Amino acid biosensor and use thereof

Abstract

The application discloses a kind of, the application can respond to serine and homoserine derived from glutamic acid coryneform 13032 NCgl0581 transcriptional regulator (GenBank: BAB97999.1) F104 position is saturated mutation, to this as foundation biological element, constructs a kind of high-efficiency amino acid biosensor, then the response amino acid concentration is coupled with fluorescence intensity signal, realizes the real-time monitoring of strain intracellular response amino acid concentration.The mutant not only has the induction response activity of serine and homoserine, and also has the induction response activity of proline and threonine.It can be applied to the genetic modification and screening of threonine and proline high-yield strain.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to an amino acid biosensor and its applications. Background Technology

[0002] LysR-type transcriptional regulators (LTTRs) are widely distributed across various genera of microorganisms, regulating diverse gene functions, including anabolic metabolism, quorum sensing, and virulence. With advancements in bioinformatics and protein structure, an increasing number of LTTRs are being discovered. Currently, the LysR family of transcriptional regulators constitutes the largest family of transcription factors in prokaryotes. Due to the sensing properties of transcriptional regulators, biosensors based on these regulators are widely used for detecting intracellular small molecule metabolite concentrations and for high-throughput screening of high-yield small molecule metabolite strains, such as high-throughput screening of high-yield amino acid strains and key enzymes in their biosynthetic pathways. [1-3] However, biosensors based on natural transcription factors often exhibit low response strength to small molecules and a limited range of amino acids, making them difficult to apply to high-throughput screening of high-amino acid-producing strains. Therefore, modifying natural transcription factors to respond to a wider range of substrates and optimizing the response strength of biosensors is a crucial problem that urgently needs to be solved.

[0003] NCgl0581 (Cgl0606) is a LysR family transcriptional regulator of Corynebacterium glutamicum. It can respond to intracellular serine and homoserine, but the response intensity is low. Existing studies have shown that it has no responsive activity to threonine and proline, and therefore cannot be used to construct highly sensitive biosensors.

[0004] [1]Binder,S.,Schendzielorz,G., N., Krumbach, K., Hoffmann, K., Bott, M. and Eggeling, L. (2012) A high-throughput approach to identify genomicvariants of bacterial metabolite producers at the single-cell level. Genomebiology, 13, R40.

[0005] [2] Kortmann, M., Mack, C., Baumgart, M. and Bott, M. (2019) Pyruvatecarboxylase variants enabling improved lysine production from glucose identified by biosensor-based high-throughput fluorescence-activated cell sorting screening. ACS Synth. Biol., 8, 274-281.

[0006] [3]Schendzielorz,G.,et al.(2014)Taking control over control:use ​​ofproduct sensing in single cells to remove flux control at key enzymes inbiosynthesis pathways.ACS Synth.Biol.,3,21-29. Summary of the Invention

[0007] The present invention aims to provide an NCgl0581 transcription factor mutant that can respond to more substrates and with increased response intensity, an amino acid biosensor containing the mutant and its construction method, and to establish a high-throughput screening method for high-yield amino acid-responsive strains and key enzymes in their biosynthetic pathways using the biosensor.

[0008] This invention selects the transcriptional regulatory factor NCgl0581 (GenBank: BAB97999.1) from Corynebacterium glutamicum 13032, which is responsive to serine and homoserine, as the research object. By performing a saturation mutation on its 104th amino acid residue, mutants responsive to threonine and proline were obtained, and the response intensity of the mutants to serine and homoserine was also increased. Using this as a basic biological element, a highly efficient amino acid biosensor was constructed, enabling high-sensitivity detection of high-yielding strains of threonine and other amino acids, as well as protein-coding genes related to amino acid synthesis. This is of great significance for constructing high-yielding strains of threonine and other amino acids, and for high-throughput screening of strains or proteins related to amino acid synthesis.

[0009] Therefore, the present invention first provides a mutant of the transcriptional regulatory factor NCgl0581, wherein the amino acid sequence of the mutant contains a mutation at position 104 of SEQ ID NO:1.

[0010] According to the present invention, the mutant has a mutation of F104I, F104L, F104M, F104V or F104Y at position 104 of the amino acid sequence shown in SEQ ID No: 1.

[0011] According to an embodiment of the present invention, the mutant has more than 70% homology with the amino acid sequence shown in SEQ ID NO.1, for example, more than 80% homology, and even more than 90%, more than 95%, or more than 98% homology.

[0012] The present invention further provides a polynucleotide encoding the NCgl0581 mutant, a recombinant expression vector containing the polynucleotide of the NCgl0581 mutant, and a recombinant strain.

[0013] The recombinant strain is derived from microorganisms of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Enterobacteria*, *Salmonella*, *Streptomyces*, *Pseudomonas*, *Brevibacterium*, or *Corynebacterium*. For example, the *Corynebacterium* microorganism may be *Corynebacterium glutamicum*. In a specific embodiment, the recombinant strain is *Corynebacterium glutamicum* ATCC 13032, *Corynebacterium glutamicum* ATCC 13869, *Corynebacterium glutamicum* ATCC 14067, or a derivative thereof.

[0014] In addition, the present invention provides an amino acid biosensor, wherein the amino acid is serine, homoserine, threonine or proline, and the biosensor contains a mutant of the NCgl0581 encoding gene; wherein the polypeptide encoded by the mutant of the NCgl0581 encoding gene has an F104I, F104L, F104M, F104V or F104Y mutation relative to the wild-type NCgl0581, and the amino acid sequence of the wild-type NCgl0581 is shown in SEQ ID No: 1.

[0015] In a specific implementation, the amino acid biosensor further includes an NCgl0581 regulatory promoter and a gene encoding a reporter marker protein, wherein the NCgl0581 mutant can specifically bind to the NCgl0581 regulatory promoter, thereby inducing and activating the expression of the reporter marker protein.

[0016] In one specific embodiment of the present invention, the nucleotide sequence of the NCgl0581 regulatory promoter is shown in SEQ ID No: 2.

[0017] In one specific embodiment of the present invention, the reporter marker protein is a fluorescent protein; more preferably, it is an enhanced yellow fluorescent protein (EYFP).

[0018] In one specific embodiment of the present invention, the amino acid biosensor is a plasmid vector; more preferably, it is an Escherichia coli-Corynebacterium glutamicum shuttle vector pTRCmob.

[0019] The present invention also provides a recombinant strain containing the above-mentioned amino acid biosensor.

[0020] The recombinant strain is derived from microorganisms of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Enterobacteria*, *Salmonella*, *Streptomyces*, *Pseudomonas*, *Brevibacterium*, or *Corynebacterium*. For example, the *Corynebacterium* microorganism may be *Corynebacterium glutamicum*. In a specific embodiment, the recombinant strain is *Corynebacterium glutamicum* ATCC 13032, *Corynebacterium glutamicum* ATCC 13869, *Corynebacterium glutamicum* ATCC 14067, or a derivative thereof.

[0021] The present invention also provides a method for preparing the above-mentioned NCgl0581 mutant, a biosensor containing the mutant, or a recombinant strain.

[0022] The present invention also provides the application of the above-mentioned NCgl0581 mutant, biosensors containing the mutant, or recombinant strains in regulating the transcriptional expression level of target genes.

[0023] The present invention also provides the application of the above-mentioned NCgl0581 mutant, biosensors containing the mutant, or recombinant strains in screening high-yielding strains of threonine and proline.

[0024] The present invention also provides the application of the above-mentioned amino acid biosensor or recombinant strains containing the above-mentioned amino acid biosensor in screening key enzymes in the threonine and proline biosynthetic pathways.

[0025] The present invention also provides a method for regulating the expression of a target gene, the method comprising the step of operatively linking the polynucleotide encoding the NCgl0581 mutant to the NCgl0581 regulatory promoter sequence and the target gene. The expression of the target gene is activated by the response of the NCgl0581 mutant to amino acid concentration.

[0026] In one specific embodiment, the promoter sequence is a polynucleotide with NCgl0581-regulated promoter activity, i.e., a promoter polynucleotide capable of specifically binding to NCgl0581 and thereby activating the expression of a reporter marker protein. In one specific embodiment, the promoter sequence is as shown in SEQ ID No: 2. The target gene includes at least one of the following: a gene encoding a protein related to amino acid synthesis, a gene encoding a gene expression regulatory protein, and a gene encoding a protein related to membrane transport.

[0027] The present invention also provides a method for screening high-yield amino acid strains, the method comprising the steps of using the NCgl0581 mutant, a polynucleotide containing the mutant, a recombinant expression vector, a recombinant strain, and a biosensor to screen high-yield basic amino acid strains.

[0028] The present invention also provides a method for screening proteins or protein-coding genes related to amino acid synthesis, wherein the method includes the steps of using the NCgl0581 mutant, polynucleotides containing the mutant, recombinant expression vectors, recombinant strains, and biosensors to screen proteins or protein-coding genes related to amino acid synthesis.

[0029] The advantage of this invention lies in providing a highly efficient and improved amino acid biosensor. This invention involves saturating the F104 position of the wild-type NCgl0581 transcription regulator (GenBank: BAB97999.1) from Corynebacterium glutamicum 13032 to obtain F104I, F104L, F104M, F104V, or F104Y mutants. These mutants serve as basic biological elements to construct a highly efficient amino acid biosensor, which then couples the responsive amino acid concentration with fluorescence intensity signals to achieve real-time monitoring of the intracellular responsive amino acid concentration of the strain. The mutants not only exhibit enhanced induction response to serine and homoserine but also possess induction response activity to proline and threonine, increasing the variety of responsive substrates. This makes it suitable for screening high-yielding strains of serine, homoserine, threonine, and proline, thus having broader application value. Furthermore, the NCgl0581 mutant of this invention is currently the only reported transcription regulator responsive to threonine, which is of great significance for high-throughput screening of high-yielding threonine strains and related proteins. Attached Figure Description

[0030] Figure 1 Screening diagram of the induced response of sensors based on NCgl0581 and its mutants to threonine and proline;

[0031] Figure 2 Biosensor pSerWT F104I Graph of proline-induced response test;

[0032] Figure 3 Biosensor pSerWT F104I Graphs of induced responses to serine, threonine, and homoserine; Detailed Implementation

[0033] Terminology Definition

[0034] The terms "polynucleotide" and "coding gene" in this invention refer to polymers composed of nucleotides. Polynucleotides can be in the form of individual fragments 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 (individual 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.

[0035] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to amino acid polymers 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).

[0036] 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 is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms.

[0037] In this invention, the terms "transcription regulatory factor" and "transcription factor" refer to a class of proteins that can control the transcription of DNA into RNA, i.e., the first stage of gene expression. They regulate the transcription process of target genes by specifically binding to DNA sequences in regulatory regions. In some embodiments, the transcription regulatory factor is the transcription regulatory factor NCgl0581.

[0038] The “NCgl0581 transcription factor” described in this invention, also commonly written as Cgl0606, is a LysR family transcription factor from Corynebacterium glutamicum that responds to intracellular serine and homoserine residues. Its amino acid sequence is shown in SEQ ID NO:1. For those skilled in the art, it could also be a polypeptide derived from Corynebacterium glutamicum with transcription factor activity that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence shown in SEQ ID NO:1. In other words, it could also be a polypeptide that still possesses transcription factor activity by arbitrarily adding 1-10 amino acid residues to both ends of the polypeptide shown in the amino acid sequence of SEQ ID NO:1.

[0039] The term "mutant" in this invention refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "wild type" or "comparative" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position. In a specific embodiment, the term "mutation" refers to an alteration of the amino acid sequence. In one specific embodiment, the term "mutation" refers to "substitution." Specifically, a "mutation" may also include the addition, deletion, or substitution of amino acids at one or more positions corresponding to the sequence shown in SEQ ID NO: 1 that do not affect the activity of the transcription factor. It is well known that changing a few amino acid residues in certain regions of a polypeptide, such as non-critical regions, does not substantially alter its biological activity; for example, sequences obtained by appropriately substituting, adding, or deleting certain amino acids do not affect their activity.

[0040] In some embodiments, the “mutation” of the present invention may be selected from “conservative mutations.” The term “conservative mutation” refers to a mutation that maintains the normal function of a protein. A representative example of a conservative mutation is a conservative substitution. The term “conservative substitution” involves replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0041] The “NCgl0581 mutant” described in this invention is a polypeptide with F104I, F104L, F104M, F104V or F104Y amino acid substitutions at position 104 of the wild-type NCgl0581 amino acid sequence. The mutant not only has an enhanced induction response to serine and homoserine, but also has induction response activity to proline and threonine. In specific embodiments, the NCgl0581 mutant of the present invention can be a polypeptide having an amino acid substitution of F104I, F104L, F104M, F104V, or F104Y at position 104 of the amino acid sequence shown in SEQ ID NO:1; it can also be a polypeptide with more than 70% homology to the amino acid sequence shown in SEQ ID NO:1, for example, more than 80% homology, or more than 90%, 95%, or 98% homology, derived from Corynebacterium glutamicum and possessing transcription factor activity, and having an amino acid substitution of F104I, F104L, F104M, F104V, or F104Y at position 104 corresponding to the amino acid sequence shown in SEQ ID NO:1; or it can be a polypeptide that still possesses transcription factor activity by arbitrarily adding 1-10 amino acid residues to both ends of the polypeptide shown in the amino acid sequence shown in SEQ ID NO:1, and having an amino acid substitution of F104I, F104L, F104M, F104V, or F104Y corresponding to the amino acid sequence shown in SEQ ID NO:1; and it can also be a polypeptide that still possesses transcription factor activity by arbitrarily adding 1-10 amino acid residues to both ends of the polypeptide shown in the amino acid sequence shown in SEQ ID NO:1, and having an amino acid substitution of F104I, F104L, F104M, F104V, or F104Y corresponding to the amino acid sequence shown in SEQ ID NO:1. The polypeptide shown in NO:1 has an amino acid substitution of F104I, F104L, F104M, F104V or F104Y at position 104.

[0042] The terms "homology," "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 percentage of identity 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 then multiplying by 100.

[0043] 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% of nucleotides. In some embodiments, the sequences are substantially identical along the entire length of any one or two compared biopolymers (e.g., polynucleotides).

[0044] The term "corresponding to" in this invention has the meaning commonly understood by those skilled in the art. Specifically, "corresponding to" means that, after homology or sequence identity alignment, one sequence corresponds to a specified position in another sequence. Therefore, for example, regarding "corresponding to the 150th amino acid residue of the amino acid sequence shown in Sequence 1," if a 6×His tag is added to one end of the amino acid sequence shown in Sequence 1, then the 150th position corresponding to the 150th position in the resulting mutant might be the 156th position.

[0045] 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.

[0046] 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 strain. "Recombinant expression vector" or "recombinant 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 disclosure 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.

[0047] The term "protein-coding gene" in this invention 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. Protein-coding genes contain a CDS (Coding Sequence) that guides the production of mRNA encoding proteins. In a specific embodiment, the protein-coding gene is the gene encoding a protein related to amino acid synthesis. For example, as those skilled in the art will know, protein-coding genes may be genes encoding aspartate kinase III LysC, genes encoding aspartate semialdehyde dehydrogenase Asd, genes encoding aspartate kinase I ThrA, genes encoding homoserine kinase ThrB, genes encoding threonine synthase ThrC, genes encoding aspartate aminotransferase AspC, genes encoding 6-phosphogluconate dehydrogenase Gnd, genes encoding glutamate kinase ProB that relieves feedback inhibition, genes encoding glutamate-5-semialdehyde dehydrogenase ProA, genes encoding pyrrole-5-carboxylic acid dehydrogenase ProC, genes encoding glutamate dehydrogenase Gdh, genes encoding pyruvate carboxylase Pyc, genes encoding phosphoenolpyruvate carboxylase Ppc, genes encoding glyceraldehyde-3-phosphate dehydrogenase GapN, and genes encoding l-proline efflux protein ThrE or SerE.

[0048] The term "biosensor" in this invention refers to a plasmid capable of binding to a certain concentration of inducer and assessing the inducer concentration by initiating the expression of a downstream reporter gene. The principle is as follows: When the concentration of the inducer amino acid (e.g., serine) is low, the transcription factor NCgl0581 cannot bind to the amino acid. When the amino acid concentration reaches a certain level, the transcription factor NCgl0581 binds to the inducer amino acid and regulates the promoter of the target gene. Therefore, if a reporter gene, such as a fluorescent protein, is connected downstream of the target gene promoter, the expression of the downstream reporter gene can be initiated. The intensity of the reporter gene's fluorescence signal can be used to assess the amino acid concentration, thereby screening for high-amino acid-producing strains.

[0049] The term "response intensity" in this invention refers to the level of transcriptional initiation of target gene transcription by a transcriptional regulatory factor under the induction of an amino acid. In some embodiments, the response intensity of the transcriptional regulatory factor induced by a specific amino acid is characterized by linking a fluorescent reporter gene downstream of the transcriptional regulatory factor and detecting the intensity of the fluorescence signal under the induction of a specific amino acid.

[0050] The term "NCgl0581-regulated promoter" in this invention refers to a promoter polynucleotide that can specifically bind to NCgl0581 and thereby activate the transcription of a reporter marker gene, and whose transcriptional activity is regulated by NCgl0581. In some embodiments, the NCgl0581-regulated promoter is not specifically limited, as long as its activity is regulated by NCgl0581. For example, the NCgl0581-regulated promoter can be the promoter shown in SEQ ID NO: 2, or a promoter that is still regulated by NCgl0581 after modification of the promoter shown in SEQ ID NO: 2, or an unknown or unidentified promoter regulated by NCgl0581.

[0051] The term "recombinant strain" in this invention refers to a mutant containing the transcriptional regulatory factor NCgl0581 disclosed herein, or any strain containing a polynucleotide encoding the mutant, or a recombinant expression vector. In this invention, the recombinant strain can be any microorganism, derived from the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Enterobacteria*, *Salmonella*, *Streptomyces*, *Pseudomonas*, *Brevibacterium*, or *Corynebacterium*. For example, the *Corynebacterium* species can be *Corynebacterium glutamicum*. In specific embodiments, the recombinant strain is *Corynebacterium glutamicum* ATCC 13032, *Corynebacterium glutamicum* ATCC 13869, *Corynebacterium glutamicum* ATCC 14067, or a derivative thereof. Specifically, the recombinant strain may be a strain that produces threonine, proline, serine, or homoserine.

[0052] For example, the recombinant strain is a recombinant strain that produces threonine. In some embodiments, for the recombinant strain that produces threonine, one or more genes selected from the following may be enhanced or overexpressed:

[0053] a) Gene encoding aspartate kinase III lysC;

[0054] b) Gene encoding aspartate semialdehyde dehydrogenase asd;

[0055] c) Gene encoding aspartate kinase I thrA;

[0056] d) Gene encoding homoserine kinase thrB;

[0057] e) Gene encoding threonine synthase thrC;

[0058] f) Gene encoding aspartate aminotransferase aspC;

[0059] g) encodes the gnd gene for 6-phosphoglucate dehydrogenase.

[0060] In some embodiments, for recombinant strains producing threonine, one or more genes selected from, but not limited to, those included in the following may be weakened or eliminated:

[0061] a) The dapA gene encoding dihydropyridine synthase;

[0062] b) The dapB gene encoding dihydropyridine dicarboxylic acid reductase;

[0063] c) The ackA gene encoding acetate kinase;

[0064] d) The pta gene encoding phosphorylated acetyltransferase;

[0065] e) Encodes the transcriptional regulatory factor tyrR gene.

[0066] For example, the recombinant strain is a recombinant strain that produces proline. In some embodiments, for the recombinant strain that produces proline, one or more genes selected from the following may be enhanced or overexpressed in the host cell that produces proline:

[0067] a) Encodes the proB gene, a glutamate kinase that relieves feedback inhibition;

[0068] b) Gene encoding proA, an enzyme in the glutamate-5-hemialdehyde dehydrogenase group;

[0069] c) Gene encoding pyrrole-5-carboxylic acid dehydrogenase proC;

[0070] d) Gene encoding glutamate dehydrogenase GDH;

[0071] e) Gene encoding pyruvate carboxylase (pyc);

[0072] f) Gene encoding gapN, an enzyme containing glyceraldehyde-3-phosphate dehydrogenase;

[0073] g) Encodes the l-proline efflux protein thrE or serE gene.

[0074] In some embodiments, the recombinant strain producing proline may also have one or more genes selected from the following that are attenuated or have reduced expression:

[0075] a) Gene encoding proline dehydrogenase / pyrrole-5-carboxylic acid dehydrogenase putA;

[0076] b) Gene encoding L-glutamate efflux protein mscCG;

[0077] c) Genes encoding the transport proteins proP / proY / proU.

[0078] The terms "transformation" and "introduction" in this invention have the meanings commonly understood by those skilled in the art, referring to 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.

[0079] 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.

[0080] The term "amino acid" or "L-amino acid" in this invention refers to the basic building block of a protein in which the amino and carboxyl groups are bonded to the same carbon atom. Exemplarily, an amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, 5-aminolevulinic acid, or derivatives of any of the above-mentioned amino acids. Furthermore, the amino acid may also be other types of amino acids in the art. In specific embodiments of this disclosure, the amino acid is threonine, proline, serine, and homoserine.

[0081] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0082] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0083] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0084] The primers used in the following examples are shown in Table 1:

[0085] Table 1

[0086]

[0087] Example 1: Modification of NCgl0581 transcriptional regulatory factor

[0088] (1) Construction of biosensors

[0089] Based on the reported eyfp gene sequence information (GenBank No. CCD28585.1), the whole gene was synthesized. Using eyfp-F / eyfp-R primers, the eyfp gene fragment was amplified by PCR. Using the *Corynebacterium glutamicum* 13032 genome as a template, and using NCgl0581-F / NCgl0581-R primers, the NCgl0581 gene (GenBank: BAB97999.1) and its regulatory promoter sequence SEQ ID NO: 2 were amplified by PCR. Using plasmid pTRCmob as a template, and using pTRCmob-Rev-F / pTRCmob-Rev-R primers, the full-length plasmid was amplified by reverse PCR to obtain a linearized vector fragment. After purification and recovery of the above three fragments, they were ligated using the ClonExpress MultiS One Step Cloning Kit recombinase produced by Nanjing Vazyme Co., Ltd. The recombinant vector pTRCmob-NCgl0581-eyfp, verified by transformation, enzyme digestion, and sequencing, was named pSerWT.

[0090] (2) Construction of a library of amino acid saturation mutants at F104 of NCgl0581

[0091] Using plasmid pSerWT as a template, PCR reverse amplification was performed using pTRCmob-Rev-F / 104-Rev-R primers to obtain a linearized vector. Using plasmid pSerWT as a template, partial NCgl0581 and eyfp genes were amplified using BH-104-F / YFP-R primers. After removing the template plasmid with Dpn I, the two fragments were ligated using recombinase and chemically transformed into E. coli DH5α, then cultured at 37°C. The next day, colonies were scraped from the plate, and the NCgl0581 F104 amino acid saturation mutant library plasmid pSerWT was extracted. Mut Electroporation was performed on competent cells of strain C. glutamicum ATCC 13032 to obtain a saturated mutant library strain 13032 (pSerWT). Mut ).

[0092] (3) Screening of the F104 amino acid residue saturation mutant library of NCgl0581 in 96 wells

[0093] Ninety-four single clones were randomly selected from the plates of the above-mentioned saturated mutant library and inoculated into 96-well plates containing 200 μL of TSB liquid medium (glucose, 5 g / L; yeast extract, 5 g / L; soybean peptone, 9 g / L; urea, 3 g / L; succinic acid, 0.5 g / L; K2HPO4·3H2O, 1 g / L; MgSO4·7H2O, 0.1 g / L; biotin, 0.01 mg / L; vitamin B1, 0.1 mg / L; MOPS, 20 g / L, containing 25 μg / mL kanamycin). Control strains 13032 (pTRCmob) and 13032 (pSerWT) were also inoculated at the same time. The plates were incubated in a shaker at 30 °C and 800 rpm for 8 h. The seed culture was transferred at 5% (v / v) to a medium containing 200 μL CGXⅡY (50 g / L glucose, 2 g / L yeast extract, 16.5 g / L NH4Cl, 5 g / L urea, 1 g / L KH2PO4, 1 g / L K2HPO4, 42 g / L MOPS, 0.25 g / L MgSO4, 0.01 g / L FeSO4·2H2O, 0.01 g / L MnSO4·H2O, 0.001 g / L ZnSO4·7H2O, 0.2 mg / L CuSO4, 0.02 mg / L NiCl2·6H2O, 0.01 g / L CaCl2, 0.03 g / L protocatechuic acid, 0.2 mg / L biotin, 0.1 mg / L vitamin C). B1, with 25 μg / mL kanamycin added, was added to a 96-well plate. Simultaneously, 0.6 M threonine or 0.6 M proline was added to each well. The plate was incubated at 30°C and 800 rpm for 12 h in a shaker. The bacterial culture was then diluted 20-fold with PBS buffer. The fluorescence intensity and OD of eYFP were detected using an ELISA reader (SpectraMax M5, Molecular Devices, λexcitation = 488 nm, λemission = 520 nm). 600nm The mutants with stronger fluorescence values ​​than the control strain 13032 (pSerWT) were sent for sequencing to analyze their mutation sites. For example... Figure 1 As shown, through 96-well plate screening and sequencing analysis, it was found that the F104I, F104L, F104M, F104V and F104Y mutants of NCgl0581 have induced response activities to threonine and proline.

[0094] Example 2. Biosensor pSerWT F104I Characterization and testing

[0095] To test the NCgl0581 mutant sensor's response to proline and its analogues, the F104I mutant obtained in the examples was selected for further performance verification. First, strains 13032 (pTRCmob), 13032 (pSerWT), and 13032 (pSerWT) were used. F104I The seed culture was inoculated into a 24-well plate containing 1 mL TSB (containing 25 μg / mL kanamycin) and cultured at 30°C and 800 rpm for 8 h. Then, the seed culture was transferred to a 96-well plate containing 200 μL CGXⅡY (containing 25 μg / mL kanamycin) at an initial OD of 0.5. 0 M, 0.2 M, 0.4 M, and 0.6 M proline were added, and the plates were cultured at 30°C and 800 rpm for 12 h. After dilution, the culture was analyzed using an ELISA reader (SpectraMax M5, Molecular Devices, λexcitation = 488 nm, λemission = 520 nm) to detect the fluorescence and OD values ​​of the bacterial culture. 600 The test results are as follows: Figure 2 As shown, it can be seen that with the increase of proline concentration, 13032(pSerWT) F104I The fluorescence value of 13032(pSerWT) gradually increased, while the fluorescence value of 13032(pSerWT) did not increase. This indicates that 13032(pSerWT) has no induced response activity to proline, while pSerWT... F104I The response was induced by proline, and the induced response gradually increased with increasing proline concentration.

[0096] The sensor's response to several other amino acids was also tested, and the results are as follows: Figure 3 As shown, with increasing amino acid concentration, the fluorescence value of 13032(pSerWT) induced by serine and homoserine gradually increased, while it showed no induced response activity to threonine. F104I It still exhibits inducible response activity to serine and homoserine, and the response intensity is higher than that of 13032(pSerWT). Furthermore, 13032(pSerWT)... F104I The mutant also exhibits responsive activity to threonine, indicating that the mutant of this invention not only increases the responsiveness to serine and homoserine, but can also effectively respond to proline and threonine.

[0097] Finally, the proline-producing strain was used to test 13032(pSerWT) F104I The response of ) was observed. First, the empty vector pTRCmob, sensor plasmid pSerWT, and pSerWT were respectively... F104IProline was introduced via electroporation to produce strain SLCgP54 (see CN112111469B), resulting in strains SLCgP54(pTRCmob), SLCgP54(pSerWT), and SLCgP54(pSerWT). F104I Strains 13032 (pTRCmob), 13032 (pSerWT), and 13032 (pSerWT) were used. F104I ), SLCgP54(pTRCmob), SLCgP54(pSerWT) and SLCgP54(pSerWT F104I The seed culture was inoculated into 24-well plates containing 1 mL TSB (containing 25 μg / mL kanamycin) and cultured at 30 °C and 800 rpm for 8 h. The seed culture was then divided according to the initial OD... 600 0.5 μg / mL of a proline fermentation medium (containing 25 μg / mL kanamycin) was transferred to a 24-well plate containing: 80 g / L glucose; 1 g / L yeast extract; 1 g / L soybean peptone; 1 g / L K₂HPO₄·3H₂O; 1 g / L (NH₄)₂SO₄; 1 g / L NaCl; 42 g / L MOPS (3-morpholinopropanesulfonic acid); 10 g / L urea; 0.45 g / L MgSO₄·7H₂O; 0.05 g / L FeSO₄·7H₂O; 0.4 mg / L biotin; and 0.1 mg / L vitamin B1. The plate was incubated at 30°C and 800 rpm for 24 h. The fermentation product was then collected. The supernatant was collected after centrifugation at 12000 rpm for 2 min. The proline yield in the supernatant was determined using the method described in CN112111469B. Meanwhile, after diluting the fermentation product by a certain factor, the fluorescence value and OD of the bacterial solution were detected using an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMax M5, Molecular Devices, λexcitation = 488nm, λemission = 520nm). 600 The test results showed that no proline accumulation was detected in strain 13032, while the extracellular proline concentration in SLCgP54 was approximately 3.5 g / L. The fluorescence values ​​of 13032 (pSerWT) and SLCgP54 (pSerWT) were the same as those of the corresponding empty vector control strain, indicating that the pSerWT sensor did not respond to intracellular proline induction; while the fluorescence values ​​of SLCgP54 (pSerWT) were... F104I The response fluorescence value is 13032 (pSerWT). F104I 1.6 times that of pSerWT indicates that pSerWT F104I Induction by intracellular proline can effectively screen for proline-producing strains.

[0098] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. sequence list <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> An amino acid biosensor and its application <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 303 <212> PRT <213> Corynebacterium glutamicum <400> 1 Met Leu Asn Leu Asn Arg Leu His Ile Leu Gln Glu Phe His Arg Leu 1 5 10 15 Gly Thr Ile Thr Ala Val Ala Glu Ser Met Asn Tyr Ser Arg Ser Ala 20 25 30 Ile Ser Gln Gln Met Ala Leu Leu Glu Lys Glu Ile Gly Val Lys Leu 35 40 45 Phe Glu Lys Ser Gly Arg Asn Leu Tyr Phe Thr Glu Gln Gly Glu Val 50 55 60 Leu Ala Ser Glu Thr His Ala Ile Met Ala Ala Val Asp His Ala Arg 65 70 75 80 Ala Ala Val Leu Asp Ser Leu Ser Glu Val Ser Gly Thr Leu Lys Val 85 90 95 Thr Ser Phe Gln Ser Leu Leu Phe Thr Leu Ala Pro Lys Ala Ile Ala 100 105 110 Arg Leu Thr Glu Lys Tyr Pro His Leu Gln Val Glu Ile Ser Gln Leu 115 120 125 Glu Val Thr Ala Ala Leu Glu Glu Leu Arg Ala Arg Arg Val Asp Val 130 135 140 Ala Leu Gly Glu Glu Tyr Pro Val Glu Val Pro Leu Val Glu Ala Ser 145 150 155 160 Ile His Arg Glu Val Leu Phe Glu Asp Pro Met Leu Leu Val Thr Pro 165 170 175 Ala Ser Gly Pro Tyr Ser Gly Leu Thr Leu Pro Glu Leu Arg Asp Ile 180 185 190 Pro Ile Ala Ile Asp Pro Asp Leu Pro Ala Gly Glu Trp Val His 195 200 205 Arg Leu Cys Arg Arg Ala Gly Phe Glu Pro Arg Val Thr Phe Glu Thr 210 215 220 Ser Asp Pro Met Leu Gln Ala His Leu Val Arg Ser Gly Leu Ala Val 225 230 235 240 Thr Phe Ser Pro Thr Leu Leu Thr Pro Met Leu Glu Ser Val His Ile 245 250 255 Gln Pro Leu Pro Gly Asn Pro Thr Arg Thr Leu Tyr Thr Ala Val Arg 260 265 270 Glu Gly Arg Gln Gly His Pro God Ile Lys God Phe Arg Arg God Leu 275 280 285 Ala His Val Ala Lys Glu Ser Tyr Leu Glu Ala Arg Leu Val Glu 290 295 300 <210> 2 <211> 72 <212> DNA <213> Corynebacterium glutamicum <400> 2 60. gacacctcca aaattgttca gtattactta actatagttg gctgtttttc gatctagtcc ttaaaggttt ct <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <400> 3 aactttaaga aggagatatc atatggtgag caagggcgag <210> 4 <211> 43 <212> DNA <213> Artificial Sequence <400> 4 tctcatccgc caaaacagcc ttacttgtac agctcgtcca tgc <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <400> 5 gacatcataa cggttctggc tcactctact agacgagcct ccaa 44 <210> 6 <211> 43 <212> DNA <213> Artificial Sequence <400> 6 gatatctcct tcttaaagtt ttatccaatg gcagcaccaa att 43 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 ggctgttttg gcggatga 18 <210> 8 <211> twenty one <212> DNA <213> Artificial Sequence <400> 8 gccagaaccg ttatgatgtc g 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 acccttgccc cgaaagccat 20 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (21)..(23) <223> n = a, g, c, or t <400> 10 atggctttcg gggcaagggt nnncagcagg gattggaagg aggt 44 <210> 11 <211> 43 <212> DNA <213> Artificial Sequence <400> 11 tctcatccgc caaaacagcc ttacttgtac agctcgtcca tgc 43

Claims

1. A mutant of the transcriptional regulatory factor NCgl0581, characterized in that, The amino acid sequence of the mutant corresponds to a mutation at amino acid residue 104 of SEQ ID NO: 1, wherein the mutation is that phenylalanine is replaced by any amino acid selected from isoleucine, leucine, methionine, valine or tyrosine.

2. A polynucleotide encoding the mutant of claim 1.

3. A recombinant expression vector containing the mutant of claim 1 or the polynucleotide of claim 2.

4. A recombinant strain containing the mutant of claim 1, the polynucleotide of claim 2, or the recombinant expression vector of claim 3, wherein the recombinant strain is Corynebacterium glutamicum.

5. An amino acid biosensor, wherein the biosensor contains the mutant of claim 1, the polynucleotide of claim 2, or the recombinant expression vector of claim 3, and the biosensor further includes an NCgl0581 regulatory promoter with a nucleotide sequence as shown in SEQ ID No: 2, and a gene encoding a reporter marker protein fluorescent protein; wherein the amino acids are threonine and proline.

6. The amino acid biosensor as described in claim 5, characterized in that, The fluorescent protein is a yellow fluorescent protein, EYFP.

7. The application of the mutant of claim 1, the polynucleotide of claim 2, the recombinant expression vector of claim 3, the recombinant strain of claim 4, or the amino acid biosensor of claim 5 or 6 in screening high-yielding strains of threonine and proline.

8. A method for screening high-yielding amino acid strains, the method comprising the step of screening high-yielding amino acid strains using the mutant of claim 1, the polynucleotide of claim 2, the recombinant expression vector of claim 3, the recombinant strain of claim 4, or the biosensor of claim 5 or 6, wherein the amino acid is threonine or proline.

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