Hybrids of glutamate synthase, host cells, and preparation methods of compounds

Abstract

This application relates to a hybrid of glutamate synthase, a host cell, and a method for preparing a compound, the hybrid comprising any one of the following: (1) an α-GS subunit of NADPH-dependent glutamate synthase and a β-domain of NADH-dependent glutamate synthase; and (2) an α-domain of NADH-dependent glutamate synthase and a β-GS subunit of NADPH-dependent glutamate synthase. The process of generating a specific product can regulate the balance of cofactors, maintain the redox balance throughout the cell, and thereby enhance the production capacity of the strain.

Description

Technical Field

[0001] This application relates to the fields of protein engineering and genetic engineering, specifically to a hybrid of glutamate synthase, a host cell, and a method for preparing the compound. Background Technology

[0002] Glutamate synthetase (GS) is an important enzyme widely found in various organisms, participating in the synthesis of glutamate. The main function of GS is to transfer an amino group from a donor to α-ketoglutarate to synthesize glutamate. It generates glutamate by transferring ammonia to α-ketoglutarate, requiring ATP and coenzymes such as NADPH and NADH. This process is a crucial step in ammonia metabolism, especially important in nitrogen metabolism in plants and microorganisms.

[0003] Maintaining intracellular cofactor balance is crucial for normal cellular metabolism, especially for industrially produced strains, where NADH / NAD ratio is important. + and NADPH / NADP + Cofactors are the most important redox carriers in cellular metabolic production. Maintaining the balance between redox rates is crucial for maintaining normal anabolism and catabolism, and is equally important for the production performance of microbial strains. Currently, promoter engineering can precisely regulate the expression of cofactor-dependent genes or modify intracellular cofactor regeneration pathways to regulate the form and concentration of intracellular cofactors, thereby directionally altering and optimizing microbial cellular metabolic functions and achieving metabolic flow targeting of metabolites (Gu Y, Lv X, Liu Y, et al. Metabolic Engineering, 2018). However, due to the complexity of cofactor metabolism and function, and the fact that cofactor balance regulation strategies cannot precisely and efficiently control intracellular redox metabolic balance during the synthesis of specific products, the production performance of microbial strains is affected. Summary of the Invention

[0004] Based on this, one embodiment of this application provides a hybrid of glutamate synthase, which alters the coenzyme preference of glutamate synthase in metabolic synthesis, enhances the endogenous coenzyme balance, and can balance the intracellular redox state without additional expression or introduction of other genes, thereby enhancing the production capacity of the strain.

[0005] The technical solution includes:

[0006] A heterozygote of glutamate synthase, said heterozygote comprising any one of the following:

[0007] (1) The α-GS subunit of NADPH-dependent glutamate synthase and the β-domain of NADH-dependent glutamate synthase; and,

[0008] (2) The α-domain of NADH-dependent glutamate synthase and the β-GS subunit of NADPH-dependent glutamate synthase.

[0009] In one embodiment, the α-GS subunit of the NADPH-dependent glutamate synthase and the β-GS subunit of the NADPH-dependent glutamate synthase are derived from Corynebacterium glutamicum.

[0010] In one embodiment, the α-domain and β-domain of the NADH-dependent glutamate synthase are derived from Saccharomyces cerevisiae.

[0011] In one embodiment, the amino acid sequence of the α-GS subunit of the NADPH-dependent glutamate synthase is as shown in SEQ ID NO: 1; or / and, the amino acid sequence of the β-GS subunit of the NADPH-dependent glutamate synthase is as shown in SEQ ID NO: 2; or / and, the amino acid sequence of the α-domain of the NADH-dependent glutamate synthase is as shown in amino acids 1 to 1642 of SEQ ID NO: 3; or / and, the amino acid sequence of the β-domain of the NADH-dependent glutamate synthase is as shown in amino acids 1643 to 2145 of SEQ ID NO: 3.

[0012] In one embodiment, the glutamate synthase hybrid comprises the amino acid fragment shown in SEQ ID NO: 1 and the fragment shown in amino acids 1643 to 2145 of SEQ ID NO: 3.

[0013] In one embodiment, the glutamate synthase hybrid comprises the fragments represented by amino acids 1 to 1642 in SEQ ID NO: 3 and the amino acid fragments represented by SEQ ID NO: 2.

[0014] Nucleic acid, which encodes the aforementioned hybrid.

[0015] Expression vector, including the nucleic acid.

[0016] Optionally, the expression vector includes one or more of plasmid vectors, shuttle vectors, bacteriophages, and viral vectors. Optionally, the plasmid vector includes one or more of the pET series, pXM series, and pEC series. Optionally, the expression vector includes one or more of pET-28a, pXMJ19, and pEC-Ptac.

[0017] The host cell contains the nucleic acid or the expression vector.

[0018] Optionally, the nucleic acid is integrated into the genome of the host cell; optionally, the host cell includes any one of Corynebacterium glutamicum, lactic acid bacteria, streptococci, Escherichia coli, yeast, Rhodococcus, Nocardia, and Bacillus subtilis.

[0019] A method for preparing a heterozygote includes using the host cell to prepare the heterozygote.

[0020] A method for preparing a compound, comprising: preparing the compound using the host cell.

[0021] Optionally, the compound includes one or more of glycosaminoglycans, glucosamine and its derivatives, amino acids and organic acids.

[0022] In one embodiment, the preparation method satisfies one or more of the following conditions:

[0023] a. The glycosaminoglycans include one or more of hyaluronic acid and chondroitin;

[0024] b. The derivative includes N-acetylglucosamine;

[0025] c. The amino acid includes one or more of glutamic acid, glutamine, ornithine, arginine, lysine, and proline; and

[0026] d. The organic acid includes one or more of pyruvic acid, acetic acid, lactic acid and succinic acid.

[0027] Compared with traditional technologies, this application has the following advantages:

[0028] The coenzyme preference pattern of the glutamate synthase hybrid in this application is reversed, enhancing the endogenous coenzyme balance capacity. It can balance the intracellular redox state without the need for additional expression or introduction of other genes, significantly improving the production capacity of industrial strains. This application also provides the use of the host cells containing the glutamate synthase hybrid; specifically, the host cells can be used to synthesize glycosaminoglycans, glucosamine, amino acids, organic acids, and organic alcohols, with a significantly increased yield compared to the control. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 A structural model of an artificial hybrid of glutamate synthase;

[0031] Figure 2 Biomass of the reconstructed strain containing an artificial hybrid of glutamate synthase;

[0032] Figure 3 To improve the yield of glycosaminoglycans synthesized by reconstructed strains containing artificial hybrids of glutamate synthase;

[0033] Figure 4 To measure the yield of glucosamine synthesized by reconstructed strains containing artificial hybrids of glutamate synthase;

[0034] Figure 5 The yield of amino acids synthesized by reconstructed strains containing artificial hybrids of glutamate synthase. Detailed Implementation

[0035] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0037] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0038] In this article, "NADPH-dependent glutamate synthase" refers to a glutamate synthase that uses NADPH as a coenzyme. Its functional unit contains an α subunit (α-GS) and a β subunit (β-GS). It exists in a heteropolymorphic active form. The α-GS subunit contains binding sites for the prosthetic group FMN and the 3Fe-4S cluster, and is mainly responsible for catalyzing the synthesis of glutamate. The β-GS subunit contains binding sites for the coenzyme NADPH and the 4Fe-4S cluster, and is mainly responsible for the reduction of FAD (flavin adenine dinucleotide) and electron transfer.

[0039] In this article, "NADH-dependent glutamate synthase" refers to glutamate synthase that uses NADH as a coenzyme. Its functional unit is a single subunit, but it consists of two catalytic domains, including an α-domain and a β-domain. The α-domain is the catalytic domain and is responsible for the synthesis of glutamate. The β-domain contains the binding site of the coenzyme NADH and is responsible for electron transfer. The two catalytic domains are connected by a flexible loop.

[0040] In this article, "glycosaminoglycans" are referred to as heteropolysaccharides, which are unbranched long-chain polymers composed of repeating complex units containing hexuronic acid (except keratin) and hexosamine. Common glycosaminoglycans include hyaluronic acid, chondroitin, and heparin.

[0041] Glutamate synthase is a coenzyme-dependent synthase that primarily uses NADH and NADPH. It catalyzes the synthesis of glutamate, consuming reduced NADH and NADPH while simultaneously producing oxidized NADPH. + and NADP + Because glutamate synthases from different sources have different cofactor preferences, altering their cofactor preferences can be a means of regulating intracellular redox metabolism. For example, glutamate synthases from Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, Streptococcus, and Lactobacillus use NADPH as a cofactor in glutamate synthesis, while glutamate synthases from Saccharomyces cerevisiae, Arabidopsis thaliana, and rice use NADH as a cofactor. Therefore, expressing glutamate synthases from different sources may alter their cofactor preferences, causing varying degrees of interference with intracellular glutamate homeostasis and redox status.

[0042] Starting with different coenzyme-dependent glutamate synthases, the inventors discovered through enzyme sequence alignment and structural analysis that NADPH-dependent glutamate synthase (GS) contains two subunits, α-GS and β-GS, existing in a heteropolymeric active form. The α-GS subunit contains binding sites for the prosthetic group FMN and the 3Fe-4S cluster, primarily responsible for catalyzing glutamate synthesis; the β-GS subunit contains binding sites for the coenzyme NADPH and the 4Fe-4S cluster, primarily responsible for the reduction of FAD (flavin adenine dinucleotide) and electron transfer. NADH-dependent glutamate synthase, on the other hand, consists of two catalytic domains: an α-domain and a β-domain. The α-domain is the catalytic domain, responsible for glutamate synthesis; the β-domain contains the binding site for the coenzyme NADH, responsible for electron transfer. The two catalytic domains are connected by a flexible loop.

[0043] Furthermore, using NADH-dependent glutamate synthase and NADPH-dependent glutamate synthase as subjects, the subunit domains responsible for binding to coenzymes (NADPH / NADH) in both glutamate synthases were hybridized and replaced.

[0044] Specifically, to alter the coenzyme preference of NADPH-dependent glutamate synthase, a novel glutamate synthase hybrid was constructed by hybridizing the α-GS subunit of glutamate synthase with the β-domain of NADH-dependent glutamate synthase. The resulting glutamate synthase hybrid exhibits a coenzyme-dependent change from NADPH to NADH compared to naturally derived glutamate synthase.

[0045] Specifically, to alter the coenzyme preference of NADH-dependent glutamate synthase, a novel artificial glutamate synthase hybrid was constructed by hybridizing the α-domain of glutamate synthase with the β-GS subunit of NADPH-dependent glutamate synthase. The resulting glutamate synthase hybrid exhibits a coenzyme-dependent change from NADH to NADPH compared to naturally derived glutamate synthase.

[0046] The glutamate synthase hybrid was obtained through molecular cloning and recombination, exhibiting a reversed coenzyme preference compared to the wild type. Various industrial strains were constructed using this glutamate synthase hybrid, which can be used to synthesize glycosaminoglycans, glucosamine, amino acids, organic acids, and organic alcohols, with yields increased by 30%-50% compared to the control.

[0047] One embodiment of this application provides a hybrid of glutamate synthase or a fragment thereof, which can regulate cofactor balance and maintain intracellular redox balance during the generation of a specific product, specifically including any one of (1) and (2):

[0048] (1) The α-GS subunit of NADPH-dependent glutamate synthase and the β-domain of NADH-dependent glutamate synthase; and,

[0049] (2) The α-domain of NADH-dependent glutamate synthase and the β-GS subunit of NADPH-dependent glutamate synthase.

[0050] In a specific example, it includes the α-GS subunit of NADPH-dependent glutamate synthase and the β-domain of NADH-dependent glutamate synthase.

[0051] In one specific example, the NADPH-dependent glutamate synthase was sourced from Corynebacterium glutamicum.

[0052] The NADPH-dependent glutamate synthase is derived from the glutamate synthase of Corynebacterium glutamicum ATCC 13032, and its amino acid sequence is as follows:

[0053] α-GS subunit (SEQ ID NO: 1):

[0054]

[0055] β-GS subunit (SEQ ID NO: 2):

[0056] MADPQGFIKYSRREPAHRPVPLRLMDHSEVYEKAPAGQIEEQAARCMDCGVPFCHEGCPLGNIIPEWNDLVRQGRWKEAYDRLHATNNFPEFTGRLCPAPCEGACVLGINDDSVTIKNVELEIVEKA FREGWVQPVIPSMSTGLSVAVVGSGPAGLAAAQQLTRAGHSVTVFERDDRLGGLMRYGVPEYKMENRWIDRRIEQMEAEGTTFQVGTSPRAAELALFDAILLATGTPVARELSVPGHDLNGIHAAMD YLTAQNRINEGDGEVSPINAKGKKVVIIGGGDTGTDCFGTALRQGAESVTQFDIRPRAPFQRADSTPWPMYPNLFRTATAHEEGEYIITGDESADEIAALGLAERAAGSTLGERKFAVNTVEFHGNN GHVTGLTGNQIRVVNGKREPIEGTEFPFEADLVLVALGFTGAEQGGLAHELGVGFDDRGRILRDSEYRSPTNSRVYIAGDNGRGQSLIVWAIAEGRACAAAIDADLMGETALPVAVAPQDVPLAV*.

[0057] In one specific example, the NADH-dependent glutamate synthase was sourced from Saccharomyces cerevisiae.

[0058] The NADH-dependent glutamate synthase is derived from the glutamate synthase of yeast (Saccharomyces cerevisiae ATCC204508 / S288c), and its amino acid sequence is shown in SEQ ID NO: 3, wherein the α-domain is located at amino acid positions 1 to 1642 in SEQ ID NO: 3, and the β-domain is located at amino acid positions 1643 to 2145 in SEQ ID NO: 3.

[0059] SEQ ID NO: 3:

[0060] GFMIHKRRHETHRDPRTRVNDW KEFTNPITKKDAKYQTARCMDCGTPFCLSDTGCPLSNIIPKFNELLFKNQWKLALDKLLETNNFPEFTFGRVCPAPC EGACTlgIIEDPVGIKSVERIIIDNAFKEGWIKPCPPSTRTGFTVGVIGSGPAGLACADMLNRAGHTVTVYERSDR CGGLLMYGIPNMKLDKAIVQRRIDLLSAEGIDFVTNTEIGKTISMDELKNKHNAVVYAIGSTIPRDLPIKGRELKN IDFAMQLLESNTKALLNKDLEIIREKIQGKKVIVVGGGDTGNDCLGTSVRHGAASVLNFELLPEPPVERAKDNPWP QWPRVMRVDYGHAEVKEHYGRDPREYCILSKEFIGNDEGEVTAIRTVRVEWKKSQSGVWQMVEIPNSEEIFEADII LLSMGFVGPELINGNDNEVKKTRRGTIATLDDSSYSIDGGKTFACGDCRRGQSLIVWAIQEGRKCAASVDKFLMDG TTYLPSNGGIVQRDYKLLKELASQV The unlined parts are α-domains, and the lined parts are β-domains.

[0061] In one specific example, the amino acid sequence of the α-GS subunit of NADPH-dependent glutamate synthase is shown in SEQ ID NO: 1.

[0062] In one specific example, the amino acid sequence of the β-GS subunit of NADPH-dependent glutamate synthase is shown in SEQ ID NO: 2.

[0063] In one specific example, the amino acid sequence of the α-domain of NADH-dependent glutamate synthase is shown as amino acids 1 to 1642 in SEQ ID NO: 3.

[0064] In one specific example, the amino acid sequence of the β-domain of NADH-dependent glutamate synthase is shown as amino acids 1643 to 2145 in SEQ ID NO: 3.

[0065] In one specific example, the glutamate synthase hybrid comprises the amino acid fragment shown in SEQ ID NO: 1 (the α-GS subunit of NADPH-dependent glutamate synthase) and the fragment shown in amino acids 1643 to 2145 of SEQ ID NO: 3 (the β-domain of NADH-dependent glutamate synthase). This glutamate synthase hybrid exhibits a reversed coenzyme preference, enabling it to utilize NADH to generate NAD. + By constructing genetically engineered strains, the yields of glycosaminoglycans, glucosamine, amino acids, and organic acids can be significantly increased by 30%-50%, demonstrating a remarkable improvement effect.

[0066] In one embodiment, the glutamate synthase hybrid comprises the fragment represented by amino acids 1 to 1642 of SEQ ID NO: 3 (the α-domain of NADH-dependent glutamate synthase) and the amino acid fragment represented by SEQ ID NO: 2 (the β-GS subunit of NADPH-dependent glutamate synthase). This glutamate synthase hybrid exhibits a reversed coenzyme preference, enabling it to utilize NADPH to generate NADP. + By constructing genetically engineered strains, the yields of pyruvate and acetic acid were significantly increased by 20%-25%, demonstrating a remarkable improvement.

[0067] One embodiment of this application also provides a nucleic acid encoding the hybrid or a fragment thereof.

[0068] In one specific example, the nucleic acid was obtained by hybrid combination based on the nucleotide sequences of wild-type glutamate synthases from different sources.

[0069] An embodiment of this application also provides an expression vector comprising the nucleic acid.

[0070] The expression vectors selected in this application can stably exist and autonomously replicate in various hosts, including prokaryotic and eukaryotic cells. In one specific example, the expression vector includes one or more of plasmid vectors, shuttle vectors, bacteriophages, and viral vectors. In one specific example, the plasmids include one or more of the pET series, pXM series, and pEC series. In one specific example, the expression vectors include one or more of pET-28a, pXMJ19, and pEC-Ptac.

[0071] In one specific example, the nucleic acid encoding the glutamate synthase hybrid was inserted into the plasmid vector through molecular biological operations such as enzyme digestion and ligation to construct a recombinant expression plasmid.

[0072] One embodiment of this application also provides a host cell containing the nucleic acid or the expression vector.

[0073] In one specific example, nucleic acids are integrated into the genome of the host cell.

[0074] In one specific example, the host cell includes a host bacterium. In one specific example, the host bacterium includes any one of Corynebacterium glutamicum, lactic acid bacteria, streptococci, Escherichia coli, yeast, Rhodococcus, Nocardia, and Bacillus subtilis.

[0075] In one specific example, the host cell is any one of Corynebacterium glutamicum, yeast, Bacillus subtilis, and Escherichia coli.

[0076] In one specific example, the nucleic acid is directly inserted into the chromosome of the host bacterium, or the expression vector is introduced into the host bacterium using the calcium chloride method or electroporation transformation method.

[0077] An embodiment of this application also provides a method for preparing the hybrid, including preparing the hybrid using the host cell.

[0078] One embodiment of this application also provides the use of the host cell in the preparation of compounds.

[0079] An embodiment of this application also provides a method for preparing a compound, comprising: preparing the compound using the host cell.

[0080] In one specific example, the host cells are fermented in a culture medium to prepare the compound.

[0081] In one specific example, the compound includes, but is not limited to, one or more of glycosaminoglycans, glucosamine and its derivatives, amino acids, and organic acids.

[0082] In one specific example, glycosaminoglycans include, but are not limited to, one or more of hyaluronic acid and chondroitin.

[0083] In one specific example, the derivatives include, but are not limited to, N-acetylglucosamine.

[0084] In one specific example, the amino acids include, but are not limited to, one or more of glutamic acid, glutamine, ornithine, arginine, lysine, and proline.

[0085] In one specific example, the organic acid includes one or more of pyruvic acid, acetic acid, lactic acid, and succinic acid.

[0086] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0087] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0088] Example 1: Design of an artificial hybrid of glutamate synthase

[0089] Glutamate synthase is a coenzyme-dependent synthase, primarily composed of NADH and NADPH. It catalyzes the synthesis of glutamate, consuming reduced NADH and NADPH while producing oxidized NAD+ and NADP+. Different sources of glutamate synthase exhibit different cofactor preferences; therefore, altering these coenzyme preferences can serve as a means of regulating intracellular redox metabolism. For example, *Corynebacterium glutamicum* consumes oxidized NAD+ during the synthesis of glycosaminoglycans, glucosamine, amino acids, and organic alcohols. + This process generates reduced NADH. Taking hyaluronic acid synthesis as an example, in order to maintain the intracellular redox state of Corynebacterium glutamicum during HA (hyaluronic acid) synthesis, the reduced NADH produced in this process needs to be converted into oxidized NAD. +This allows for the endogenous cycling of coenzymes. However, glutamate synthase from Corynebacterium glutamicum is NADPH-dependent and cannot achieve endogenous coenzyme cycling. Therefore, an artificial hybrid of glutamate synthase was designed to modify the coenzyme-biased nature of naturally derived glutamate synthase. Specifically, the α-GS subunit of the NADPH-dependent glutamate synthase from Corynebacterium glutamicum is hybridized with the β-domain of the NADH-dependent glutamate synthase from yeast to construct a novel artificial hybrid of glutamate synthase 1 (structural model shown in figure 1). Figure 1 As shown in the diagram, the obtained artificial hybrid glutamate synthase 1 converts the coenzyme-dependent form of naturally derived glutamate synthase from NADPH to NADH. Alternatively, by hybridizing the β-GS subunit of NADPH-dependent glutamate synthase from Corynebacterium glutamicum with the α-domain of NADH-dependent glutamate synthase from yeast, artificial hybrid glutamate synthase 2 is obtained, which converts the coenzyme-dependent form of naturally derived glutamate synthase from NADH to NADPH.

[0090] Example 2: Cloning of the artificial heterozygote 1 gene of glutamate synthase and construction of recombinant plasmid

[0091] The NADPH-dependent glutamate synthase from naturally derived Corynebacterium glutamicum ATCC 13032 consists of two subunits, α-GS and β-GS, while the NADH-dependent glutamate synthase from yeast (Saccharomyces cerevisiae ATCC 204508 / S288c) also contains α- and β-domains. The sequence of the artificial hybrid glutamate synthase is composed of the α-GS domain of the NADPH-dependent glutamate synthase and the β-domain of the NADH-dependent glutamate synthase. Therefore, using the NADPH-dependent glutamate synthase from Corynebacterium glutamicum and the NADH-dependent glutamate synthase from yeast as templates, primers were designed to amplify the α-GS and β-domains, respectively. The sequence of the artificial hybrid glutamate synthase was then obtained through gene recombination.

[0092] The amino acid sequence of the α-GS subunit of the NADPH-dependent glutamate synthase in Corynebacterium glutamicum is as follows:

[0093] α-GS subunit (SEQ ID NO: 1):

[0094]

[0095] The amino acid sequence of the β-domain of yeast NADH-dependent glutamate synthase is as follows:

[0096] GFMIHKRRHETHRDPRTRVNDWKEFTNPITKKDAKYQTARCMDCGTPFCLSDTGCPLSNIIPKFNELLFKNQWKLALDKLLETNNFPEFTGRVCPAPCEGACTLGIIEDPLGIKSVERIIIDNAFK EGWIKPCPPSTRTGFTVGVIGSGPAGLACADMLNRAGHTVTVYERSDRCGGLLMYGIPNMKLDKAIVQRRIDLLSAEGIDFVTNTEIGKTISMDELKNKHNAVVYAIGSTIPRDLPIKGRELKNID FAMQLLESNTKALLNKDLEIIREKIQGKKVIVVGGGDTGNDCLGTSVRHGAASVLNFELLPEPPVERAKDNPWPQWPRVMRVDYGHAEVKEHYGRDPREYCILSKEFIGNDEGEVTAIRTVRVEWK KSQSGVWQMVEIPNSEEIFEADIILLSMGFVGPELINGNDNEVKKTRRGTIATLDDSSYSIDGGKTFACGDCRRGQSLIVWAIQEGRKCAASVDKFLMDGTTYLPSNGGIVQRDYKLLKELASQV*

[0097] Gene synthesis of α-GS: Primers 1 and 2 were designed, and the gene was amplified using a high-fidelity enzyme.

[0098] The sequence of primer 1 for α-GS amplification is: caggaaacagaattaattaagcttatgccagtgttgaaatcag, SEQ ID NO: 4; the sequence of primer 2 for α-GS amplification is: gtttgtggatcataaaacccattagctcactgcctccatg, SEQ ID NO: 5.

[0099] The 50 μL PCR system includes:

[0100] 1 μL genomic template (approximately 10 ng / μL); 25 μL high-fidelity enzyme; 2.5 μL primer 1 (10 μM); 2.5 μL primer 2 (10 μM); 19 μL ddH2O.

[0101] Gene synthesis of β-domain: Primers 1 and 2 were designed, and gene amplification was performed using a high-fidelity enzyme.

[0102] Primer 1 for β-domain amplification: tgggttttatgatccacaaacgtcg, SEQ ID NO:6; Primer 2 for β-domain amplification: cggtacccggggatcctctagagtcgacttagacttgactagctaattc, SEQ ID NO:7.

[0103] The 50 μL PCR system includes:

[0104] 1 μL genomic template (approximately 10 ng / μL); 25 μL high-fidelity enzyme; 2.5 μL primer 3 (10 μM); 2.5 μL primer 4 (10 μM); 19 μL ddH2O.

[0105] The conditions for the PCR reaction are as follows:

[0106] (1) Pre-denaturation at 98 ℃ for 1 min;

[0107] (2) Denaturation at 98 ℃ for 30 s;

[0108] (3) Anneal at ℃ for 10 s (Tm-5 of primers);

[0109] (4) Extend at 72 ℃ for 7 min;

[0110] The above steps (2)-(4) are performed for a total of 30 cycles, and finally extended at 72 ℃ for 10 min.

[0111] The gene product obtained by PCR amplification was recovered by gel excision and used for later use. The recovered PCR product was linked with the plasmid backbone using the Gbsion kit and transformed into E. coli cells. Single colony sequencing confirmed that the glutamate artificial heterozygote gene was correctly cloned, and the plasmid was extracted. Finally, the recombinant plasmid containing the glutamate artificial heterozygote gene was obtained.

[0112] Example 3 Construction of recombinant plasmid for artificial hybrid glutamate synthase 2

[0113] The NADPH-dependent glutamate synthase from naturally occurring Corynebacterium glutamicum ATCC 13032 consists of two subunits, α-GS and β-GS, while the NADH-dependent glutamate synthase from yeast (Saccharomyces cerevisiae ATCC 204508 / S288c) also contains both α- and β-domains. The sequence of the artificial hybrid glutamate synthase 2 is composed of the β-GS domain of the NADPH-dependent glutamate synthase and the α-domain of the NADH-dependent glutamate synthase. Therefore, primers were designed using the NADPH-dependent glutamate synthase from Corynebacterium glutamicum and the NADH-dependent glutamate synthase from yeast as templates to amplify the β-GS and α-domains, respectively. The sequence of the artificial hybrid glutamate synthase was then obtained through gene recombination.

[0114] The amino acid sequence of the β-GS subunit of the NADPH-dependent glutamate synthase in Corynebacterium glutamicum is as follows:

[0115] β-GS subunit (SEQ ID NO: 2):

[0116] MADPQGFIKYSRREPAHRPVPLRLMDHSEVYEKAPAGQIEEQAARCMDCGVPFCHEGCPLGNIIPEWNDLVRQGRWKEAYDRLHATNNFPEFTGRLCPAPCEGACVLGINDDSVTIKNVELEIVEK AFREGWVQPVIPSMSTGLSVAVVGSGPAGLAAAQQLTRAGHSVTVFERDDRLGGLMRYGVPEYKMENRWIDRRIEQMEAEGTTFQVGTSPRAAELALFDAILLATGTPVARELSVPGHDLNGIHAAM DYLTAQNRINEGDGEVSPINAKGKKVVIIGGGDTGTDCFGTALRQGAESVTQFDIRPRAPFQRADSTPWPMYPNLFRTATAHEEGEYIITGDESADEIAALGLAERAAGSTLGERKFAVNTVEFHGN NGHVTGLTGNQIRVVNGKREPIEGTEFPFEADLVLVALGFTGAEQGGLAHELGVGFDDRGRILRDSEYRSPTNSRVYIAGDNGRGQSLIVWAIAEGRACAAAIDADLMGETALPVAVAPQDVPLAV*

[0117] The amino acid sequence of the α-domain of yeast NADH-dependent glutamate synthase is as follows:

[0118]

[0119] Gene synthesis of β-GS: Primers 1 and 2 were designed, and the gene was amplified using a high-fidelity enzyme.

[0120] The sequence of primer 1 for β-GS amplification is: gcgaaaggattgaaaaaacacgtggtatggccgacccacaaggattcatc, SEQ ID NO: 8; the sequence of primer 2 for β-GS amplification is: ctagacagccagcggcacgtcctgcaagcttgtcgacggagctcgaattc, SEQ ID NO: 9.

[0121] The 50 μL PCR system includes:

[0122] 1 μL genomic template (approximately 10 ng / μL); 25 μL high-fidelity enzyme; 2.5 μL primer 1 (10 μM); 2.5 μL primer 2 (10 μM); 19 μL ddH2O.

[0123] Gene synthesis of α-domain: Primers 1 and 2 were designed, and gene amplification was performed using a high-fidelity enzyme.

[0124] Primer 1 for α-domain amplification: tggacagcaaatgggtcgcggatccatgccagtgttgaaatcag, SEQ ID NO:10; Primer 2 for α-domain amplification: accacgtgttttttcaatcctttcgct, SEQ ID NO:11.

[0125] The 50 μL PCR system includes:

[0126] 1 μL genomic template (approximately 10 ng / μL); 25 μL high-fidelity enzyme; 2.5 μL primer 3 (10 μM); 2.5 μL primer 4 (10 μM); 19 μL ddH2O.

[0127] The conditions for the PCR reaction are as follows:

[0128] (1) Pre-denaturation at 98 ℃ for 1 min;

[0129] (2) Denaturation at 98 ℃ for 30 s;

[0130] (3) Anneal at ℃ for 10 s (Tm-5 of primers);

[0131] (4) Extend at 72 ℃ for 7 min;

[0132] The above steps (2)-(4) are performed for a total of 30 cycles, and finally extended at 72 ℃ for 10 min.

[0133] The gene product obtained by PCR amplification was recovered by gel excision and used for later use. The recovered PCR product was linked with the plasmid backbone using the Gbsion kit and transformed into E. coli cells. Single colony sequencing confirmed that the glutamate artificial heterozygote gene was correctly cloned, and the plasmid was extracted. Finally, a recombinant plasmid containing the glutamate artificial heterozygote 2 gene was obtained.

[0134] Example 4 Construction of the suicide plasmid of artificial hybrid 1 of glutamate synthase

[0135] Primers were designed based on the α-GS and β-GS subunit sequences of glutamate synthase in the genome of Corynebacterium glutamicum to amplify homologous arm sequences, which were then linked with the β-domain of glutamate synthase derived from yeast using Gbsion. The linked fragment was constructed into the pk18mob suicide plasmid and double-exchange homologous recombination was performed.

[0136] The primers used are:

[0137] Upstream homologous arm primer 1: atgattacgaattcgcttcggacacgatcac, SEQ ID NO:12;

[0138] Upstream homologous arm primer 2: gtttgtggatcataaaacccattagctcactgcctccatg, SEQ ID NO:13.

[0139] Downstream homologous primer 1: tctaacttgggtagaaaatgctagaaac, SEQ ID NO:14;

[0140] Downstream homologous primer 2: gcaggtcgactctagaggatcccacgttcagggctgttcg, SEQ ID NO:15.

[0141] Suicide plasmid backbone primer 1: gggcgaacagccctgaacgtgggatcctctagagtcgacctgcaggca, SEQ ID NO:16;

[0142] Suicide plasmid backbone primer 2: gggtgatcgtgtccgaagcgaattcgtaatcatgtcatagctgtt, SEQ ID NO:17.

[0143] Double swap steps:

[0144] 1. The constructed target suicide plasmid was electroporated into Corynebacterium glutamicum and heat-shocked at 46 °C for 6 min. The plating was then spread on kanamycin-resistant LBHIS plates, and colony PCR was performed to verify successful single crossover.

[0145] 2. Resuspend the successfully exchanged single colony in 200 μL of sterile water, spread it on LB agar plates (antibiotic-free) containing 150 g / L sucrose, and pick single colonies to verify whether the replacement was successful.

[0146] Example 5: Construction of genetically engineered bacteria for artificial hybrid glutamate synthase 1

[0147] The suicide plasmid prepared in Example 4 was transformed into competent cells of Corynebacterium glutamate using the heat shock method. The artificial heterozygote gene of glutamate synthase was replaced into the genome using the homologous recombination double exchange method, and genetically engineered bacteria (Cg1) with artificial heterozygote of glutamate synthase at the genome level were obtained.

[0148] Example 6: Construction of genetically engineered bacteria for artificial hybrid glutamate synthase 2

[0149] The recombinant expression vector prepared in Example 3 was transformed into competent E. coli cells using the heat shock method. The cells were then plated on solid plates containing antibiotics (50 μg / mL) and incubated overnight at 37 °C. Single colonies were picked and transferred to LB liquid culture containing 50 μg / mL kanamycin and incubated at 37 °C for 12 h. The colonies were then sent for sequencing. The correct clones were stored at -70 °C to obtain the genetically engineered bacteria (Cg2) containing artificial heterozygotes of glutamate synthase.

[0150] Example 7: Fermentation production of glycosaminoglycans by genetically engineered bacteria of glutamate synthase artificial hybrid 1

[0151] Genetically engineered bacterial colonies containing artificial hybrids of glutamate synthase were picked from LB agar plates and transferred to LBG medium containing the appropriate antibiotics. The colonies were cultured overnight at 30 °C and 200 rpm. At a 5% inoculum size, the seed culture was added to a 300 mL Erlenmeyer flask containing 50 mL of fermentation medium (glucose (40 g / L), ammonium sulfate (30 g / L), corn steep liquor powder (20 g / L), potassium dihydrogen phosphate (1 g / L), dipotassium hydrogen phosphate (0.5 g / L), magnesium sulfate heptahydrate (5 g / L), ferrous sulfate heptahydrate (0.01 g / L), and manganese sulfate heptahydrate (0.01 g / L; pH adjusted to 7.2), along with the appropriate antibiotics. After culturing at 28 °C for 3 h, IPTG was added to a final concentration of 1 mM to induce expression. The colonies were then cultured at 30 °C and 200 rpm for 48 h, after which biomass and glycosaminoglycan concentrations were measured.

[0152] Biomass determination method: 50 μL of *Corynebacterium glutamicum* fermentation broth was pipetted and diluted to 3 mL with 2950 μL of water. The diluted broth was then transferred to a glass cuvette, and the absorbance at 600 nm was measured using a UV-Vis spectrophotometer. This absorbance was taken as OD600. Calibration was performed using the absorbance of water as zero. Results are as follows: Figure 2 As shown.

[0153] The method for determining glycosaminoglycans is as follows: Take an appropriate amount of fermentation broth, add 3 times the volume of ethanol, mix well, and let stand for 2 hours. Centrifuge, discard the supernatant, retain the precipitate, and dry at room temperature for 1 hour. Add an equal volume of deionized water and accelerate the decomposition using a vortex mixer. Centrifuge to discard the bacterial cells and insoluble impurities, and obtain the supernatant, which is the glycosaminoglycan aqueous solution.

[0154] The concentration of glycosaminoglycans was then determined using the CTAB (trimethylhexylammonium bromide) turbidimetric method. The reaction system consisted of the sample, acetate buffer, and CTAB solution. After a reaction of 5 min, the OD400 absorbance was measured. The concentration was calculated using the formula: Glycosaminoglycan (g / L) = OD400 + 0.009.

[0155] The results are as follows Figure 3 As shown, the reconstructed strain (Cg1) containing an artificial heterozygote of glutamate synthase has an improved effect on the synthesis of hyaluronic acid and chondroitin compared with the wild-type strain (Cg0), with the yields increasing by 30% and 20%, respectively.

[0156] Example 8: Fermentation production of glucosamine by genetically engineered bacteria of artificial hybrid glutamate synthase 1

[0157] Genetically engineered bacterial colonies containing artificial hybrids of glutamate synthase were picked from LB agar plates and transferred to LBG medium containing the corresponding antibiotics. The colonies were then incubated at 30°C. o Incubate overnight at 200 rpm at C. Add 5% inoculum to a 300 mL Erlenmeyer flask containing 50 mL of fermentation medium (glucose (40 g / L), ammonium sulfate (30 g / L), corn steep liquor powder (20 g / L), potassium dihydrogen phosphate (1 g / L), dipotassium hydrogen phosphate (0.5 g / L), magnesium sulfate heptahydrate (5 g / L), ferrous sulfate heptahydrate (0.01 g / L), manganese sulfate heptahydrate (0.01 g / L). Adjust pH to 7.2.) and add the appropriate antibiotics. Incubate at 28°C. o After culturing for 3 h, IPTG at a final concentration of 1 mM was added to induce expression. The mixture was then incubated at 30 °C and a shaker speed of 200 rpm for 48 h, after which the concentrations of glucosamine and its derivatives were determined.

[0158] Determination of glucosamine and its derivatives: Glucosamine and its derivatives in the fermentation broth were determined by high-performance liquid chromatography (HPLC). The detection method was as follows: the mobile phase was 0.5 mM dilute sulfuric acid, the column was a BIO-Rad HPX-87H column, and the column temperature was 65°C. o C, the mobile phase flow rate was 0.5 mL / min, the sample uptake was 20 μL, the detection was performed using a differential detector, and finally the content of each substance in the culture medium was calculated based on the standard curve of the standard product.

[0159] The results are as follows Figure 4 As shown, the reconstructed strain (Cg1) containing an artificial hybrid of glutamate synthase showed an improved yield of glucosamine and N-acetylglucosamine compared to the wild-type strain (Cg0), with yields increasing by 35% and 25%, respectively.

[0160] Example 9: Fermentation production of amino acids by genetically engineered bacteria of artificial hybrid glutamate synthase 1

[0161] Genetically engineered bacterial colonies containing artificial hybrids of glutamate synthase were picked from LB agar plates and transferred to LBG medium containing the corresponding antibiotics. The colonies were then incubated at 30°C. o Incubate overnight at 200 rpm at C. Add 5% inoculum to a 300 mL Erlenmeyer flask containing 50 mL of fermentation medium (glucose (40 g / L), ammonium sulfate (30 g / L), corn steep liquor powder (20 g / L), potassium dihydrogen phosphate (1 g / L), dipotassium hydrogen phosphate (0.5 g / L), magnesium sulfate heptahydrate (5 g / L), ferrous sulfate heptahydrate (0.01 g / L), manganese sulfate heptahydrate (0.01 g / L). Adjust pH to 7.2.) and add the appropriate antibiotics. Incubate at 28°C. o At C, after culturing for 3 h, IPTG was added to a final concentration of 1 mM to induce expression. At 30... o C. Incubate at 200 rpm for 48 h, then measure the concentration of amino acids.

[0162] Methods for determining amino acid content: The amino acid content in the fermentation broth was determined by derivatization. First, the target amino acid was derivatized and modified. The modified derivative was then quantitatively analyzed by high performance liquid chromatography (HPLC). The detection method was as follows: the mobile phase was 0.5 mM dilute sulfuric acid, the column was an AQ-C18 (250 mm × 6.6 mm, 5 μm) column, the column temperature was 35 ℃, the mobile phase flow rate was 1.0 mL / min, the sample uptake was 10 μL, and the detection was performed using a UV detector. Finally, the content of each substance in the culture medium was calculated based on the standard curve of the standard sample.

[0163] The results are as follows Figure 5As shown, the reconstructed strain (Cg1) containing an artificial heterozygote of glutamate synthase showed improved synthesis of glutamate, glutamine, ornithine, arginine, lysine and proline compared to the wild-type strain (Cg0), with increases of 25%, 30%, 35%, 15%, 20% and 35%, respectively.

[0164] Example 10: Fermentation of organic acids by genetically engineered bacteria of artificial hybrid glutamate synthase 1

[0165] Genetically engineered bacterial colonies containing artificial hybrids of glutamate synthase were picked from LB agar plates and transferred to LBG medium containing the corresponding antibiotics. The colonies were then incubated at 30°C. o Incubate overnight at 200 rpm at C. Add 5% inoculum to a 300 mL Erlenmeyer flask containing 50 mL of fermentation medium (glucose (40 g / L), ammonium sulfate (30 g / L), corn steep liquor powder (20 g / L), potassium dihydrogen phosphate (1 g / L), dipotassium hydrogen phosphate (0.5 g / L), magnesium sulfate heptahydrate (5 g / L), ferrous sulfate heptahydrate (0.01 g / L), manganese sulfate heptahydrate (0.01 g / L). Adjust pH to 7.2.) and add the appropriate antibiotics. Incubate at 28°C. o After culturing for 3 h, IPTG at a final concentration of 1 mM was added to induce expression. The mixture was then incubated at 30 °C and a shaker speed of 200 rpm for 48 h, after which the concentration of organic acids was measured.

[0166] Determination of organic acid content: The content of organic acids in the fermentation broth was quantitatively analyzed by high performance liquid chromatography (HPLC). The mobile phase was 5 mM sulfuric acid aqueous solution, and the column used was an HPX-87H (9 μm, 300 mm × 7.8 mm) column at a temperature of 65°C. The temperature was set at ℃, the mobile phase flow rate was 0.4 mL / min, the sample uptake was 10 μL, and a differential detector was used for detection. Finally, the peak values ​​of the data were calculated based on the standard curve to determine the content of each substance in the culture medium. The reconstructed strain (Cg1) containing the artificial heterozygote of glutamate synthase showed improved synthesis of lactic acid and succinic acid compared to the wild-type strain (Cg0), with increases of 25% and 50%, respectively.

[0167] Example 11 Fermentation production of organic acids by genetically engineered bacteria of glutamate synthase artificial hybrid 2

[0168] Genetically engineered bacterial colonies containing artificial hybrids of glutamate synthase were picked from LB agar plates and transferred into LB agar containing the corresponding antibiotics. The colonies were then incubated at 37°C. oIncubate overnight at 200 rpm at C. Add 1% inoculum to 50 mL of fermentation medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl), and add the appropriate antibiotics. Incubate at 37°C. o After culturing for 3 h, IPTG at a final concentration of 1 mM was added to induce expression. The mixture was then incubated at 30 °C and a shaker speed of 200 rpm for 48 h, after which the concentration of organic acids was measured.

[0169] Determination of organic acid content: The content of organic acids in the fermentation broth was quantitatively analyzed by high performance liquid chromatography (HPLC). The mobile phase was 5 mM sulfuric acid aqueous solution, and the column used was an HPX-87H (9 μm, 300 mm × 7.8 mm) column at a temperature of 65°C. The temperature was set at ℃, the mobile phase flow rate was 0.4 mL / min, the sample uptake was 10 μL, and a differential detector was used for detection. Finally, the content of each substance in the culture medium was calculated based on the standard curve. The reconstructed strain (Cg2) containing the artificial hybrid of glutamate synthase showed improved pyruvate and acetic acid synthesis compared with the wild-type strain (Cg0), with increases of 20% and 25%, respectively.

[0170] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0171] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A hybrid of glutamate synthetase, characterized in that, the hybrid of glutamate synthetase comprises the fragment of amino acid shown in SEQ ID NO: 1 and the fragment of amino acid shown in SEQ ID NO: 3 from 1643 to 2145; or, the hybrid of glutamate synthetase comprises the fragment of amino acid shown in SEQ ID NO: 3 from 1 to 1642 and the fragment of amino acid shown in SEQ ID NO:

2.

2. Nucleic acid, characterized in that which encodes the hybrid of claim 1.

3. An expression vector, characterized in that, comprising the nucleic acid of claim 2.

4. The expression vector of claim 3, comprising one or more of a plasmid vector, a shuttle vector and a viral vector; the viral vector comprises a bacteriophage.

5. The expression vector of claim 4, wherein, the plasmid vector comprises one or more of pET series, pXM series and pEC series.

6. The expression vector of claim 4 or 5, wherein, the expression vector comprises one or more of pET-28a, pXMJ19 and pEC-Ptac.

7. A host cell characterized in that, comprising the nucleic acid of claim 2 or the expression vector of any one of claims 3-6.

8. The host cell of claim 7, wherein, the nucleic acid is integrated into the genome of the host cell.

9. The host cell of claim 7 or 8, wherein, the host cell comprises any one of Corynebacterium glutamicum, lactic acid bacteria, Streptococcus, Escherichia coli, yeast, Rhodococcus, Nocardia and Bacillus subtilis.

10. A method for the preparation of a hybrid, characterized in that, comprising preparing the hybrid using the host cell of any one of claims 7-9.

11. A method of preparing a compound, characterized by, comprising: preparing a compound using the host cell of any one of claims 7-9; the compound is selected from one or more of glycosaminoglycan, glucosamine and its derivatives, amino acid and organic acid; wherein: a. the glycosaminoglycan is selected from one or more of hyaluronic acid and chondroitin; b. the derivative is N-acetylglucosamine; c. the amino acid is selected from one or more of glutamic acid, glutamine, ornithine, arginine, lysine and proline; and d. the organic acid is selected from one or more of pyruvic acid, acetic acid, lactic acid and succinic acid.

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