Method for constructing amino acid-producing strain and use thereof

Abstract

The present application belongs to the field of microbiology and molecular biology, and the amino acid production of the amino acid production strain is improved by overexpression of Clp protease, which has important industrial application prospect for large-scale industrial production of amino acids.

Description

Technical Field

[0001] This invention belongs to the fields of microbiology and molecular biology, specifically relating to a method for constructing amino acid-producing strains, and a method for producing amino acids using this method. Background Technology

[0002] Corynebacterium glutamicum ( Corynebacterium glutamicum Corynebacterium glutamicum is currently the main strain used in microbial fermentation for amino acid production. With the continuous development of biotechnology, there are increasing reports on the use of genetic modification techniques to improve the amino acid production of Corynebacterium glutamicum, such as the modification of amino acid synthesis pathways, the selection of different substrates, and the enhancement of amino acid efflux pathways. At present, the acid production capacity of industrial production strains has reached a high level. Under the current circumstances, how to discover modifiable genetic targets to further improve the acid production capacity of strains has become a goal that is constantly pursued by those skilled in the art.

[0003] ATP-dependent proteases, such as Clp, play a crucial role in cellular protein quality control systems and cellular process regulation. ClpC encodes the Clp ATPase subunit, while ClpB, an ATP-dependent molecular chaperone, participates in metabolic pathways and plays a vital role in microorganisms under stress. ClpX and ClpP are involved in many important microbial functions, including stress responses and energy metabolism. However, research on these genes has largely focused on pathogenic bacteria, with limited studies on these genes in industrial-scale microorganisms, primarily in *Escherichia coli*. Existing research mainly focuses on protein structure, catalytic activity, and binding sites, with limited research on the impact of the Clp protease system on industrial microbial fermentation. Since industrial bacteria endure various stresses during fermentation, energy metabolism and maintaining normal growth and reproduction under stress are extremely important. Therefore, the role of the Clp protease system in industrial bacterial fermentation requires further investigation. Studies have shown that the Clp protease system plays an important role in high temperature, pH, and ethanol stress, and some of its genes may also be involved in regulating NADPH synthesis and glucose consumption during fermentation (Huang, MZ, et al., Role of the ClpX from Corynebacterium crenatum involved in stress responses and energy metabolism. Appl. Microbiol. Biotechnol.2020, 104:5505-5517.). For example, studies have shown that ClpB-deficient Corynebacterium mutants exhibit reduced growth under heat stress, and also affect glucose consumption and the production of L-arginine, L-glutamate, and lactic acid during fermentation (Huang, MZ, et al., Role of ClpB From Corynebacterium crenatum in Thermal Stress and ArginineFermentation. Front. Microbiol. (2020, 11.). Current research on the Clp protease system in industrial fermentation mainly focuses on improving or reducing the tolerance of gene knockout strains under extreme conditions.

[0004] However, there is still no research on the effects of gene overexpression in the Clp system on amino acid synthesis and secretion, especially in industrial model strains such as Corynebacterium glutamicum. Summary of the Invention

[0005] The purpose of this invention is to provide a novel method for constructing an amino acid-producing strain, and a method for constructing the strain and producing amino acids using this method. This invention has discovered that enhanced activity of these proteases is beneficial for improving the amino acid production capacity of the strain, thus leading to this invention.

[0006] In a first aspect, the present invention provides a method for constructing an amino acid-producing strain, comprising enhancing the activity of Clp proteases in the strain. The Clp proteases are ClpB, ClpC, ClpP1, and ClpP2 proteases.

[0007] In a further embodiment, the Clp protease is selected from at least one of the following:

[0008] A. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1-4;

[0009] B. A polypeptide mutant comprising the amino acid sequence shown in SEQ ID NO: 1-4, wherein the polypeptide mutant has substitution, duplication, deletion or addition of one or more amino acids at one or more positions of the sequence shown in SEQ ID NO: 1-4, and has the activity of a lysine efflux protein;

[0010] C. A polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% of the amino acid sequence shown in SEQ ID NO: 1-4, and having Clp protease activity.

[0011] Optionally, the ClpB protein is the protein with the amino acid sequence SEQ ID NO: 1, the ClpC protein is the protein with the amino acid sequence SEQ ID NO: 2, the ClpP1 protein is the protein with the amino acid sequence SEQ ID NO: 3, and the ClpP2 protein is the protein with the amino acid sequence SEQ ID NO: 4.

[0012] In some embodiments, the enhanced protein activity, enhanced expression level of protein-coding genes, enhanced enzyme activity, and enhanced expression level of enzyme-coding genes in recombinant microorganisms include recombinant microorganisms modified by the following genetic engineering methods: introducing strong promoters or strong ribosome binding sites into the cells of microorganisms; introducing recombinant expression vectors of non-integrated proteins; introducing recombinant expression vectors of chromosomal integrated proteins; altering the promoter, translation regulatory region, or coding region codon of a coding gene to enhance transcription or translation; altering the coding gene sequence to enhance the stability of its mRNA or stabilize the structure of the encoded protein; or any other method that enhances the activity of a gene by modifying its coding region and its adjacent upstream and downstream regions.

[0013] In this invention, "amino acid producing strain" refers to any type of strain capable of producing amino acids, and its sources include, but are not limited to, Escherichia coli spp. Escherichia Erwinia ( ) Erwinia ), Serratia ( Serratia ), Providencia spp. Providencia ), Enterobacteriaceae ( Enterobacteria Salmonella ( Salmonella Streptomyces ( Streptomyces ), Pseudomonas spp. Pseudomonas ), genus *Brucea* ( Brevibacterium ) or Corynebacterium spp. ( Corynebacterium In some embodiments, the amino acid-producing strain is derived from Corynebacterium glutamicum or Escherichia coli. In some preferred embodiments, the amino acid-producing strain is derived from Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC 14067, or a derivative thereof.

[0014] In some embodiments, the lysine-producing strain can be a strain expressing the feedback-relieved aspartate kinase LysC based on Corynebacterium glutamicum ATCC 13032. Prior art has reported LysC strains that relieve feedback inhibition, such as LysCT311I, LysCS301F, LysCL301K, LysCL301M, LysCFBR, LysCI293Y, LysCI293Q, LysCD294F, LysCT307G, etc. Furthermore, the lysine-producing strain can also be other types of strains capable of producing lysine.

[0015] In some embodiments, the lysine-producing strain may also have one or more genes selected from the following that are attenuated or have reduced expression:

[0016] a. Encoding alcohol dehydrogenase adhE Gene;

[0017] b. Encoding acetate kinase ackA Gene;

[0018] c. Encoding phosphorylated acetyltransferases pta Gene;

[0019] d. Encoding lactate dehydrogenase ldhA Gene;

[0020] e. Encoding formic acid transporter focA Gene;

[0021] f. Encoding pyruvate-formate lyase pflB Gene;

[0022] g. Encoding pyruvate oxidase poxB Gene;

[0023] h. Encoding a bifunctional enzyme of aspartate kinase I / homoserine dehydrogenase I thrA Gene;

[0024] i. Encoding homoserine kinase thrB Gene;

[0025] j. Encoding lysine decarboxylase ldcC Genes; and

[0026] h. Encoding lysine decarboxylase cadA Gene.

[0027] In some embodiments, the lysine-producing strain may also have one or more genes selected from the following enhanced or overexpressed:

[0028] a. Encoding dihydropyridine synthase that relieves lysine feedback inhibition dapA Gene;

[0029] b. Encoding dihydropyridinedicarboxylate reductase dapB Gene;

[0030] c. Encoding diaminopimelic acid dehydrogenase ddh Gene;

[0031] d. Encoding tetrahydropyridinedicarboxylate succinylase dapD and encoding succinyldiaminopimelic acid deacylase dapE ;

[0032] e. Encoding aspartate-semialdehyde dehydrogenase asd Gene;

[0033] f. Encoding p-phosphoenolpyruvate carboxylase pc Gene;

[0034] g. Encoding nicotinamide adenine dinucleotide transhydrogenase pntAB Gene;

[0035] i. Transport proteins encoding lysine lysE Gene.

[0036] In some embodiments, the glutamate-producing strain may be a strain modified from Corynebacterium glutamicum ATCC 13032 or Corynebacterium glutamicum ATCC13869, wherein such modifications include, but are not limited to, enhancement or overexpression of one or more genes selected from the following:

[0037] a. Encoding mechanosensitive channel proteins yggB Gene;

[0038] b. Encoding phosphotransketase fxpk Gene;

[0039] c. Encoding pyruvate carboxylase pyc Gene;

[0040] d. Encoding glutamate dehydrogenase gdh Gene;

[0041] e. The gene encoding carbonic anhydrase.

[0042] In some embodiments, the glutamate-producing strain may also have one or more genes selected from the following that are attenuated or have reduced expression:

[0043] a. Encoding α-ketoglutarate dehydrogenase odhA Gene;

[0044] b. Genes encoding transcriptional regulation amtR Gene;

[0045] c. Encoding transcription repressors acnR Gene.

[0046] Secondly, the present invention provides an amino acid-producing strain, which is constructed by the above method, wherein the Clp protease activity of the strain is enhanced. In a specific embodiment, the Clp protease is ClpB, ClpC, ClpP1, and ClpP2 proteins.

[0047] In a further embodiment, the Clp protease is selected from at least one of the following:

[0048] A. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1-4;

[0049] B. A polypeptide mutant comprising the amino acid sequence shown in SEQ ID NO: 1-4, wherein the polypeptide mutant has substitution, duplication, deletion or addition of one or more amino acids at one or more positions of the sequence shown in SEQ ID NO: 1-4, and has the activity of a lysine efflux protein;

[0050] C. A polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% of the amino acid sequence shown in SEQ ID NO: 1-4, and having Clp protease activity.

[0051] Optionally, the ClpB protein is the protein with the amino acid sequence SEQ ID NO: 1, the ClpC protein is the protein with the amino acid sequence SEQ ID NO: 2, the ClpP1 protein is the protein with the amino acid sequence SEQ ID NO: 3, and the ClpP2 protein is the protein with the amino acid sequence SEQ ID NO: 4.

[0052] In some embodiments, the enhanced protein activity, enhanced expression level of protein-coding genes, enhanced enzyme activity, and enhanced expression level of enzyme-coding genes in recombinant microorganisms include recombinant microorganisms modified by the following genetic engineering methods: introducing strong promoters or strong ribosome binding sites into the cells of microorganisms; introducing recombinant expression vectors of non-integrated proteins; introducing recombinant expression vectors of chromosomal integrated proteins; altering the promoter, translation regulatory region, or coding region codon of a coding gene to enhance transcription or translation; altering the coding gene sequence to enhance the stability of its mRNA or stabilize the structure of the encoded protein; or any other method that enhances the activity of a gene by modifying its coding region and its adjacent upstream and downstream regions.

[0053] In specific embodiments, the amino acid-producing strains include, but are not limited to, those derived from the genus *Escherichia* (…). Escherichia Erwinia ( ) Erwinia ), Serratia ( Serratia ), Providencia spp. Providencia ), Enterobacteriaceae ( Enterobacteria Salmonella ( Salmonella Streptomyces ( Streptomyces ), Pseudomonas spp. Pseudomonas ), genus *Brucea* ( Brevibacterium ) or Corynebacterium spp. ( Corynebacterium The microorganisms are: Enterobacter spp. Escherichia coli, and Corynebacterium spp. Corynebacterium glutamicum.

[0054] In a further preferred embodiment, the starting strain is Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC 14067, or a derivative strain of any of the above.

[0055] Thirdly, the present invention provides a method for producing amino acids, the method comprising producing a target amino acid using an amino acid-producing strain of the second aspect, optionally including a step of separating the target amino acid from a fermentation broth.

[0056] This invention improves the amino acid yield of amino acid-producing strains by overexpressing the Clp protease, which has significant industrial application prospects for large-scale industrial production of amino acids. This invention also provides a novel method for constructing amino acid-producing strains. Detailed Implementation

[0057] Terminology Definition

[0058] When used in conjunction with the term “comprising” in the claims and / or specification, the words “a” or “an” may mean “one”, but may also mean “one or more”, “at least one”, and “one or more”.

[0059] As used in the claims and specification, the words “comprising,” “having,” “including,” or “containing” mean included or open-ended and do not exclude additional, uncited elements or method steps.

[0060] Throughout the application, the term “about” means: a value includes the standard deviation of the error of the apparatus or method used to determine that value.

[0061] While the disclosure supports the definition of the term "or" as merely a substitute and "and / or", the term "or" in the claims means "and / or" unless expressly stated as merely a substitute or as mutually exclusive among substitutes.

[0062] As used in this disclosure, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, and may be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).

[0063] The Clp protease complex described in this invention is an ATP-dependent protease system, generally composed of a catalytic subunit ClpP (ClpP1 and ClpP2 in Corynebacterium glutamicum) and regulatory subunits with ATPase activity (ClpX, ClpC, and ClpB in Corynebacterium glutamicum). In specific embodiments, the amino acid sequence of the ClpB protein is shown in SEQ ID NO: 1, the amino acid sequence of the ClpC protein is shown in SEQ ID NO: 2, the amino acid sequence of the ClpP1 protein is shown in SEQ ID NO: 3, and the amino acid sequence of the ClpP2 protein is shown in SEQ ID NO: 4.

[0064] The term "wild type" in this invention refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory setting is naturally occurring.

[0065] The term "microorganism" in this invention refers to a general term for tiny organisms that are difficult to observe with the naked eye, including bacteria, fungi, etc. Because microorganisms have a large surface area to volume ratio, they can rapidly exchange substances with the external environment and produce metabolic products. In this invention, "microorganism" specifically refers to fermentative microorganisms capable of fermentation and culture to produce metabolic products such as sugars, lipids, amino acids, and nucleotides.

[0066] In this invention, "wild-type microorganisms" can refer to microorganisms that can be found in nature without artificial modification. Furthermore, wild-type microorganisms also include derivative microorganisms that have undergone certain modifications or alterations. It should be noted that in this invention, a microorganism can be considered wild-type as long as it has not undergone any of the following modifications: introduction of ClpB protein or enhancement of its expression level; introduction of a mutant that maintains the activity of ClpB protein or enhancement of its expression level; introduction of ClpC protein or enhancement of its expression level; introduction of a mutant that maintains the activity of ClpC protein or enhancement of its expression level; introduction of ClpP1 protein or enhancement of its expression level; introduction of a mutant that maintains the activity of ClpP1 protein or enhancement of its expression level; introduction of ClpP2 protein or enhancement of its expression level; introduction of a mutant that maintains the activity of ClpP2 protein or enhancement of its expression level.

[0067] The term "recombinant microorganism" in this invention refers to a modified microorganism obtained through recombination using genetic engineering methods. Embodiments include, but are not limited to, increasing the copy number of protein-coding genes, modifying the sequence of protein-coding genes, introducing exogenous recombinant genes, etc. The term "recombinant gene" refers to a gene that is not naturally occurring, and includes protein-coding sequences operatively linked to expression control sequences. Embodiments include, but are not limited to, introducing exogenous genes into microorganisms, endogenous protein-coding sequences operatively linked to heterologous promoters, and genes having modified protein-coding sequences. Recombinant genes are stored in the genome of microorganisms, plasmids in microorganisms, or bacteriophages in microorganisms.

[0068] In this invention, the term "coding gene" refers to a DNA molecule capable of guiding protein synthesis according to certain rules. The process by which a protein-coding gene guides protein synthesis generally includes transcription using double-stranded DNA as a template and translation using mRNA as a template. A coding gene contains a CDS (Coding Sequence) that guides the production of mRNA encoding the protein. Exemplarily, the protein-coding gene in this invention is a gene encoding an amino acid efflux protein. In some embodiments, the protein-coding gene in this invention is a gene encoding a polypeptide with the sequence shown in SEQ ID NO: 1-4 or a mutant gene that retains Clp protease activity.

[0069] In some embodiments, the enhanced protein activity, enhanced expression level of protein-coding genes, enhanced enzyme activity, and enhanced expression level of enzyme-coding genes in recombinant microorganisms include recombinant microorganisms modified by the following genetic engineering methods: introducing strong promoters or strong ribosome binding sites into the cells of microorganisms; introducing recombinant expression vectors of non-integrated proteins; introducing recombinant expression vectors of chromosomal integrated proteins; altering the promoter, translation regulatory region, or coding region codon of a coding gene to enhance transcription or translation; altering the coding gene sequence to enhance the stability of its mRNA or stabilize the structure of the encoded protein; or any other method that enhances the activity of a gene by modifying its coding region and its adjacent upstream and downstream regions.

[0070] In some embodiments, the recombinant microorganism of the present invention is a recombinant microorganism obtained by genetically engineering one or more of the following microorganisms: Escherichia coli ( Escherichia Erwinia ( ) Erwinia ), Serratia ( Serratia ), Providencia spp. Providencia ), Enterobacteriaceae ( Enterobacteria Salmonella ( Salmonella Streptomyces ( Streptomyces ), Pseudomonas spp. Pseudomonas ), genus *Brucea* ( Brevibacterium ) or Corynebacterium spp. ( Corynebacterium Microorganisms.

[0071] The term "polynucleotide" in this invention refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a single fragment or as a component of a larger nucleotide sequence structure, derived from a nucleotide sequence isolated at least once in number or concentration, and capable of being recognized, manipulated, and recovered using standard molecular biology methods (e.g., using cloning vectors). This also includes an RNA sequence (i.e., A, U, G, C) when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), where "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (single fragments or entire fragments), or it can be a component or part of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0072] The terms "sequence identity" and "identity percentage" in this invention refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotide or polypeptide and scoring the number of positions in the aligned polynucleotide or polypeptide containing the same nucleotide or amino acid residues, comparing this to the number of positions in the aligned polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides may differ at a position, for example, by containing different nucleotides (i.e., substitution or mutation) or deleted nucleotides (i.e., nucleotide insertion or deletion in one or two polynucleotides). Polypeptides may differ at a position, for example, by containing different amino acids (i.e., substitution or mutation) or deleted amino acids (i.e., amino acid insertion or deletion in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the identity percentage can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0073] In some embodiments, when comparing and aligning two or more sequences or subsequences using sequence comparison algorithms or by visual inspection to measure maximum correspondence, the two or more sequences or subsequences have a “sequence identity” or “percentage of identity” of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotides. In some embodiments, the sequences are substantially identical along the entire length of any one or two compared biopolymers (e.g., polynucleotides).

[0074] The term "expression" in this invention includes any step involving RNA and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0075] The term "vector" in this invention refers to a DNA construct containing a DNA sequence operatively linked to a suitable control sequence for expressing a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure for expressing, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence transcribed into mRNA and translated into a protein; and iii) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies DNA. In this invention, "recombinant expression vector" and "recombinant vector" may be used interchangeably.

[0076] The term "transformation" in this invention has the meaning commonly understood by those skilled in the art, namely, the process of introducing exogenous DNA into a host. The methods of transformation include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0077] The term "culture" in this invention can be performed according to conventional methods in the art, including but not limited to plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, and various culture conditions such as temperature, time, and pH of the culture medium can be appropriately adjusted according to actual conditions.

[0078] Unless otherwise defined in this invention or clearly indicated by the background, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Example

[0079] Materials and Methods

[0080] The DNA polymerase used in the embodiments of the present invention was purchased from Fastpfu by TransGen Biotech Ltd. in Beijing; the restriction endonuclease and DNA ligase were purchased from Fermentas.

[0081] Yeast powder and peptone were purchased from Oxoid, UK; glycine and IPTG were purchased from Promega; agar powder and antibiotics were purchased from Beijing Solarbio; glucose, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, urea, and other commonly used chemical reagents were purchased from Sinopharm.

[0082] The plasmid extraction kit and the agarose gel electrophoresis recovery kit were both purchased from Shanghai Sangon Biotech, and all related operations were strictly performed in accordance with the instructions.

[0083] Plasmid construction and sequencing validation were performed by Genewiz.

[0084] Trans T1 competent cells were purchased from Beijing TransGen Biotech Co., Ltd.

[0085] LB medium composition: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, and 2% agar powder added to the solid medium.

[0086] TSB medium composition: glucose 5 g / L, yeast extract 5 g / L, soybean peptone 9 g / L, K2HPO4·3H2O 1 g / L, Urea 3 g / L, succinic acid 0.5 g / L, MgSO4·7H2O 0.1 g / L, biotin 10 μg / L, V B1 0.1 mg / L, MOPS 20 g / L.

[0087] Lysine and glutamic acid were analyzed using the SBA-40D biosensor analyzer manufactured by Shandong Academy of Sciences.

[0088] Example 1. Construction of Clp protease system expression vector

[0089] Based on the genome sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI, primers clpB-F / R, clpC-F / R, clpX-F / R, clpP1-F / R, and clpP2-F / R were designed based on the protein coding sequences of ClpB, ClpC, ClpP1, ClpP2, and ClpX shown in SEQ ID NO: 1-5. Primer pEC-F / R was designed using pEC-XK99E plasmid as a template. PCR amplification yielded a plasmid backbone containing the trc promoter. The PCR amplification parameters were: 94°C for 5 min; 94°C for 20 s, 55°C for 20 s, 72°C for 1.5 min, 30 cycles; extension at 72°C for 5 min. After fragment recovery, homologous recombination was performed, and the pEC-XK99E plasmid was ligated. The ligation product was then transformed into Trans T1 competent cells, plated on kanamycin-resistant plates, and cultured overnight. Positive clones were selected for colony PCR and sequencing verification. The correct recombinant vectors were named pEC-clpB, pEC-clpC, pEC-clpX, pEC-clpP1, and pEC-clpP2, respectively.

[0090] Example 2. Effect of Clp protease system expression on lysine synthesis

[0091] To verify that expression of the Clp protease system gene in Corynebacterium glutamicum promotes lysine synthesis and secretion, the above-mentioned vector and its control vector pEC-XK99E were transformed into lysine-producing Corynebacterium glutamicum strain SCgL40 (WO2021143727A1). The preparation and transformation of competent cells followed the procedures outlined in *Molecular Cloning: A Laboratory Manual* by J. Sambrook et al. The transformation products were plated on kanamycin-resistant TSB plates, incubated overnight, and positive clones were picked for plasmid extraction and verification, yielding recombinant strains SCgL40 / pEC-XK99E, SCgL40 / pEC-clpB, SCgL40 / pEC-clpC, SCgL40 / pEC-clpX, SCgL40 / pEC-clpP1, and SCgL40 / pEC-clpP2.

[0092] Single colonies of the recombinant bacteria were inoculated into 5 mL of TSB liquid medium containing 25 µg / mL kanamycin and cultured at 30°C and 220 rpm for 8–12 h. The culture was then transferred to 24-well plates containing 800 µL of fermentation medium, 25 µg / mL kanamycin, and 0.005 mM IPTG at an initial OD of 0.1 and induced at 30°C and 800 rpm for 19 h. Fermentation broth was collected during the fermentation process for lysine content determination.

[0093] The fermentation results of the recombinant strains are shown in Table 1. Compared with the control strain, the lysine production of strains SCgL40 / pEC-clpB, SCgL40 / pEC-clpC, SCgL40 / pEC-clpP1, and SCgL40 / pEC-clpP2 increased by 20.51%, 33.33%, 41.03%, and 33.33%, respectively, while the lysine production of strain SCgL40 / pEC-clpX decreased significantly. These results indicate that enhancing the expression of the Clp protease system can significantly increase the lysine production during fermentation.

[0094] Table 1 Effect of Clp protease on lysine production

[0095] Strain Lysine (g / L) SCgL40 / pEC-XK99E 1.95±0.19 SCgL40 / pEC-clpB 2.35±0.1 SCgL40 / pEC-clpC 2.6±0.16 SCgL40 / pEC-clpP1 2.75±0.1 SCgL40 / pEC-clpP2 2.6±0 SCgL40 / pEC-clpX 0.6±0.1

[0096] Example 3. Effect of Clp protease system expression on glutamate synthesis

[0097] To verify the effectiveness of Clp protease system overexpression in the production of other target compounds, we conducted a test using glutamate as an example. We prepared competent cells of the high-yielding glutamate strain Z188 (NCBI Reference Sequence: NZ_AKXP00000000.1) using a method reported in the literature (Ruan Y, et al. Biotechnol. Lett., 2015, 37:2445-2452.). We then electroporated 1 μg of different expression vectors pEC-clpB, pEC-clpC, pEC-clpX, pEC-clpP1, pEC-clpP2, and the control vector pEC-XK99E into the prepared Z188 competent cells. The cells were then plated on TSB solid medium containing 25 μg / mL kanamycin and cultured at 30°C for 24 hours. After h, positive clones were picked and plasmids were extracted for verification, and recombinant strains Z188 / pEC-clpB, Z188 / pEC-clpC, Z188 / pEC-clpP1, Z188 / pEC-clpP2 and Z188 / pEC-XK99E were obtained respectively.

[0098] To test the effect of Clp protease component overexpression on glutamate production by *Corynebacterium glutamicum*, fermentation tests were performed on the recombinant strains obtained above. The seed culture medium consisted of: glucose, 50 g / L; phosphate, 0.7 g / L; ammonium sulfate, 10 g / L; MgSO4·7H2O, 0.8 g / L; corn steep liquor powder, 3 g / L; urea, 10 g / L; peptone, 1 g / L; yeast extract, 0.5 g / L; initial pH 7.0. The fermentation medium, compared to the seed culture medium, did not contain peptone or yeast extract, but instead included 84 g / L MOPS. The strains were first inoculated into the seed culture medium and cultured for 14 h. The culture was then used as seed culture in 24-well plates containing 800 μL of fermentation medium per well. Initial OD... 600 The culture temperature was controlled at 0.3, and the culture was carried out at 30°C for 29 h. The shaking speed of the well plate was 800 rpm. Each strain was replicated in triplicate. After fermentation, the glutamate yield and glucose consumption were measured, and the glucose-to-glutamate conversion rate was calculated. The results are shown in Table 2. When the IPTG concentration was 0.005 mM, the glutamate yield and conversion rate of strains Z188 / pEC-clpB, Z188 / pEC-clpC, Z188 / pEC-clpP1, and the control strain increased by more than 30%, while the glutamate yield of strains Z188 / pEC-clpX and Z188 / pEC-clpP2 decreased significantly. Therefore, the Clp protease system overexpression discovered in this invention has good application prospects in the production of glutamate and other compounds.

[0099] Table 2 Effect of Clp protease on glutamate production

[0100] Strain <![CDATA[OD 600 ]]> Glutamic acid (g / L) Sugar acid conversion rate (g / g, %) Z188 / pEC-XK99E 9.32 3.03 8.44 Z188 / pEC-clpB 10.03 3.97 10.63 Z188 / pEC-clpC 6.08 4.67 16.79 Z188 / pEC-clpP1 9.12 3.97 10.34 Z188 / pEC-clpP2 9.25 2.03 5.87 Z188 / pEC-clpX 8.82 1.73 4.90

[0101] To verify whether Clp protease overexpression can increase the yield of other products, those skilled in the art can introduce the expression vectors pEC-clpB, pEC-clpC, pEC-clpX, pEC-clpP1, and pEC-clpP2 obtained in the embodiments of the present invention into the production strains of the target products, for example, the above plasmids can be introduced into existing 5-aminolevulinic acid production strains (CN103981203BA, CN109722459A), proline production strains (CN112111469B), etc., to obtain engineered strains with increased yield.

[0102] The primer sequences used are as follows:

[0103] pEC-F:ctgcaggcatgcaagcttgg (SEQ ID NO: 6)

[0104] pEC-R: TCTCAACTCCTTTGGCCTGT (SEQ ID NO: 7)

[0105] clpB-F: acaggccaaaggagttgagaATGAGTTCATTCAATCCAACTAC (SEQ ID NO: 8)

[0106] clpB-R: ccaagcttgcatgcctgcagTTAGACCGCCCTGGAAACGT (SEQ ID NO: 9)

[0107] clpC-F: acaggccaaaggagttgagaATGTTCGAGAGGTTTACCGA (SEQ ID NO: 10)

[0108] clpC-R: ccaagcttgcatgcctgcagCTACTCCTTGCTTGGAGCAG (SEQ ID NO: 11)

[0109] clpP1-F: acaggccaaaggagttgagaTTGGGTACGTTCATTTGGTT (SEQ ID NO: 12)

[0110] clpP1-R: ccaagcttgcatgcctgcagTTAAAGAAATAACGCTTTTCG (SEQ ID NO: 13)

[0111] clpP2-F: acaggccaaaggagttgagaTTGACTGTTTTCATGAGCGA (SEQ ID NO: 14)

[0112] clpP2-R: ccaagcttgcatgcctgcagCTAGTTGCTGATTGGGCCTT (SEQ ID NO: 15)

[0113] clpX-F: acaggccaaaggagttgagaATGAATCCCGAATTTATTCA (SEQ ID NO: 16)

[0114] clpX-R: ccaagcttgcatgcctgcagTCAAAGTTGCTTAAGCAAAC (SEQ ID NO: 17). <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> Construction methods and applications of amino acid-producing strains <130> <160> 17 <170>PatentIn version 3.5 <210> 1 <211> 852 <212>PRT <213>Corynebacterium glutamicum <400> 1 MSSFNPTTKTNEAMQAALQQASSAGNPDIRPAHLLAAILEQTDGVAAPVLMATGVDPKEILAEAKKLVASYPKASGANMANPNFNRDALNAFTAAQELAGELGDEYVSTEVLLAGIARGKSDAADLLTNKGATYDAIKEAFPSVRGSQRVTTQDPEGQFQALEKYSTDLTKLAREGKIDPVIGRDQEIRRVVQVLSRRTKNNPVLIGEPGVGKTAIVEGLARRIVAGDVPESLKGKTLISLDLGSMVAGAKYRGEFEERLKAVLDEIKGANGEVVTFIDELHTIVGAGASGESAMDAGNMIKPLLARGELRLVGATTLNEYRKYIEKDAALERRFQQVYVGEPTVEDAIGILRGLKERYEVHHGVRIQDSALVAAAELSNRYITSRFLPDKAIDLVDEAASRLRMEIDSSPQEIDELERIVRRLEIEEMALSKESDAASKERLEKLRSELADEREKLSELKARWQNEKTAIDDVREMKEELEALRSESDIAERDGNYGRVAELRYGRIPELEKQIEDAESKVEVNENAMLTEEVTPDTIADVVSAWTGIPAGKMMQGETEKLLNMERVLGNRVVGQLEAVTAVSDAVRRSRAGVADPNRPTGSFLFLGPTGVGKTELAKAVAEFLFDDDRAMIRIDMSEYGEKHSVARLVGAPPGYVGYDQGGQLTEAVRRRPYTVVLFDEVEKAHPDVFDILLQVLDEGRLTDGQGRTVDFRNTILILTSNLGAGGTREQMMDAVKMAFKPEFVNRLDDVVIFDRLSPEQLTSIVDIQIKQLTDRLAGRRLNLRVSDSAKAWLAERGYDPAYGARPLRRLIQQAIGDTLAKELLAGNVRDGDGVLVDVADGGQKLDVSRAV 852 <210>2 <211> 925 <212>PRT <213>Corynebacterium glutamicum <400>2 MFERFTDRARRVIVLAQEEARMLNHNYIGTEHILLGLIHEGEGVAAKALESMGISLDAVRQEVEEIIGQGSQPTTGIPFTPRAKKVLELSLREGLQMGHKYIGTEFLLLGLIREGEGVAAQVLVKLGADLRVRQQVIQLLSGYEGGQGGSPEGQGAPTGGDAVGAGAAPGGRPSSGSGPGERSTSLVLDQFGRNLTQAAKDGKLDPVVGRDKEIERIMQVLSRRTKNNPVLIGEPGVGKTAVVEGLALDIVNGKVPETLKDKQVYSLDLGSLVAGSRYRGDFEERLKKVLKEINQRGDIILFIDEIHTLVGAGAAEGAIDAASLLKPKLARGELQTIGATTLDEYRKHIEKDAALERRFQPVQVPEPSVDLTVEILKGLRDRYEAHHRVSITDGALTAAAQLADRYINDRFLPDKAVDLIDEAGARMRIKRMTAPSSLREVDERIADVRREKEAAIDAQD FEKAAGLRDKERKLGEERSEKEKQWRSGDLEDIAEVGEEQIAEVLANWTGIPVFKLTEAESSRLLNMEELHKRIIGQDEAVKAVSRAIRRTRGLKDPKRPSGSFIFAGPSGVGKTELSKALAGFLFGDDSSLIQIDMGEFHDRFTASRLFGAPPGYVGYEEGGQLTEKVRRKPFSVVLFDEIEKAHKEIYNTLLQVLEDGRLTDGQGRIVDFKNTVLIFTSNLGTADISKAVGLGFSGSSETDSDAQYDRMKNKVHDELKKHFRPEFLNRIDEIVVFHQLTKDQIVQMVDLLIGRVSNALAEKDMSIELTEKAKDLLANRGFDPVLGARPLRRTIQREIEDQMSEKILFGEIGAGEIVTVDVEGWDGESKDTDRAKFTFTPRPKPMPEGKFSEISVEAAEAIQDVDSAADGDVPETDSLSDILETLEKFEEDVENGTDIDQVSGDYYGTDDQGGTAPSKE 925 <210> 3 <211> 201 <212> PRT <213>Corynebacterium glutamicum <400>3 MTVFMSDIRMAAQGGPGFGNDVFDRLLSERIIFLGSQVDDEIANKLCAQILLLSAEDPTRDISLYINSPGGSVTAGMAIYDTMKYSPCDIATYGMGLAASMGQFLLSGGTKGKRFALPHARIMMHQPSAGVGGTAADIAIQAEQFAATKREMAQLIAEHTGQTFEQISKDSDRDRWFTAQEAKDYGLVDHVITLAEGPISN 201 <210>4 <211> 492 <212>PRT <213>Corynebacterium glutamicum <400>4 MSNGFQMPTSRYVLPSFIEQSAYGTKETNPYAKLFEERIIFLGTQVDDTSANDIMAQLLVLEGMDPDRDITLYINSPGGSFTALMAIYDTMQYVRPDVQTVCLGQAASAAAVLLAAGAPGKRAVLPNSRVLIHQPATQGTQGQVSDLEIQAAEIERMRRLMETTLAEHTGKTAEQIRIDTDRDKILTAEEALEYGIVDQVFDYRKLKR 208 <210>5 <211> 426 <212>PRT <213>Corynebacterium glutamicum <400>5 MARMQESADLLKCSFCGKSQKQVKKLIAGGAVYICDECIELCNEIIEEELGQAQHDEQERNELPKPSEISAFLDTYVIGQDPAKRILSVAVYNHYKRLRASETIGRRRNDEPETELVKSNILMLGPTGSGKTFLAQTLAKLLDVPFAIADATSLTEAGYVGEDVENILLKLLQAADFDVERAQRGIIYIDEVDKISRKSENPSITRDVSGEGVQQALLKILEGTVAAIPPQGGRKHPNQDFIQLDTTNILFIVAGAFSGLEKVIADRNGKKGLGFGVEVSSKKEEANIVDIFKDVLPEDLVKFGLIPEFIGRLPVVATVSNLDQKSLVKVLTEPRNSLVKQYRRLFEMDDAVLTFTDDALEEIANQALERKTGARGLRAIMEEILVPIMYDLPDRKDVGEVIINGAVARGEAEPEMLEAVAEEKTA 426 <210>6 <211> 20 <212>DNA <213>Artificial sequence <400>6 ctgcaggcatgcaagcttgg20 <210>7 <211> 20 <212>DNA <213>Artificial sequence <400>7 tctcaactcctttggcctgt20 <210>8 <211>43 <212>DNA <213>Artificial sequence <400>8 acaggccaaaggagttgagaatgagttcattcaatccaactac 43 <210>9 <211> 40 <212>DNA <213>Artificial sequence <400>9 ccaagcttgcatgcctgcagttagaccgccctggaaacgt 40 <210> 10 <211> 40 <212> DNA <213> Artificial sequence <400> 10 acaggccaaaggagttgagaatgttcgagaggtttaccga 40 <210> 11 <211> 40 <212> DNA <213> Artificial sequence <400> 11 ccaagcttgcatgcctgcagctactccttgcttggagcag 40 <210> 12 <211> 40 <212> DNA <213> Artificial sequence <400> 12 acaggccaaaggagttgagattgggtacgttcatttggtt 40 <210> 13 <211> 41 <212> DNA <213> Artificial sequence <400> 13 ccaagcttgcatgcctgcagttaaagaaataacgcttttcg 41 <210> 14 <211> 40 <212> DNA <213> Artificial sequence <400> 14 acaggccaaaggagttgagattgactgttttcatgagcga 40 <210> 15 <211> 40 <212> DNA <213> Artificial sequence <400> 15 ccaagcttgcatgcctgcagctagttgctgattgggcctt40 <210> 16 <211> 40 <212> DNA <213> Artificial sequence <400> 16 acaggccaaaggagttgagaatgaatcccgaatttattca 40 <210> 17 <211> 40 <212> DNA <213> Artificial sequence <400> 17 ccaagcttgcatgcctgcagtcaaagttgcttaagcaaac 40

Claims

1. A method for constructing an amino acid-producing strain, comprising overexpressing the Clp protease gene in a starting strain; The amino acid producing strain is Corynebacterium glutamicum; the amino acid is selected from lysine and glutamic acid. When the amino acid is lysine, the Clp protease is a polypeptide selected from any of the amino acid sequences shown in SEQ ID NO: 1-4; when the amino acid is glutamic acid, the Clp protease is a polypeptide selected from any of the amino acid sequences shown in SEQ ID NO: 1-3.

2. The construction method as described in claim 1, wherein overexpression is achieved by modifying the strain using the following genetic engineering methods: introducing a strong promoter or a strong ribosome binding site into the strain; introducing a recombinant expression vector for non-integrative proteins; introducing a recombinant expression vector for chromosomal integrated proteins; or altering the promoter, translation regulatory region, or coding region codon of the coding gene to enhance transcription or translation.

3. The construction method as described in claim 1, characterized in that, The starting strains of Corynebacterium glutamicum are Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, and Corynebacterium glutamicum ATCC 14067.

4. An amino acid-producing strain, characterized in that, Compared with wild-type microorganisms, the strain overexpresses the Clp protease gene; the amino acid producing strain is Corynebacterium glutamicum; the amino acid is selected from lysine and glutamic acid; For lysine, the Clp protease is a polypeptide selected from any of the amino acid sequences shown in SEQ ID NO: 1-4; for glutamic acid, the Clp protease is a polypeptide selected from any of the amino acid sequences shown in SEQ ID NO: 1-3.

5. The production strain as described in claim 4, characterized in that, The starting strains of Corynebacterium glutamicum are Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, and Corynebacterium glutamicum ATCC 14067.

6. The recombinant strain obtained by the construction method according to claim 1, 2 or 3, or the production strain according to claim 4 or 5, in the production of amino acids; wherein the amino acids are selected from lysine and glutamic acid; When the amino acid is lysine, the Clp protease is a polypeptide selected from any of the amino acid sequences shown in SEQ ID NO: 1-4; when the amino acid is glutamic acid, the Clp protease is a polypeptide selected from any of the amino acid sequences shown in SEQ ID NO: 1-3.

7. A method for producing an amino acid, comprising fermenting a target amino acid using an amino acid-producing strain as described in claim 4 or 5; wherein the target amino acid is selected from lysine and glutamic acid; When the target amino acid is lysine, the Clp protease is a polypeptide selected from any of the amino acid sequences shown in SEQ ID NO: 1-4; when the target amino acid is glutamic acid, the Clp protease is a polypeptide selected from any of the amino acid sequences shown in SEQ ID NO: 1-3.

8. The production method as described in claim 7, characterized in that, It also includes the step of separating the target amino acid from the fermentation broth.