an L-arginine producing bacterium selected from a bacterium in which a repressor protein gene in the genome is knocked out. argR of Corynebacterium glutamicum, Corynebacterium blunt-toothed and Escherichia coli.

5. A microorganism capable of synthesizing L-arginine, characterized in that: Expressing the gene as claimed in claim 2 or 3, the microorganism is selected from the group consisting of a microorganism in which a repressor protein gene in the genome is knocked out. argR of Corynebacterium glutamicum, Corynebacterium blunt-toothed and Escherichia coli.

6. The microorganism according to claim 5, characterized in that The original acetylglutamate kinase encoding gene in the microbial genome is replaced by the gene as claimed in claim 2 or 3; or the gene as claimed in claim 2 or 3 is integrated into the microbial genome.

7. The microorganism according to claim 5, characterized in that The integration of the gene refers to the process of transforming a plasmid containing the gene into the microorganism, or directly cloning the gene into the genome of the microorganism.

8. Use of microorganisms in the production of L-arginine as claimed in claim 5.

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