CN113201538B - Polynucleotides with promoter activity and use thereof in the production of target compounds - Google Patents

Abstract

The present disclosure relates to polynucleotides having promoter activity and their use in the production of target compounds, in particular, the present disclosure relates to polynucleotides having promoter activity, transcription expression cassettes comprising polynucleotides having promoter activity, recombinant expression vectors, recombinant host cells, and methods of regulating transcription of target genes, methods of producing proteins, and methods of producing target compounds. The polynucleotide having promoter activity provided by the present disclosure is a high-salt, high-osmotic-pressure-inducible promoter having enhanced promoter activity under an environment of elevated salt concentration and osmotic pressure. The polynucleotide is operably connected with the target gene, so that the expression strength of the target gene in a high-salt and high-osmotic-pressure stress environment can be remarkably improved, a downstream product can be stably and efficiently produced, and the problems that high inducers such as IPTG (isopropyl-beta-thiogalactoside) and the like are added at present and toxicity is caused to a strain are effectively solved.

Description

Polynucleotides with promoter activity and use thereof in the production of target compounds

technical field

The present disclosure belongs to the technical field of biotechnology and genetic engineering, and in particular relates to a polynucleotide with promoter activity, a transcription expression cassette, a recombinant expression vector, a recombinant host cell comprising the polynucleotide with promoter activity, and a control target gene Methods of transcription, methods of preparing proteins, and methods of producing target compounds.

Background technique

Corynebacterium, especially the non-pathogenic Corynebacterium glutamicum, is one of the most commonly used strains in the fermentation industry due to its strong amino acid synthesis ability. It is widely used in the industrial production of proteins, amino acids, organic acids and other chemicals. The expression regulation and optimization of the target gene is the key to improve the synthesis of protein or product, and the promoter element is an important tool for regulating gene expression. At present, a series of strong promoters ^[1-2] or constitutive promoters ^[3] have been identified or developed in Corynebacterium glutamicum, which are used to regulate the expression of key genes in metabolic pathways. Although the above promoter elements can finely regulate the expression of target genes, however, for the synthesis of some toxic proteins or metabolites, the use of constitutive promoters is still relatively limited. In addition, expression from strong promoters or constitutive promoters often imposes a large metabolic burden on engineered strains.

Inducible promoters can control the timing of transcription initiation and are therefore more conducive to strain regulation and redistribution of metabolic flux. At present, inducible promoters such as tac and trc are widely used in the metabolic regulation of C. glutamicum. However, the above promoters often require additional expensive inducers, such as IPTG, and the addition of these inducers will also cause certain toxicity to the strain, or cause greater interference to the fermentation system. Therefore, the development of an autoinduction system under industrial fermentation conditions is crucial for the construction of industrial strains. At this stage, some studies have reported auto-inducible promoters in C. glutamicum, such as lysine-inducible promoters ^[4] , growth process-regulated promoters ^[5-6] and so on. However, the number of the above-mentioned auto-inducible promoters is still relatively small, and the response conditions are relatively narrow, so they cannot be widely used in more fermentation systems and product synthesis.

Under the strict control of pH and dissolved oxygen in the fermenter, the production of bulk chemicals such as glutamic acid and lysine by Corynebacterium glutamicum can reach a level of 100 g/L or even more than 200 g/L ^{[7-8 ]} , therefore, the accumulation of high concentrations of products or intermediate metabolites and the continuous flow of substrates in the late fermentation stage will inevitably lead to high salt and high osmotic pressure. At the same time, high-salt and hypertonic conditions are also an environmental inducement factor that almost all industrial strains will face in the late fermentation stage ^[9] . At present, in C. glutamicum, there is no high-salt and hypertonic inducible promoter to regulate the target gene. Application precedent for expression.

Therefore, identifying high-salt-hypertonic inducible promoters and developing and constructing an auto-inducible system for high-salt-hypertonic conditions in the late fermentation stage can not only increase the available auto-inducible systems, but also provide universal auto-induction elements for the development of all industrial strains , which has also become a key problem that needs to be solved urgently in the development of industrial strains of Corynebacterium glutamicum.

Citation:

[1] CN101605890A.

[2] Becker, J., et al., Metab. Eng., 2011, 13, 159-168.

[3] Rytter, J.V., et al., Appl. Microbiol. Biotechnol., 2014, 98, 2617-2623.

[4] CN101087881A.

[5] Kim, M.J., et al., Appl. Microbiol. Biotechnol., 2016, 100, 4473-4483.

[6] Ma, Y.C., et al., Microb. Cell Fact., 2018, 17.

[7] Xu, J.Z., et al., Microb Cell Fact, 2020, 19, 39.

[8] Hu Hongtong et al., China Brewing, 2018, 37(10), 51-56.

[9] Varela C et al. Applied Microbiology And Biotechnology 2003, 60(5): 547-555.

SUMMARY OF THE INVENTION

Invention to solve problem

In view of the technical problems existing in the prior art, for example, the use of inducible promoters such as tac and trc in the process of industrial fermentation needs to add expensive inducers such as IPTG, and the addition of the inducer causes the problem of toxicity to strains, for this reason, The present disclosure provides a polynucleotide with promoter activity, and the aforementioned polynucleotide exhibits enhanced promoter activity in an environment with elevated salt concentration and osmotic pressure. The operative connection of the polynucleotide and the protein-coding gene related to amino acid synthesis can realize the high-efficiency expression of the protein-coding gene in a high-salt and high-osmotic pressure environment, which effectively solves the problem that the current inducible promoter needs to add a high-inducing agent, and Inducers pose a problem of toxicity to the strain.

solution to the problem

(1) A polynucleotide with promoter activity, wherein the polynucleotide is selected from any one of the following groups (i)-(iv):

(i) comprising the nucleotide sequence shown in any of SEQ ID NOs: 1-3;

(ii) comprising the reverse complement of the nucleotide sequence shown in any of SEQ ID NOs: 1-3;

(iii) the reverse complement of a sequence capable of hybridizing to the nucleotide sequence shown in (i) or (ii) under high stringency hybridization conditions or very high stringency hybridization conditions;

(iv) the nucleotide sequence shown in (i) or (ii) has at least 80%, optionally at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, most preferably at least 99% % sequence identity to the sequence.

(2) The polynucleotide having a promoter activity according to (1), wherein the polynucleotide has an increased promoter activity in an environment with an increased salt concentration or osmotic pressure.

(3) A transcription expression cassette, wherein the transcription expression cassette comprises the polynucleotide with promoter activity according to (1) or (2); optionally, the transcription expression cassette further contains a protein encoding A gene, the protein-coding gene is operably linked to the polynucleotide having promoter activity.

(4) A recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide having promoter activity according to (1) or (2), or the transcription expression cassette according to (3).

(5) A recombinant host cell, wherein the recombinant host cell comprises the transcription expression cassette according to (3), or the recombinant expression vector according to (4).

(6) The recombinant host cell according to (5), wherein the host cell is derived from the genus Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia; preferably, the host The cell is Corynebacterium glutamicum or Escherichia coli; more preferably, the host cell is Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC13869 or Corynebacterium glutamicum ATCC 14067.

(7) A polynucleotide according to (1) or (2), a transcription expression cassette according to (3), a recombinant expression vector according to (4), and according to (5) or (6) Use of the recombinant host cell in at least one of the following:

(a) regulating the transcription level of a gene, or preparing a reagent or kit for regulating the transcription level of a gene;

(b) preparing a protein, or preparing a reagent or kit for preparing a protein;

(c) producing the target compound, or preparing a reagent or kit for producing the target compound.

(8) The use according to (7), wherein the protein is selected from a gene expression regulatory protein or a protein related to the synthesis of a target compound.

(9) The use according to (7) or (8), wherein the target compound includes at least one of amino acids and organic acids; alternatively, the amino acids include lysine, glutamic acid and threonine At least one of amino acids, and the organic acid includes at least one of citric acid and succinic acid.

(10) A method for regulating transcription of a target gene, wherein the method comprises the step of operably linking the polynucleotide with promoter activity according to any one of (1) to (2) and the target gene.

(11) A method for preparing a protein, comprising using the transcription expression cassette according to (3), the recombinant expression vector according to (4), or any one of (5)-(6) The step of expressing the protein in the recombinant host cell; optionally, the protein is a protein related to the synthesis of the target compound or a gene expression regulatory protein;

Optionally, the method further comprises the step of isolating or purifying the protein.

(12) A method for producing a target compound, comprising using the transcription expression cassette described in (3), the recombinant expression vector described in (4), or the recombinant expression described in any one of (5)-(6). The host cell expresses a protein related to the synthesis of the target compound or a gene expression regulatory protein, and the step of producing the target compound in the presence of the protein related to the synthesis of the target compound or the gene expression regulatory protein;

Optionally, the target compound includes at least one of amino acids and organic acids; optionally, the amino acids include at least one of lysine, glutamic acid and threonine, and the organic acid includes lemon at least one of acid and succinic acid;

Optionally, the protein is a protein related to lysine synthesis; optionally, the protein related to lysine synthesis includes aspartate kinase, aspartate semialdehyde dehydrogenase, aspartate Acid ammonia lyase, dihydrodipicolinate synthase, dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate Acid deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, lysine transporter, transketolase, diaminopimelate One or more combinations of dehydrogenase and pyruvate carboxylase;

Optionally, the method further comprises the step of isolating or purifying the target compound.

effect of invention

In one embodiment, the present disclosure provides a polynucleotide having promoter activity that is a high-salt, high-osmolarity-inducible promoter with enhanced initiation in an environment of elevated salt concentration and osmotic pressure subactivity. The polynucleotide is operably linked to the target gene, which can significantly improve the expression intensity of the target gene under the stress environment of high salt and high osmotic pressure, and then produce the downstream products stably and efficiently, which effectively solves the problem of adding IPTG and other expensive inducers. , and the problem of toxicity to the strain.

In another embodiment, the present disclosure provides transcriptional expression cassettes, recombinant expression vectors, and recombinant host cells comprising the above-described polynucleotides having promoter activity. In transcription expression cassettes, recombinant expression vectors, and recombinant host cells, the polynucleotide with promoter activity is operably linked to the protein-coding gene, which can improve the expression strength of the protein-coding gene under the stress environment of high salt and high osmotic pressure. .

In another embodiment, the present disclosure provides a method for producing amino acids, using the above-mentioned polynucleotides with promoter activity, which can improve the expression of proteins related to amino acid synthesis under stress conditions, while attenuating the expression of other pathway proteins, The metabolic flow is more enriched in the direction of amino acid synthesis, so as to produce amino acids stably and efficiently, and achieve the purpose of excessive accumulation of amino acids.

In another embodiment, when L-Lysine is produced by the above method, L-Lysine can be produced stably and efficiently in an environment of high salt and high osmotic pressure.

Description of drawings

Figure 1 shows the induction of NCgl1418 promoter by hyperosmolarity;

Fig. 2 shows the induction effect of different concentrations of sodium sulfate on NCgl1418 promoter;

Figure 3 shows the induction of NCgl1418 promoter by high concentrations of sugar;

Fig. 4 shows the promoter strength comparison result of P _NCgl1418 and P _tuf ;

Figure 5 shows the activity comparison results of NCgl1418 promoters of different lengths.

Detailed ways

When used in conjunction with the term "comprising" in the claims and/or specification, the words "a (a)" or "an (an)" may mean "one", but may also mean "one or more", "at least one" one" and "one or more than one".

As used in the claims and specification, the words "comprising", "having", "including" or "comprising" are meant to be inclusive or open ended and do not exclude additional, unrecited elements or methods step.

Throughout this application, the term "about" means that a value includes the standard deviation of the error of the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as only an alternative and "and/or", the term "or" in the claims means that unless expressly stated to be only an alternative or mutually exclusive between alternatives "and / or".

When used in the claims or specification, a selected/alternative/preferred "numerical range" includes both the numerical endpoints at both ends of the range and, with respect to the aforementioned numerical endpoints, all natural numbers covered in the middle of the numerical endpoints.

The term "polynucleotide" in this disclosure refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments or part of a larger nucleotide sequence structure derived from a nucleotide sequence that has been isolated at least once in quantity or concentration, and can be obtained by standard Molecular biological methods (eg, using cloning vectors) identify, manipulate, and recover sequences and their component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (ie A, T, G, C), this also includes an RNA sequence (ie A, U, 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 may be a component or component of a larger nucleotide structure, such as an expression vector or polycistronic sequence. Polynucleotides include DNA, RNA and cDNA sequences.

The terms "sequence identity" and "percent identity" in this disclosure refer to the percentage of nucleotides or amino acids that are identical (ie, identical) 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 polynucleotides or polypeptides and The number of positions containing the same nucleotide or amino acid residue is scored and compared to the number of positions containing different nucleotide or amino acid residues in the aligned polynucleotides or polypeptides. Polynucleotides can differ at a position, eg, by containing different nucleotides (ie, substitutions or mutations) or deletions of nucleotides (ie, insertions of nucleotides or deletions of nucleotides in one or both polynucleotides). Polypeptides can differ at one position, for example, by containing different amino acids (ie, substitutions or mutations) or missing amino acids (ie, amino acid insertions or amino acid deletions in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in a polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

In some specific embodiments, the polynucleotide with promoter activity comprises the reverse complement of the nucleotide sequence shown in any of SEQ ID NOs: 1-3, and the polynucleotide maintains high salt, high Osmotic pressure-inducible promoter activity.

In some specific embodiments, the polynucleotide having promoter activity comprises the nucleotide sequence shown in any of SEQ ID NOs: 1-3 under high stringency hybridization conditions or very high stringency hybridization conditions or the reverse complement of the sequence to which its reverse complement hybridizes, and the polynucleotide maintains a high salt, high osmotic pressure-inducible promoter activity.

In some specific embodiments, the polynucleotide having promoter activity comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity, and multinucleated The glucosinolate maintains a high-salt, high-osmolarity-inducible promoter activity.

As used in this disclosure, the term "high stringency conditions" refers to 5X SSPE (saline sodium phosphate EDTA) at 42°C for probes of at least 100 nucleotides in length following standard Southern blotting procedures , 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide prehybridized and hybridized for 12 to 24 hours. Finally the carrier material was washed three times for 15 minutes at 65°C using 2X SSC, 0.2% SDS.

As used in this disclosure, the term "very high stringency conditions" refers to 5X SSPE (saline sodium phosphate EDTA) at 42°C for probes of at least 100 nucleotides in length following standard Southern blotting procedures ), 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide and prehybridized for 12 to 24 hours. Finally, the carrier material was washed three times for 15 minutes at 70°C using 2X SSC, 0.2% SDS.

The term "complementary" as used herein refers to hybridization or base pairing between nucleotides or nucleotides, such as between two strands of a double-stranded DNA molecule or an oligonucleotide primer and a sequenced or amplified between primer binding sites on single-stranded nucleotides, etc.

The term "promoter" in the present disclosure refers to a nucleic acid molecule that is generally located upstream of the coding sequence of a gene of interest, provides a recognition site for RNA polymerase, and is located 5' upstream of the mRNA transcription initiation site. It is a nucleic acid sequence that is not translated, and RNA polymerase binds to this nucleic acid sequence to initiate transcription of the target gene. In the synthesis of ribonucleic acid (RNA), promoters can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription), including core promoter regions and regulatory regions, like "Switches" that determine the activity of genes, which in turn control which proteins cells start producing.

The term "promoter core region" in the present disclosure refers to a nucleic acid sequence located in the prokaryotic promoter region, which is the core sequence region that exerts the function of the promoter, mainly including the -35 region, the -10 region, the -35 region and the -10 region. The region between the regions and the transcription initiation site, the -35 region is the recognition site of RNA polymerase, and the -10 region is the binding site of RNA polymerase.

In some specific embodiments, the polynucleotides having promoter activity of the present disclosure can be used to initiate the expression of protein-encoding genes. In other embodiments, the polynucleotides of the present disclosure having promoter activity can be used to initiate the expression of non-coding genes.

The term "expression" in this disclosure includes any step involving RNA production and protein production, including but not limited to:

Transcription, post-transcriptional modification, translation, post-translational modification and secretion.

The term "target gene" in the present disclosure refers to any gene that is linked to the polynucleotide having promoter activity in the present disclosure to regulate its transcription level.

In some embodiments, a gene of interest refers to a gene encoding a protein of interest in a microorganism. Exemplarily, the target gene is a gene encoding an enzyme related to the biosynthesis of the target compound, a gene encoding an enzyme related to reducing power, a gene encoding an enzyme related to glycolysis or the TCA cycle, or a gene encoding an enzyme related to the target compound. Genes that release related enzymes, etc.

The term "target compound" in the present disclosure can be selected from at least one of amino acids and organic acids, and can also be selected from other kinds of compounds that may be obtained by biosynthesis in the art.

In some embodiments, the compound of interest is an "amino acid" or "L-amino acid." "Amino acid" or "L-amino acid" generally refers to the basic building blocks of proteins in which the amino and carboxyl groups are bound to the same carbon atom. Exemplarily, the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid , asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline in one or more combinations, or Other classes of amino acids in the art.

In some embodiments, the target compound is an organic acid. The organic acid may be an organic compound having an acidity, for example, those compounds including carboxyl groups and sulfonic acid groups. Exemplarily, the organic acid includes one or more of lactic acid, acetic acid, succinic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, citric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, and valeric acid. combination, or other kinds of organic acids in the art.

The term "protein-coding gene" in the present disclosure refers to a synthetic DNA molecule that can guide proteins through certain rules. The process of protein-coding genes instructing protein synthesis generally includes transcription using double-stranded DNA as a template and translation using mRNA as a template. process. Protein-coding genes contain CDS sequences (Coding Sequence) that can direct the production of protein-coding mRNAs.

Exemplary, protein-coding genes include, but are not limited to, those used to encode proteins associated with the synthesis of target compounds. In some embodiments, protein-coding genes involve those used to encode proteins associated with the synthesis of L-lysine. For proteins related to the synthesis of L-lysine, including aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydrogenase Picolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate One or more of aminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, lysine transporter, transketolase, diaminopimelate dehydrogenase and pyruvate carboxylase The combination. In some embodiments, a protein-coding gene is involved in encoding a protein associated with the synthesis of organic acids, exemplary, a protein-coding gene for encoding a protein associated with the synthesis of citrate, or for encoding a protein associated with the synthesis of succinate . In other embodiments, the protein-coding gene involves a protein associated with gene editing, such as the Cpf1 protein. The polynucleotide with promoter activity of the present disclosure can be suitable for improving the expression of the target gene under the stress environment of high salt and high osmotic pressure, so as to realize the efficient production of the target product.

The term "gene expression regulatory protein" in the present disclosure includes, but is not limited to, exogenous gene expression regulatory tool proteins, such as dCas9 protein, dCpf1 protein required for CRISPRi regulation, Hfq protein required for sRNA regulation, etc., as well as endogenous or exogenous transcriptional regulation factors, which in turn regulate the expression of key genes in metabolic pathways.

The term "transcriptional expression cassette" in the present disclosure refers to a type of expression element that includes a transcriptional regulatory element and a target gene, and utilizes the transcriptional regulatory element to regulate the expression of the target gene. In the present disclosure, transcriptional regulatory elements include promoters, and on this basis, elements such as enhancers, silencers, and insulators may also be included. In the present disclosure, the target gene is specifically a protein-coding gene. The target gene and the polynucleotide are "operably linked", which means that the polynucleotide with promoter activity is functionally linked to the target gene to initiate and mediate the transcription of the target gene, and the operably linked manner can be Use any means described by those skilled in the art.

The term "vector" in this disclosure refers to a DNA construct that contains DNA sequences operably linked to appropriate control sequences to express a gene of interest in a suitable host. A "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors may include, for example, i) collections of genetic elements that have regulatory effects on gene expression, such as promoters and enhancers; ii) structural or coding sequences that are transcribed into mRNA and translated into protein; and iii) appropriate transcription and transcriptional subunits of translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical and any vector can be used, including plasmids, viruses, phages and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, such as from vaccinia, adenovirus, chicken DNA from viruses such as pox, baculovirus, SV40, and pseudorabies.

The term "host cell" in the present disclosure means any cell type that is amenable to transformation, transfection, transduction, etc. with a transcription initiation element or expression vector comprising a polynucleotide of the present disclosure. The term "recombinant host cell" encompasses a host cell that differs from the parental cell after introduction of a transcription initiation element or recombinant expression vector, in particular by transformation.

The term "transformation" in the present disclosure has the meaning commonly understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.

The host cell of the present disclosure may be a prokaryotic cell or a eukaryotic cell, as long as it is a cell capable of introducing the polynucleotide having promoter activity of the present disclosure. In one embodiment, the host cell refers to a prokaryotic cell, in particular the host cell is derived from a microorganism suitable for the fermentative production of amino acids, such as Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia. Preferably, the host cell is Corynebacterium glutamicum derived from the genus Corynebacterium. Among them, the Corynebacterium glutamicum can be Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC14067 and the like.

The host cells of the present disclosure can be cultured according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, etc., and can be appropriately adjusted according to actual conditions Various culture conditions such as temperature, time and pH of the medium, etc.

As an auto-inducible promoter, the polynucleotide with promoter activity in the present disclosure can show enhanced promoter activity in the environment of high salt and high osmotic pressure, thereby avoiding the need to add the present invention in the fermentation environment. High-priced and toxic inducers such as IPTG commonly used in the field.

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

High salt, high osmotic pressure inducible promoter

In the present disclosure, Corynebacterium glutamicum ATCC13032 strain was cultivated in CGXII medium with and without the addition of 0.6M NaCl or lysine sulfate, cells were harvested in mid-logarithmic growth, total RNA was extracted and analyzed by transcriptome sequencing. The promoter of the NCgl1418 gene whose transcription level was significantly increased under hypertonic conditions was selected as the most candidate high-salt-hypertonic inducible promoter.

In some specific embodiments, the NCgl1418 promoter is selected from any one of the following group consisting of (i)-(iv):

(i) comprising the nucleotide sequence shown in any one of SEQ ID NOs: 1-3.

(ii) comprising the reverse complement of the nucleotide sequence shown in any of SEQ ID NOs: 1-3.

(iii) the reverse complement of a sequence capable of hybridizing to the nucleotide sequence shown in (i) or (ii) under high stringency hybridization conditions or very high stringency hybridization conditions;

(iv) the nucleotide sequence shown in (i) or (ii) has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Sequences of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

In some specific embodiments, the NCgl1418 promoter has increased promoter activity in a high salt environment. In some specific embodiments, the NCgl1418 promoter has increased promoter activity in a hyperosmotic environment.

In the present disclosure, "high-salt environment" can be inorganic salt ions such as high-concentration Na ₂ SO ₄ , NaCl, K ₂ SO ₄ , KCl, etc. in the medium, or products such as lysine in the fermentation broth as the fermentation time prolongs, or Increased concentrations due to accumulation of certain intermediate metabolites (e.g., lysine sulfate, etc.), or due to substrate influx (e.g., substrates such as ammonium sulfate), or other Arbitrary salt concentration. In some specific embodiments, a "high salt environment" refers to a salt concentration above 0.2M; in some more specific embodiments, a "high salt environment" refers to a salt concentration between 0.2-0.8M. For example, the salt concentration is 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M.

In the present disclosure, "hyperosmotic environment" refers to increased osmotic pressure in response to increased salt concentration.

In some preferred embodiments, the NCgl1418 promoter exhibits higher promoter activity or higher transformation efficiency in a "high salt environment" formed with sulfate and hydrochloride; in some preferred embodiments , the sulfate is Na ₂ SO ₄ or K ₂ SO ₄ , lysine sulfate, and the hydrochloride is lysine hydrochloride, sodium chloride or potassium chloride.

Construction of recombinant expression vector

In some specific embodiments, the present disclosure uses the ATCC 13032 genome as a template and uses primers 1418-F and 1418-R to amplify the promoter sequence (SEQ ID NO: 1) of the NCgl1418 gene by PCR; pXM-gfp As a template, primers pGFP-F and pGFP-R were used to amplify pXM-gfp to obtain a vector fragment with the lacI gene and tac promoter removed; the above fragments were recombined to obtain a recombinant expression vector pXM-P _NCgl1418 -gfp.

In some specific embodiments, the present disclosure uses pXM-P _NCgl1418 -gfp as a template, using primer pairs 1418-203-F/R, 1418-145-F/R and 1418-94-F/R, respectively, by PCR Amplify to obtain NCgl1418 promoter fragments with 203bp, 145bp and 94bp; after the above three fragments are recovered, use T4 PNK to phosphorylate the vector fragment, and construct a new vector by self-circularization, which is named pXM-P ₂₀₃ respectively. -gfp, _pXM -P145-gfp and _pXM -P94-gfp.

In some specific embodiments, the present disclosure uses the ATCC 13032 genome as a template and uses primers 1418-DF and 1418-DR to amplify the promoter sequence of the NCgl1418 gene by PCR. Taking pXM-07 as a template, firstly, using primers pXM07-F1 and pXM07-R1, a vector fragment with dCpf1 was obtained by PCR amplification; Vector fragment 2 of the starting point; take pEC-26 as a template, use primers pEC26-F and pEC26-R, and obtain crRNA array fragments targeting gltA, pgi, hom and pck genes through PCR amplification; after the above-mentioned fragments are recovered, carry out Recombinant ligation to obtain recombinant expression vector pXM-P _NCgl1418 -dCpf1.

In some specific embodiments, the present disclosure uses the ATCC 13032 genome as a template and uses primers 1418-LF and 1418-LR to amplify the promoter sequence of the NCgl1418 gene and the DNA sequence of the lysE gene by PCR. Using pEC-XK99E as a template, primers pEC-F and pEC-R were used to amplify a vector fragment by PCR to obtain a vector fragment. The above three fragments were recovered and then recombined to obtain a recombinant expression vector pEC-P _NCgl1418 -lysE.

A specific source of Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032 (Corynebacterium glutamicum ATCC 13032, genome sequence of ATCC 13032: NC_003450.3).

The production process of amino acids

(1) In the present disclosure, a polynucleotide with promoter activity is operably linked to a protein encoding gene related to amino acid synthesis or a gene expression regulatory protein encoding gene to obtain a protein that can be related to amino acid synthesis or a gene expression regulatory protein. The recombinant expression vector is used to transform the host cell to obtain the recombinant host cell.

(2) The recombinant host cells are fermented and cultured, and amino acids are collected from the recombinant host cells or the culture solution of the recombinant host cells to complete the amino acid production process.

In the above-mentioned production process, because the polynucleotide has improved promoter activity, in the recombinant host cell, the transcriptional activity of the encoding gene of the protein or gene expression control protein related to amino acid synthesis is improved, and the protein or gene related to amino acid synthesis is expressed. The expression of regulatory proteins was increased, and the production of amino acids was significantly increased.

In some specific embodiments, no inducer is added in the steps of the method for preparing amino acids employed in the present disclosure. In a specific embodiment, in the steps of the method for preparing amino acids employed in the present disclosure, IPTG is not added.

For produced amino acids, the amino acids include lysine, glutamic acid, threonine, and the like; for produced organic acids, the organic acids include citric acid, succinic acid, and the like.

For a protein related to amino acid synthesis or a gene expression regulatory protein, the protein related to amino acid synthesis is a protein related to lysine synthesis. In some specific embodiments, the protein related to amino acid synthesis is the lysine transporter LysE, and increasing the expression of LysE with a polynucleotide with promoter activity can promote the extracellular emission and extracellular accumulation of lysine. In some specific embodiments, the gene expression regulatory protein is dCpf1, and dCpf1 can target and regulate target genes such as gltA, pgi, hom or pck; the polynucleotide with promoter activity can increase the expression of dCpf1 under high-salt environment conditions, The weakening degree of the target gene is improved, and the synthesis and substrate utilization of lysine are further promoted.

In some specific embodiments, the host cell is Corynebacterium glutamicum, which is an important strain for the production of L-lysine, a high-salt, high-osmolarity-inducible polynucleotide After the transformation of Corynebacterium glutamicum, transcription expression cassette or recombinant expression vector, the expression of proteins related to lysine synthesis in Corynebacterium glutamicum is significantly increased, specifically in the environment of high salt and high osmotic pressure The expression level of C. glutamicum was significantly increased, which greatly improved the ability of C. glutamicum to accumulate L-lysine through long-term fermentation.

In some specific embodiments, the host cell is Corynebacterium glutamicum modified as follows: the aspartokinase encoding gene in Corynebacterium glutamicum has introduced a T311I mutant coding sequence.

Methods for manipulating microorganisms are known in the art, such as Modern Methods in Molecular Biology (OnlineISBN: 9780471142720, John Wiley and Sons, Inc.), Microbial Metabolic Engineering: Methods and Protocols (Qiong Cheng Ed ., Springer) and Systems Metabolic Engineering: Methods and Protocols (Hal S. Alper Ed., Springer).

In some specific embodiments, the culture conditions of the recombinant host cells are as follows: the recombinant host cells are inoculated with TSB medium containing corresponding antibiotics, cultured overnight at 30° C. at 220 r/min, with or without addition of 0.6 according to the initial OD of 0.3. Lysine fermentation medium with M sodium sulfate (to simulate the high-salt and hypertonic environment caused by the accumulation of high-concentration products in the later stage of fermentation), the culture system is 1 mL of 24-well plate, and the fermentation is terminated after culturing for 24 hours at 30 °C and 800 r/min. Residual glucose content, OD ₆₀₀ and lysine production.

For lysine fermentation medium, the formula is: glucose 80g/L, yeast powder 8g/L, urea 9g/L, K ₂ HPO ₄ 1.5g/L, MOPS 42g/L, FeSO ₄ 0.01g/L, MnSO ₄ 0.01 g/L, MgSO ₄ 0.6 g/L, chloramphenicol at a final concentration of 5 μg/mL, and/or kanamycin at a final concentration of 25 μg/mL.

In some specific embodiments, amino acids can be recovered from recombinant host cells or culture broth of recombinant cells by methods commonly used in the art, including but not limited to: filtration, anion exchange chromatography, crystallization or HPLC.

Example

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given for illustrative purposes only, since after reading this detailed description, Various changes and modifications will become apparent to those skilled in the art.

The experimental techniques and experimental methods used in the present embodiment, unless otherwise specified, are conventional technical methods, such as the experimental methods that do not specify specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as suggested by the manufacturer. Materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

The primer sequences used for plasmid construction in the examples are as follows:

Example 1. Screening of inducible promoters

Corynebacterium glutamicum ATCC13032 strain was grown in CGXII medium with and without the addition of 0.6M NaCl or lysine sulfate, cells were harvested in mid-logarithmic growth, total RNA was extracted and analyzed by transcriptome sequencing. The promoter of the NCgl1418 gene whose transcription level was significantly increased under hypertonic conditions was selected as the most candidate high-salt-hypertonic inducible promoter.

Example 2. Construction of recombinant vectors comprising promoter sequences

Primers 1418-F (SEQ ID NO: 6) and 1418-R (SEQ ID NO: 7) were designed according to the genome sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI (NC_003450.3). The promoter sequence (SEQ ID NO: 1) of the NCgl1418 gene was obtained by PCR amplification using the ATCC13032 genome as a template. The PCR amplification parameters were: 95°C for 5 min, 95°C for 30s, 65-55°C for 30s, 72°C for 1 min, cycle 10 times, 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, cycle 25 times, and extend at 72 °C for 10 min. At the same time, using the pXM-gfp reported in the literature as the template ^[10] , using the primers pGFP-F and pGFP-R, the vector fragment with the lacI gene and the tac promoter removed was obtained by PCR amplification. The PCR amplification parameters were: 95 ℃ 10min , 95 ℃ for 30 s, 65-55 ℃ for 30 s, 72 ℃ for 3 min, cycle 10 times, 95 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 3 min, cycle 25 times, and 72 ℃ for 10 min. After the above two fragments were recovered, the Vazyme Clon Express Multies one-step recombination kit was used for recombination ligation, and the ligation product was transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR verification. , and the correct transformants were confirmed by sequencing, and the obtained recombinant vector was named pXM-P _NCgl1418 -gfp. At the same time, the vector fragment was phosphorylated by T4 PNK, and the control vector pXM-con was obtained by self-circularization construction.

Example 3. Induction of NCgl1418 promoter by high salt

The above recombinant vector pXM-P _NCgl1418 -gfp and control vector pXM-con were transformed into Corynebacterium glutamicum ATCC13032 to obtain recombinant strain and control strain. The above strains were respectively inoculated into TSB medium containing 5 μg/mL chloramphenicol, and cultured overnight at 30°C and 220 r/min. Among them, the components of TSB liquid medium are (g/L): glucose, 5g/L; yeast powder, 5g/L; soy peptone, 9g/L; urea, 3g/L; succinic acid, 0.5g/L; K ₂ HPO ₄ ·3H ₂ O, 1 g/L; MgSO ₄ ·7H ₂ O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; MOPS, 20 g/L. According to the initial OD 0.5, the CGXIIY medium with or without the addition of 0.6M different salts was respectively transferred, and the culture system was 1 mL of 24-well plate, and the GFP fluorescence intensity and OD ₆₀₀ of different strains were detected after culturing for 18 h at 30 °C and 800 r/min. The relative intensity of the NCgl1418 promoter under different conditions was characterized by the fluorescence intensity of the unit cell (subtracting the fluorescence intensity of the control strain under the same conditions). The formula of CGXIIY medium is: glucose 50g/L, NH ₄ Cl 16.5g/L, urea 5g/L, KH ₂ PO ₄ 1g/L, K ₂ HPO ₄ 1g/L, MOPS 42g/L, MgSO ₄ 0.25g /L, FeSO ₄ ·2H ₂ O 0.01g/L, MnSO ₄ ·H ₂ O 0.01g/L, ZnSO ₄ ·7H ₂ O 0.001g/L, CuSO ₄ 0.2mg/L, NiCl·6H ₂ O 0.02mg /L, CaCl ₂ 0.01g/L, protocatechuic acid 0.03g/L, biotin 0.2mg/L, vitamin B 10.1mg/L, and the final concentration of chloramphenicol was 5μg/mL. The test results are shown in Figure 1. The data show that the addition of 0.6M different salts can induce the conversion rate of the NCgl1418 promoter and the expression of the reporter gene (4.1-7.7 times), so it is determined that the promoter is induced by high salt.

Example 4. Induction of NCgl1418 promoter by different osmotic pressures

Using the same method as Example 3, the induction effects of different concentrations of sodium sulfate on the NCgl1418 promoter were detected. The results are shown in Figure 2. The data show that the strength of the NCgl1418 promoter increases with the increase of the sodium sulfate concentration, and it exhibits an obvious gradient-induced activity within a certain range.

Example 5. Response of NCgl1418 promoter to high glucose

Using the same method as Example 3, the induction effect of high concentration sucrose (150 g/L) on the NCgl1418 promoter was detected. The results are shown in Figure 3. The data showed that the addition of high concentration of sucrose did not significantly enhance the inducible activity of the NCgl1418 promoter, indicating that the promoter did not respond to all hypertonic stress, but specifically responded to high concentrations of salt ions.

Example 6. Comparison of NCgl1418 promoter and tuf promoter strength

At present, it is known that the endogenous strong promoter of common C. glutamicum is P _tuf , therefore, we further compared the NCgl1418 promoter with this promoter to verify the strength of the NCgl1418 promoter. Primers tuf-F (SEQ ID NO: 10) and tuf-R (SEQ ID NO: 11) were designed according to the genome sequence of Corynebacterium glutamicum ATCC 13032 (NC_003450.3) published by NCBI. Using the ATCC13032 genome as a template, the promoter sequence with the tuf gene (SEQ ID NO: 5) was obtained by PCR amplification. The PCR amplification parameters were: 95 °C for 5 min, 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, cycle 30 times, and extended at 72 °C for 10 min. Meanwhile, using pXM-P _NCgl1418 -gfp as a template, using primers tuf-pGFP-F (SEQ ID NO: 12) and tuf-pGFP-R (SEQ ID NO: 13), the RBS containing P _NCgl1418 was obtained by PCR amplification The PCR amplification parameters are: 95℃ for 10min, 95℃ for 30s, 65-55℃ for 30s, 72℃ for 3min, cycle 10 times, 95℃ for 30s, 55℃ for 30s, 72℃ for 3min, cycle 25 times, 72℃ Extend for 10min. After the above fragments were recovered, the Vazyme Clon Express Multies recombination kit was used for recombination ligation, and the ligation products were transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR verification, and the correct results were obtained. The transformants were confirmed by sequencing, and the obtained recombinant vector was named pXM-P _tuf -gfp. The recombinant vector was transformed into Corynebacterium glutamicum ATCC13032 to obtain a recombinant strain. Using a method similar to Example 3, the strength of the _NCgl1418 promoter was compared with that of Ptuf under high salt or normal medium conditions. The results are shown in Figure 4. The data show that the promoter strength of NCgl1418 is lower than that of P _tuf under normal osmotic pressure, but under the condition of high osmotic pressure (adding 0.6M sodium sulfate), its transcriptional activity is basically the same as that of P _tuf , indicating that in high salt Under osmotic pressure conditions, the promoter has a high strength and can be used for the efficient inducible expression of target genes.

Example 7. Activity of NCgl1418 promoters of different lengths

Using the pXM-P _NCgl1418 -gfp constructed above as a template, primer pairs 1418-203-F/R (SEQ ID NO: 14, 15), 1418-145-F/R (SEQ ID NO: 16, 17) and 1418-94-F/R (SEQ ID NO: 18, 19), obtained by PCR amplification with 203bp (SEQ ID NO:2), 145bp (SEQ ID NO:3) and 94bp (SEQ ID NO:4) NCgl1418 promoter fragment, PCR amplification parameters are: 95°C 10min, 95°C 30s, 65-55°C 30s, 72°C 3min, cycle 10 times, 95°C 30s, 55°C 30s, 72°C 3min, cycle 25 times, 72 ℃ extended for 10min. After the above-mentioned three fragments are recovered, the vector fragment is phosphorylated by T4PNK, and a new vector is obtained by self-cyclization construction, which is named as pXM-P ₂₀₃ -gfp, pXM-P ₁₄₅ -gfp and pXM-P ₉₄ -gfp respectively. .

The above recombinant vectors pXM-P ₂₀₃ -gfp, pXM-P ₁₄₅ -gfp and pXM-P ₉₄ -gfp were respectively transformed into Corynebacterium glutamicum ATCC 13032 to obtain recombinant strains. Using a method similar to Example 3, the strengths of the NCgl1418 promoter of different lengths were compared under conditions of high salt (supplemented with 0.6M sodium sulfate) and normal medium, respectively. The results are shown in Figure 5. The data show that although the 94bp NCgl1418 promoter contains the core sequence (-35 region and -10 region), it basically loses the normal function of the promoter; The induction strength has decreased, but it can still reach more than 74% of the activity of the 243bp promoter; the promoter with a length of 203bp basically maintains the activity of the 243bp promoter under high salt osmotic pressure, which is 94% of the activity of the 243bp promoter; above The results indicated that the promoter activity of the NCgl1418 promoter and the activity under high salt osmotic pressure conditions at least required a DNA sequence with a length of 145 bp shown in SEQ ID NO: 3.

Example 8. Application of NCgl1418 promoter to regulate dCpf1 expression in lysine synthesis

Primers 1418-DF (SEQ ID NO: 20) and 1418-DR (SEQ ID NO: 21) were designed according to the genome sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI (NC_003450.3). Using the ATCC 13032 genome as a template, the promoter sequence of the NCgl1418 gene was obtained by PCR amplification. The PCR amplification parameters were: 95 °C for 10 min, 95 °C for 30 s, 65-55 °C for 30 s, 72 °C for 1 min, cycle 10 times, 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, cycle 25 times, and 72 °C for 10 min. At the same time, using the pXM-07 reported in the literature as a template ^[11] , firstly using primers pXM07-F1 (SEQ ID NO: 22) and pXM07-R1 (SEQ ID NO: 23), a vector fragment with dCpf1 was obtained by PCR amplification 1. PCR amplification parameters are: 95℃ for 10min, 95℃ for 30s, 65-55℃ for 30s, 72℃ for 3min, cycle 10 times, 95℃ for 30s, 55℃ for 30s, 72℃ for 3min, cycle 25 times, 72℃ for 10min extension Then utilize primer pXM07-F2 (SEQ ID NO: 24) and pXM07-R2 (SEQ ID NO: 25), obtain the carrier fragment two with origin of replication by PCR amplification, PCR amplification parameter is: 95 ℃ of 10min, 95 ℃30s, 65-55℃30s, 72℃3min, cycle 10 times, 95℃30s, 55℃30s, 72℃3min, cycle 25 times, 72℃extension 10min. Using the pEC-26 reported in the literature as a template ^[11] , using primers pEC26-F (SEQ ID NO: 26) and pEC26-R (SEQ ID NO: 27), target gltA, pgi, hom and pck were obtained by PCR amplification Gene crRNA array fragment, PCR amplification parameters are: 95°C 10min, 95°C 30s, 65-55°C 30s, 72°C 1min, cycle 10 times, 95°C 30s, 55°C 30s, 72°C 1min, cycle 25 times, Extend for 10 min at 72°C. After the above three fragments were recovered, the Vazyme Clon Express Multies one-step recombination kit was used for recombination ligation, and the ligation product was transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR. After verification, the correct transformants were confirmed by sequencing, and the obtained recombinant vector was named pXM-P _NCgl1418 -dCpf1. At the same time, using pXM-07 as a template, using primers pXM07-F3 (SEQ ID NO:28) and pXM07-R3 (SEQ ID NO:29), the vector fragment three was obtained by PCR amplification, and the PCR amplification parameters were: 95°C 10min, 95°C 30s, 65-55°C 30s, 72°C 3min, cycle 10 times, 95°C 30s, 55°C 30s, 72°C 3min, cycle 30 times, 72°C extension 10min; the above fragments are recovered and the obtained NCgl1418 The promoter sequence fragment of the gene was recombined and ligated by the Vazyme ClonExpress Multies one-step recombination kit, and the ligation product was transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR verification, and The correct transformants were confirmed by sequencing to obtain the control vector pXM-dCpf1-con.

According to the lysine strain construction method disclosed in the literature ^[12] , the threonine at position 311 of the aspartokinase (encoded by the lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 was mutated by homologous recombination technology based on pK18mobsacB to Isoleucine was constructed to obtain a strain SCgL30 with certain lysine synthesis ability. The above recombinant vectors pXM-P _NCgl1418 -dCpf1 and pXM-dCpf1-con were transformed into SCgL30 strains to obtain recombinant strains and control strains. The above strains were respectively inoculated into TSB medium containing 5 μg/mL chloramphenicol, and cultured overnight at 30°C at 220 r/min. According to the initial OD0.3, 0.6M sodium sulfate was added or not added (simulating the accumulation of high-concentration products in the later stage of fermentation). The lysine fermentation medium of high-salt and hypertonic environment caused by high-salt and hypertonic environment), the culture system is 1 mL of 24-well plate, 30 ° C, 800 r/min culture for 24 h, the fermentation is terminated, the residual glucose content, OD ₆₀₀ and lysine production are detected. . The lysine fermentation medium formula is: glucose 80g/L, yeast powder 8g/L, urea 9g/L, K ₂ HPO ₄ 1.5g/L, MOPS 42g/L, FeSO ₄ 0.01g/L, MnSO ₄ 0.01 g/L, MgSO ₄ 0.6g/L, and the final concentration of chloramphenicol was 5μg/mL. The test results are shown in Table 1. The data show that without the addition of sodium sulfate, the lysine production and glucose conversion rate of the target gene-attenuated strain were increased by 23% and 25%, respectively, compared with the control strain, indicating that the NCgl1418 promoter can regulate the expression of the target gene dCpf1. , to achieve the weakening function of the system on the target gene. In the high-salt condition with the addition of 0.6M sodium sulfate, the lysine production and the glucose conversion rate of the target gene-attenuated strain were increased by 49% and 40%, respectively, compared with the control strain, indicating that the expression intensity of dCpf1 was higher under high-salt conditions. The weakening degree of the target gene is improved, and the synthesis and substrate utilization of lysine are further promoted.

Table 1 Application effect of NCgl1418 promoter regulating dCpf1 expression in lysine synthesis

Example 9. Application of NCgl1418 promoter to regulate the expression of lysine transporter LysE in lysine synthesis

According to the genome sequence (NC_003450.3) of Corynebacterium glutamicum ATCC 13032 published by NCBI, primers 1418-LF (SEQ ID NO: 30) and 1418-LR (SEQ ID NO: 31) were designed respectively, lysE -F (SEQ ID NO:32) and lysE-R (SEQ ID NO:33). Using the ATCC 13032 genome as a template, the promoter sequence of the NCgl1418 gene and the DNA sequence of the lysE gene were obtained by PCR amplification. The PCR amplification parameters were: 95 °C for 5 min, 95 °C for 30 s, 65-55 °C for 30 s, 72 °C for 1 min, cycle 10 times, 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, 25 cycles, and 72 °C for 10 min. At the same time, using the pEC-XK99E reported in the literature as a template ^[13] , using primers pEC-F (SEQ ID NO: 34) and pEC-R (SEQ ID NO: 35), a vector fragment was obtained by PCR amplification, and PCR amplification The parameters are: 95°C for 10 min, 95°C for 30s, 65-55°C for 30s, 72°C for 3min, cycle 10 times, 95°C for 30s, 55°C for 30s, 72°C for 3min, cycle 25 times, and extend at 72°C for 10min. After the above three fragments were recovered, the Vazyme Clon Express Multies one-step recombination kit was used for recombination ligation, and the ligation products were transformed into Trans T1 competent cells, coated with kanamycin-resistant plates and cultured overnight, and positive clones were picked for colonization. PCR was verified, and the correct transformants were confirmed by sequencing, and the obtained recombinant vector was named pEC-P _NCgl1418 -lysE.

The above recombinant vectors pEC-P _NCgl1418 -lysE and pEC-XK99E were respectively transformed into Corynebacterium glutamicum ScgL30 to obtain recombinant strains and control strains. The application effect of the LysE expression strain regulated by the NCgl1418 promoter in lysine synthesis was verified by the method as in Example 8 (the antibiotic was replaced with kanamycin with a final concentration of 25 μg/mL). The results are shown in Table 2. The data showed that when sodium sulfate was not added, the lysine production and glucose conversion rate of the LysE expression strain regulated by the NCgl1418 promoter were 2.35 g/L and 0.039 g/g, which were 38% and 40% higher than those of the control strain, respectively. In the high-salt condition of 0.6M sodium sulfate, the lysine production and glucose conversion rate of the LysE-expressing strain reached 3.05g/L and 0.063g/g, which were 49% and 59% higher than those of the control strain, respectively, indicating that the high-salt environment conditions The lower LysE expression was more intense, which promoted the efflux and extracellular accumulation of lysine.

Table 2 Application effect of NCgl1418 promoter regulating LysE expression in lysine synthesis

The above two strategies were further combined to obtain a recombinant strain in which the NCgl1418 promoter simultaneously regulates the expression of LysE and dCpf1. Using the method as in Example 8 (the antibiotics were replaced with chloramphenicol with a final concentration of 5 μg/mL and kanamycin with a final concentration of 25 μg/mL) to verify the application effect of the above recombinant strains in lysine synthesis, the results are shown in Table 3. The data showed that without the addition of sodium sulfate, the lysine production of the target gene co-expression strain regulated by the NCgl1418 promoter increased from 1.75g/L to 3.15g/L (an 80% increase), and the glucose conversion rate increased from 0.03g/g to 0.053g/g (an increase of 78%); while in the high-salt condition with 0.6M sodium sulfate added, the lysine production and glucose conversion rate of the target gene co-expression strain reached 4.20g/L and 0.1g/g, respectively. Compared with the control strain, it has increased by 115% and 127%, and the effect is very significant.

Table 3 Application effect of NCgl1418 promoter in lysine synthesis by simultaneously regulating the expression of dCpf1 and LysE

Citation:

[10] Sun DH et al., Journal of Industrial Microbiology & Biotechnology 2019, 46(2): 203-208.

[11] Li MY et al., Frontiers in Bioengineering and Biotechnology, 2020, 8:357.

[12] Becker, J., et al., Metab. Eng., 2011, 13, 159-168.

[13] O Kirchner, et al. Journal of Biotechnology, 2003, 104:287-299.

All technical features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may also be replaced by other features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a series of equivalent or similar features.

Furthermore, from the foregoing description, those skilled in the art can readily appreciate from this disclosure its key features, and without departing from the spirit and scope of the present disclosure, many modifications can be made to the invention to adapt to various different purposes and conditions of use, and therefore such modifications are also intended to fall within the scope of the appended claims.

sequence listing

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aatactgctc agacgcgtgt c 21

<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 28

gtgtcaattt atcaagaatt tgttaataaa 30

<210> 29

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 29

cccgtctcac tggtgaaaag 20

<210> 30

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 30

agcggcatgc atttacgttt aaaactcgcg atgaagtag 39

<210> 31

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 31

agatttccat gatcaccatc attggccttt ctcgaattgg g 41

<210> 32

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 32

atggtgatca tggaaatctt cattac 26

<210> 33

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 33

gtctgtttcc tgtgtgaaac taacccatca acatcagttt gatg 44

<210> 34

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 34

tttcacacag gaaacagacc atg 23

<210> 35

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 35

aacgtaaatg catgccgctt c 21

Claims (23)

1. A polynucleotide having promoter activity, wherein the polynucleotide is selected from any one of the group consisting of:

(i) as shown in SEQ ID NO: 1;

(ii) as shown in SEQ ID NO: 2-3, or a nucleotide sequence shown in any sequence;

(iii) as shown in SEQ ID NO:1, or the reverse complement of the nucleotide sequence set forth in SEQ ID NO: 2-3, or a reverse complement of the nucleotide sequence set forth in any one of seq id nos.

2. The polynucleotide having promoter activity according to claim 1, wherein the polynucleotide has increased promoter activity in an environment of increased salt concentration or osmotic pressure.

3. A transcription expression cassette comprising the polynucleotide having promoter activity according to claim 1 or 2.

4. The transcriptional expression cassette of claim 3, further comprising a protein-encoding gene operably linked to the polynucleotide having promoter activity.

5. A recombinant expression vector comprising a polynucleotide having promoter activity according to claim 1 or 2, or a transcription expression cassette according to any one of claims 3-4.

6. A recombinant host cell comprising the transcription expression cassette according to any one of claims 3-4, or the recombinant expression vector according to claim 5.

7. The recombinant host cell according to claim 6, wherein said host cell is derived from Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Escherichia.

8. The recombinant host cell according to claim 7, wherein said host cell is Corynebacterium glutamicum or Escherichia coli.

9. The recombinant host cell according to claim 7, wherein said host cell is Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, or Corynebacterium glutamicum ATCC 14067.

10. A polynucleotide according to claim 1 or 2, a transcription cassette according to any one of claims 3-4, a recombinant expression vector according to claim 5, a use of a recombinant host cell according to any one of claims 6-9 in at least one of:

(a) regulating the transcription level of a gene, or preparing a reagent or a kit for regulating the transcription level of a gene;

(b) preparing a protein, or preparing a reagent or kit for preparing a protein;

(c) producing a compound of interest, or preparing a reagent or kit for producing a compound of interest.

11. The use according to claim 10, wherein the protein is selected from a gene expression regulatory protein or a protein associated with synthesis of a target compound.

12. The use of claim 10, wherein the target compound comprises at least one of an amino acid and an organic acid.

13. The use of claim 12, wherein the amino acid comprises at least one of lysine, glutamic acid, threonine, and the organic acid comprises at least one of citric acid and succinic acid.

14. A method for regulating transcription of a target gene, wherein the method comprises the step of operably linking the polynucleotide having promoter activity according to any one of claims 1 to 2 to the target gene.

15. A method for producing a protein comprising the step of expressing said protein using the transcription cassette according to any one of claims 3-4, the recombinant expression vector according to claim 5, or the recombinant host cell according to any one of claims 6-9.

16. The method for producing a protein according to claim 15, wherein the protein is a protein involved in synthesis of a target compound or a gene expression regulatory protein.

17. The method for producing a protein according to claim 15, wherein said method further comprises a step of isolating or purifying said protein.

18. A method for producing a target compound, comprising the step of expressing a protein involved in the synthesis of the target compound or a gene expression regulatory protein using the transcription expression cassette according to any one of claims 3 to 4, the recombinant expression vector according to claim 5, or the recombinant host cell according to any one of claims 6 to 9, and producing the target compound in the presence of the protein involved in the synthesis of the target compound or the gene expression regulatory protein.

19. The method for producing a target compound according to claim 18, wherein the target compound comprises at least one of an amino acid and an organic acid.

20. The method for producing a target compound according to claim 19, wherein the amino acid includes at least one of lysine, glutamic acid, and threonine, and the organic acid includes at least one of citric acid and succinic acid.

21. The method for producing a target compound according to claim 18, wherein the protein is a protein involved in lysine synthesis.

22. The method for producing a target compound according to claim 21, wherein the protein involved in lysine synthesis comprises one or a combination of two or more of aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydropyridinedicarboxylate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, lysine transporter, transketolase, diaminopimelate dehydrogenase, and pyruvate carboxylase.

23. The method for producing a target compound according to claim 18, wherein the method further comprises a step of isolating or purifying the target compound.

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