WO2022257757A1 - 基于dapB基因的具有启动子活性的多核苷酸及其用途 - Google Patents

基于dapB基因的具有启动子活性的多核苷酸及其用途 Download PDF

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WO2022257757A1
WO2022257757A1 PCT/CN2022/094734 CN2022094734W WO2022257757A1 WO 2022257757 A1 WO2022257757 A1 WO 2022257757A1 CN 2022094734 W CN2022094734 W CN 2022094734W WO 2022257757 A1 WO2022257757 A1 WO 2022257757A1
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gene
acid
polynucleotide
protein
promoter
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French (fr)
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孙际宾
刘娇
郑平
周文娟
孙冠男
陈久洲
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中国科学院天津工业生物技术研究所
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Priority to BR112023024799A priority patent/BR112023024799A2/pt
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Definitions

  • the disclosure belongs to the technical field of biotechnology and genetic engineering, and specifically relates to a polynucleotide with promoter activity, a transcription expression cassette comprising a polynucleotide with promoter activity, a recombinant expression vector, a recombinant host cell, and a promoter mutation
  • the method of constructing the body, the method of regulating the transcription of the target gene, the method of preparing the protein and the method of producing the target compound is a promoter mutation.
  • Microbial fermentation can produce a variety of target compounds, such as amino acids, organic acids, etc.
  • target compounds such as amino acids, organic acids, etc.
  • These target compounds can be widely used in the fields of medicine, food, animal feed, and cosmetics, and have huge economic value.
  • amino acids, organic acids, etc. how to increase the output of target compounds and realize the industrialized large-scale production of target compounds is an important problem that needs to be solved urgently.
  • Breeding high-yield fermenting microorganisms is an important means to increase the industrial production of target compounds. Compared with traditional mutation breeding techniques, genetic engineering breeding techniques have been widely used due to their strong pertinence and high efficiency. Numerous studies have shown that the efficient expression of key genes in the synthesis pathway of target compounds is the key to improving the yield and conversion rate of target compounds.
  • Transformation of key genes in microbial metabolic pathways through genetic engineering is an important method to increase the fermentation yield of target compounds.
  • the promoter is an important regulatory element that affects gene expression, and fine regulation of the promoter can optimize the conversion rate of the target compound. Promoters with different expression intensities can meet the needs of different expression intensities of different genes, thereby increasing the yield and conversion rate of target compounds.
  • the present disclosure provides a polynucleotide with promoter activity, which is a mutant comprising a polynucleotide of the sequence shown in SEQ ID NO: 1.
  • the mutation provided by the present disclosure The promoter activity of the body is significantly improved, which provides an expression regulatory element with great application potential for the transformation of the target gene. Operably linking the mutant to the target gene can effectively increase the expression of the target gene, thereby effectively increasing the yield and conversion rate of the target compound.
  • the present disclosure provides a polynucleotide having promoter activity, wherein the polynucleotide is selected from any one of the following groups (i)-(iv):
  • a mutant comprising a polynucleotide of the sequence shown in SEQ ID NO: 1, said mutant has a mutation at one or more positions in the 75th-95th positions of the sequence shown in SEQ ID NO: 1 of nucleotides;
  • a polynucleotide comprising 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;
  • the polynucleotide shown in any one of (i)-(iv) is not TCTGAACGGGTACGTCTAGAC at the 75th-95th nucleotide sequence of the sequence shown in SEQ ID NO: 1; and, with SEQ ID NO: 1 Compared with the polynucleotide of the sequence shown, the polynucleotide shown in any one of (i)-(iv) has enhanced promoter activity.
  • the mutant compared with the polynucleotide of the sequence shown in SEQ ID NO: 1, the mutant has 3-12 times more Increased promoter activity.
  • mutant corresponds to the nucleotide sequence at positions 75-95 of the sequence shown in SEQ ID NO: 1 is selected from the following Any item in the group consisting of (P dapB -1)-(P dapB -3):
  • the nucleotide sequence of the mutant is selected from the sequences shown in any one of SEQ ID NO: 2-4.
  • the present disclosure also provides a transcriptional expression cassette, wherein the transcriptional expression cassette comprises the polynucleotide having promoter activity according to the present disclosure; optionally, the transcriptional expression cassette also contains a target gene, the A target gene is operably linked to the polynucleotide having promoter activity; preferably, the target gene is a protein-coding gene.
  • the present disclosure also provides a recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide having promoter activity described in the present disclosure, or the transcriptional expression cassette described in the present disclosure.
  • the present disclosure also provides a recombinant host cell, wherein the recombinant host cell comprises the transcriptional expression cassette described in the present disclosure, or the recombinant expression vector described in the present disclosure.
  • the host cell is derived from the genus Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia; preferably, the The host cell is Corynebacterium glutamicum or Escherichia coli; more preferably, the host cell is Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 14067 or Corynebacterium glutamicum Derivative strains of Bacillus.
  • the present disclosure also provides a polynucleotide with promoter activity according to the present disclosure, the transcriptional expression cassette according to the present disclosure, the recombinant expression vector according to the present disclosure, the recombinant host according to the present disclosure Use of cells in at least one of the following:
  • the protein is selected from gene expression regulatory proteins, proteins related to the synthesis of target compounds or proteins related to membrane transport.
  • the target compound includes at least one of amino acid and organic acid
  • the amino acids include one or a combination of two or more of the following: proline, hydroxyproline, lysine, glutamic acid, threonine, glycine, alanine, valine, Leucine, Isoleucine, Serine, Cysteine, Glutamine, Methionine, Aspartic Acid, Asparagine, Arginine, Histidine, Phenylalanine, Tyrosine , tryptophan, 5-aminolevulinic acid or derivatives of any of the above amino acids;
  • the organic acid includes one or a combination of two or more of the following: citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, oxaloacetic acid, tartaric acid, propionic acid, hexenoic acid , capric acid, caprylic acid, valeric acid, malic acid or derivatives of any of the above organic acids.
  • the present disclosure also provides a method for constructing a promoter mutant, wherein the construction method includes the following steps:
  • Mutation step mutating the polynucleotide of the sequence shown in SEQ ID NO: 1, so that one or more positions in the 75th-95th positions of the sequence shown in SEQ ID NO: 1 have mutated nucleotides;
  • Screening step screening mutants of polynucleotides with improved promoter activity compared with the polynucleotide of the sequence shown in SEQ ID NO: 1 to obtain promoter mutants.
  • the mutation step includes: mutating the polynucleotide of the sequence shown in SEQ ID NO: 1, so that the first polynucleotide of the sequence shown in SEQ ID NO: 1
  • the nucleotides at positions 75-95 are mutated into the following nucleotide sequence: NNNNNNNNNNNNNTANNN; wherein, N is selected from A, T, C or G;
  • the promoter mutant has an increased promoter activity of 3-12 times or more.
  • the present disclosure also provides a method for regulating transcription, wherein the method includes the step of operably linking the polynucleotide having promoter activity described in the present disclosure to a target RNA or a target gene.
  • the target RNA includes at least one of tRNA and sRNA
  • the target gene includes a gene encoding a protein related to the synthesis of the target compound, a gene encoding a gene expression regulatory protein, and a protein encoding a protein related to membrane transport. at least one of the genes;
  • the target gene includes at least one of the following: pyruvate carboxylase gene, phosphoenolpyruvate carboxylase gene, ⁇ -glutamyl kinase gene, glutamate semialdehyde dehydrogenase gene , pyrroline-5-carboxylate reductase gene, amino acid transport protein gene, ptsG system related gene, pyruvate dehydrogenase gene, homoserine dehydrogenase gene, oxaloacetate decarboxylase gene, gluconate repressor protein gene, glucose Dehydrogenase gene, aspartate kinase gene, aspartate semialdehyde dehydrogenase gene, aspartate ammonia lyase gene, dihydrodipicolinate synthase gene, dihydropicolinate reductase gene , succinyldiaminopimelate aminotransferase gene, tetrahydropyridinedica
  • the present disclosure also provides a method for preparing a protein, wherein the method includes expressing the protein using the transcriptional expression cassette described in the present disclosure, the recombinant expression vector described in the present disclosure, or the recombinant host cell described in the present disclosure The step;
  • the protein is a protein related to the synthesis of the target compound, a protein related to membrane transport, or a gene expression regulatory protein;
  • the method further comprises the step of isolating or purifying the protein.
  • the present disclosure also provides a method for producing a target compound, wherein the method comprises using the transcriptional expression cassette described in the present disclosure, the recombinant expression vector described in the present disclosure, or the recombinant host cell expression and target compound described in the present disclosure
  • a protein related to compound synthesis, a protein related to membrane transport, or a gene expression regulatory protein producing the target compound in an environment where the protein related to the synthesis of the target compound, the protein related to membrane transport, or the gene expression regulatory protein exists step;
  • the target compound includes at least one of amino acid and organic acid
  • the amino acids include one or a combination of two or more of the following: lysine, glutamic acid, threonine, proline, hydroxyproline, glycine, alanine, valine, Leucine, Isoleucine, Serine, Cysteine, Glutamine, Methionine, Aspartic Acid, Asparagine, Arginine, Histidine, Phenylalanine, Tyrosine , tryptophan, 5-aminolevulinic acid or derivatives of any of the above amino acids;
  • the organic acid includes one or a combination of two or more of the following: citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, oxaloacetic acid, tartaric acid, propionic acid, hexenoic acid , capric acid, caprylic acid, valeric acid, malic acid or any of the above organic acid derivatives;
  • the protein related to the synthesis of the target compound is a protein related to the synthesis of L-amino acid; optionally, the protein related to the synthesis of L-amino acid includes pyruvate carboxylase, phosphoenolpyruvate carboxylase Amylase, ⁇ -glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, amino acid transport protein, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxalyl Acetate decarboxylase, gluconate repressor, glucose dehydrogenase, aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydro Dipicolinate reductase, dihydropicolinate reductase
  • the method further comprises the step of isolating or purifying the target compound.
  • the polynucleotide with promoter activity provided by the present disclosure is a mutant of the promoter of the dihydrodipicolinate reductase gene (dapB gene), compared with the promoter of the wild-type dapB gene, The promoter activity of the mutant was significantly increased. After the mutant is operably linked to the target gene, the expression efficiency of the target gene can be significantly improved, which provides an expression element with great application potential for the transformation of key genes in the synthesis pathway of the target compound. Applying the mutant to the production of the target compound can significantly increase the conversion rate of the target compound, and provides a strong promoter with great application potential for the industrial fermentation of target compounds such as amino acids and organic acids.
  • target compounds such as amino acids and organic acids.
  • the polynucleotide with promoter activity provided by the present disclosure has a promoter activity that is 3-12 times higher than that of the wild-type dapB gene promoter.
  • the present disclosure provides transcriptional expression cassettes, recombinant expression vectors, and recombinant host cells, comprising the aforementioned polynucleotides with promoter activity.
  • the polynucleotide with promoter activity is operably linked to the target gene, which can achieve high-efficiency expression of key genes in the synthesis pathway of the target compound.
  • the present disclosure provides a method for preparing proteins, which can increase the expression of proteins related to the synthesis of amino acids, organic acids, etc. or gene expression regulation proteins, thereby achieving efficient production of target compounds.
  • the present disclosure provides a method for producing a target compound.
  • the expression efficiency of a protein related to the synthesis of the target compound can be improved, thereby effectively improving the yield and transfer of the target compound. rate, to achieve large-scale industrial production of the target compound.
  • Fig. 1 shows the plasmid map of pEC-XK99E-P dapB -rfp
  • Figure 2 shows the fluorescence results of mutant clones grown on plate medium.
  • the selected/optional/preferred “numeric range” not only includes the numerical endpoints at both ends of the range, but also includes all natural numbers covered in the middle of the numerical endpoints relative to the aforementioned numerical endpoints.
  • dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reductive reaction of dihydrodipicolinate to form hexahydrodipicolinate.
  • Dihydrodipicolinate reductase is encoded by the dapB gene.
  • the dapB gene of the present disclosure is derived from Corynebacterium glutamicum.
  • Phosphopyruvate carboxylase catalyzes the conversion of phosphoenolpyruvate (PEP) to oxaloacetate and is encoded by the ppc gene.
  • Pyruvate carboxylase catalyzes the reversible carboxylation of pyruvate to form acetyl oxalate and is encoded by the pyc gene.
  • polynucleotide refers to a polymer composed of nucleotides.
  • a polynucleotide may be in the form of an individual fragment or an integral part of a larger nucleotide sequence structure derived from a nucleotide sequence that has been isolated at least once in number or concentration, capable of being separated by standard Molecular biology methods (eg, using cloning vectors) identify, manipulate and restore sequences and their component nucleotide sequences.
  • 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”.
  • polynucleotide refers to a polymer of nucleotides removed from other nucleotides (individual fragments or entire fragments), or which may be a building block or constituent of a larger nucleotide structure, as expressed vector or polycistronic sequence.
  • Polynucleotides include DNA, RNA and cDNA sequences.
  • wild-type refers to an object that can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory is naturally occurring.
  • wild-type promoter in the present disclosure refers to the promoter of the wild-type dapB gene, that is, the polynucleotide of the sequence shown in SEQ ID NO:1.
  • mutant refers to a polynucleotide or polypeptide comprising an alteration (i.e. , Polynucleotides for substitution, insertion and/or deletion, wherein substitution refers to replacing a nucleotide occupying a position with a different nucleotide. Deletion refers to removing a nucleotide occupying a position. Insertion refers to Nucleotides are added adjacent to and immediately after the nucleotide occupying the position.
  • the "mutation" in the present disclosure is “substitution”, which is a mutation caused by the substitution of a base in one or more nucleotides by another different base, also known as a base substitution mutation (substitution) or point mutation (point mutation).
  • sequence shown in SEQ ID NO: 1 is the promoter sequence of the dapB gene, which includes the promoter core region with the nucleotide sequence of "ACGGTCAGTTAGGTATGGATATCAGCACCTTCTGAACGGGTACGTC TAGACT GGTGGGCG", where the underlined position is the -10 region sequence.
  • the mutant in the present disclosure introduces a mutated nucleotide near the position of the -10 region, and it is found that after the mutation is introduced at the above position, the promoter activity of the mutant is significantly enhanced, and a new type of strong promoter is obtained, which is Efficient synthesis of target compounds provides abundant expression regulatory elements.
  • the polynucleotide having promoter activity refers to a mutant comprising a polynucleotide of the sequence shown in SEQ ID NO: 1, and the mutant is in the sequence shown in SEQ ID NO: 1
  • One or more positions in positions 75-95 have mutated nucleotides, and do not include polynucleotides in which positions 75-95 of the sequence shown in SEQ ID NO: 1 are mutated to TCTGAACGGGTACGTCTAGAC.
  • the mutant has improved promoter activity.
  • the mutant of the polynucleotide comprising the sequence shown in SEQ ID NO: 1 in the present disclosure, compared with the polynucleotide comprising the sequence shown in SEQ ID NO: 1, has more than 3-12 times of improvement promoter activity.
  • the mutant compared with the polynucleotide comprising the sequence shown in SEQ ID NO: 1, the mutant has 3.7, 3.8, 11.2 times increased promoter activity.
  • promoter refers to a nucleic acid molecule, usually located upstream of the coding sequence of the target gene, providing a recognition site for RNA polymerase, and located in the 5' direction of the mRNA transcription start site. upstream. It is a nucleic acid sequence that is not translated, and RNA polymerase binds to this nucleic acid sequence to initiate the transcription of the target gene.
  • RNA ribonucleic acid
  • the promoter can interact with transcription factors that regulate gene transcription, and control the initiation time and degree of expression of gene expression (transcription), including the core promoter region and regulatory region, just like “Switches” determine the activity of genes, which in turn control which proteins the cell starts producing.
  • the term "promoter core region” refers to a nucleic acid sequence located in the prokaryotic promoter region, which is the core sequence region that functions as a promoter, mainly including the -35 region, the -10 region, and the -35 region The region between the -10 region and the transcription start site, the -35 region is the recognition site of RNA polymerase, and the -10 region is the binding site of RNA polymerase.
  • the polynucleotide with promoter activity of the present disclosure comprises the promoter core region of the dapB gene, and introduces a mutant mutant near the position of the -10 region of the promoter core region, so as to obtain The promoter activity of the dapB gene was significantly increased.
  • sequence identity and “percent identity” 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 comparing the sequence identity in the aligned polynucleotides or polypeptides. The number of positions containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotide or polypeptide containing a different nucleotide or amino acid residue.
  • Polynucleotides may differ at one position, for example, by containing different nucleotides (ie substitutions or mutations) or missing nucleotides (ie nucleotide insertions or nucleotide deletions in one or both polynucleotides).
  • Polypeptides may 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 the polynucleotide or polypeptide.
  • Percent identity can be calculated, for example, 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.
  • two or more sequences or subsequences share at least 80%, 81%, 82%, 83% when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection. %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% core "sequence identity" or "percent identity" of nucleotides.
  • the sequences are substantially identical over the entire length of either or both compared biopolymers (eg, polynucleotides).
  • the term “complementary” refers to hybridization or base pairing between nucleotides or between nucleotides, such as between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a Between primer binding sites on single-stranded nucleotides for sequencing or amplification, etc.
  • highly stringent conditions refers to, for probes of at least 100 nucleotides in length, following standard Southern blotting procedures, at 42°C in 5X SSPE (saline sodium phosphate EDTA) , 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA, and 50% formamide prehybridize and hybridize for 12 to 24 hours. Finally the support material was washed three times at 65°C for 15 minutes each in 2X SSC, 0.2% SDS.
  • 5X SSPE saline sodium phosphate EDTA
  • very high stringency conditions means, for probes of at least 100 nucleotides in length, following standard Southern blotting procedures, at 42°C in 5X SSPE (saline sodium phosphate EDTA ), 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA, and 50% formamide for prehybridization and hybridization for 12 to 24 hours. Finally the support material was washed three times at 70°C for 15 minutes each in 2X SSC, 0.2% SDS.
  • 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 having promoter activity of the present disclosure can be used to initiate the expression of non-coding genes.
  • RNA production and protein production includes any step involved in RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • transcriptional expression cassette is a recombinant expression element comprising a polynucleotide having promoter activity.
  • the transcriptional regulatory element for regulating the target gene may also include elements such as enhancers, silencers, and insulators.
  • the target gene in the present disclosure is specifically a protein-coding gene.
  • "Operably linking" a target gene to a polynucleotide having promoter activity refers to functionally linking a polynucleotide having promoter activity to the target gene to initiate and mediate the transcription of the target gene.
  • the way of ground connection can be any way described by those skilled in the art.
  • vector refers to a DNA construct containing DNA sequences operably linked to appropriate control sequences to express a gene of interest in a suitable host.
  • "Recombinant expression vector” refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide.
  • Recombinant expression vectors can include, for example, a collection of genetic elements comprising i) 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 translation initiation and termination sequences of the transcriptional subunit.
  • Recombinant expression vectors are constructed in any suitable manner.
  • vectors are not critical, and any vector may be used, including plasmids, viruses, phage 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, from sources such as vaccinia, adenovirus, chicken DNA from viruses such as pox, baculovirus, SV40, and pseudorabies.
  • "recombinant expression vector” and "recombinant vector” can be used interchangeably.
  • target RNA includes functional RNAs that play a role in genetic coding, translation, regulation, gene expression, and the like.
  • the target RNA linked to the polynucleotide having promoter activity may be any functional RNA in the art.
  • the target RNA is tRNA or sRNA.
  • the target RNA can also be sgRNA, crRNA, tracrRNA, miRNA, siRNA and other types of RNA.
  • target gene refers to any gene linked to the polynucleotide having promoter activity in the present disclosure to regulate its transcription level.
  • the target gene is a gene encoding a protein related to the synthesis of the target compound. In some embodiments, the target gene is a gene encoding a gene expression regulatory protein. In some embodiments, the gene of interest is a gene encoding a protein associated with membrane transport.
  • the target gene is an encoding gene of an enzyme related to the biosynthesis of the target compound, an encoding gene of an enzyme related to reducing power, an encoding gene of an enzyme related to glycolysis or TCA cycle, or a gene related to the release of the target compound Related enzyme coding genes and so on.
  • the target gene includes at least one of the following genes: pyruvate carboxylase gene, phosphoenolpyruvate carboxylase gene, ⁇ -glutamyl kinase gene, glutamate semialdehyde dehydrogenase gene, Pyrroline-5-carboxylate reductase gene, amino acid transport protein gene, ptsG system related gene, pyruvate dehydrogenase gene, homoserine dehydrogenase gene, oxaloacetate decarboxylase gene, gluconate repressor protein gene, glucose dehydrogenase gene Hydrogenase gene, aspartate kinase gene, aspartate semialdehyde dehydrogenase gene, aspartate ammonia lyase gene, dihydrodipicolinate synthase gene, dihydropicolinate reductase gene, succinyldiaminopimelate aminotransferase gene, tetra
  • 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 amino and carboxyl groups are bound to the same carbon atom.
  • the amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methyl Thionine, Aspartic Acid, Asparagine, Glutamic Acid, Lysine, Arginine, Histidine, Phenylalanine, Tyrosine, Tryptophan, Proline, Hydroxyproline , 5-aminolevulinic acid or any one of the amino acid derivatives mentioned above.
  • amino acids may also be other kinds of amino acids in the art.
  • the target compound is an organic acid.
  • the organic acid may be an organic compound having acidity, for example, those compounds including carboxyl and sulfonic acid groups.
  • the organic acid includes one or more of the following: lactic acid, acetic acid, succinic acid, butyric acid, palmitic acid, oxalic acid, oxaloacetic acid, tartaric acid, citric acid, propionic acid, hexenoic acid, capric acid, octanoic acid , valeric acid, malic acid or any one of the above organic acid derivatives.
  • the organic acid can also be other types of organic acids in the art.
  • protein-coding gene in this disclosure refers to a synthetic DNA molecule that can guide proteins through certain rules.
  • the process of protein-coding genes guiding protein synthesis generally includes the transcription process using double-stranded DNA as a template and the translation using mRNA as a template. process.
  • a protein-coding gene contains a CDS sequence (Coding Sequence), which can guide the production of mRNA encoding a protein.
  • CDS sequence Coding Sequence
  • the protein-encoding gene includes but not limited to the gene encoding the protein related to the synthesis of the target compound.
  • the protein-encoding gene relates to the gene encoding the protein related to the synthesis of L-amino acid.
  • proteins related to the synthesis of L-amino acids include, but are not limited to, pyruvate carboxylase, phosphoenolpyruvate carboxylase, ⁇ -glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrrole One or both of phenoline-5-carboxylate reductase, amino acid transporter, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, gluconate repressor, glucose dehydrogenase combination of the above.
  • the protein related to the synthesis of L-amino acid is the protein related to the synthesis of L-lysine, for the protein related to the synthesis of L-lysine, including aspartokinase, aspartate Acid semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, dihydropicolinate reductase, succinyldiaminopimelic acid transamination Enzyme, tetrahydropyridine succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate One or a combination of two or more of dehydrogenase, lysine transport protein, transketolase, diaminopimelate dehydrogenase and pyruvate carboxylase.
  • the protein-coding gene relates to a protein-coding gene related to the synthesis of organic acids.
  • the protein-coding gene is used to encode a protein related to the synthesis of oxaloacetate, a protein related to the synthesis of citric acid, or Encodes proteins involved in the synthesis of succinic acid.
  • the protein-coding gene relates to a gene encoding a related enzyme that promotes oxaloacetate synthesis.
  • the protein coding gene is the ppc gene encoding phosphoenolpyruvate carboxylase, or the pyc gene encoding pyruvate carboxylase. According to the report in the existing literature [1] , the production of 5-aminolevulinic acid can be increased after the expression of related enzymes that promote the synthesis of oxaloacetate is enhanced.
  • gene expression regulatory protein in the present disclosure includes 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, and then regulate the expression of key genes in metabolic pathways.
  • host cell in the present disclosure means any cell type that is amenable to transformation, transfection, transduction, etc., with a transcriptional initiation element or expression vector comprising a polynucleotide of the present disclosure.
  • recombinant host cell encompasses a host cell that differs from the parental cell after the introduction of a transcription initiation element or a recombinant expression vector, in particular by transformation.
  • 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 transformation method includes any method for introducing nucleic acid into cells, and these methods include but are 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 the polynucleotide having promoter activity of the present disclosure can be introduced into the cell.
  • the host cell refers to a prokaryotic cell, specifically, the host cell is derived from a microorganism suitable for fermentative production of amino acids and organic acids, such as Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia belongs to.
  • the host cell is Corynebacterium glutamicum derived from the genus Corynebacterium.
  • Corynebacterium glutamicum can be Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067 etc., and the mutant strain that produces amino acid especially lysine prepared by above-mentioned bacterial strain or derivative strains of Corynebacterium glutamicum.
  • the host cell in the present disclosure may be any type of strain capable of producing amino acids, including wild-type strains and recombinant strains.
  • the host cell is a lysine-producing host cell.
  • the host cell for producing lysine may be a strain expressing aspartokinase that releases feedback inhibition based on Corynebacterium glutamicum ATCC 13032.
  • the lysine-producing host cells may be other types of strains capable of producing lysine.
  • one or more genes selected from the following in the lysine-producing host cell are attenuated or have reduced expression:
  • the cadA gene encoding lysine decarboxylase.
  • one or more genes selected from the following are enhanced or overexpressed in the lysine-producing host cell:
  • the dapA gene encoding the dihydrodipyridine synthetase that relieves lysine feedback inhibition
  • dapD encoding tetrahydrodipicolate succinylase and dapE encoding succinyldiaminopimelate deacylase
  • pntAB gene encoding nicotinamine adenine dinucleotide transhydrogenase
  • the host cell is a threonine-producing host cell.
  • the threonine-producing host cell is a strain expressing aspartokinase LysC that relieves feedback inhibition based on Corynebacterium glutamicum ATCC 13032.
  • the threonine-producing host cells may also be other types of bacterial strains capable of producing threonine.
  • one or more genes selected from the following are enhanced or overexpressed in the threonine-producing host cell:
  • the host cell is an isoleucine-producing host cell.
  • the isoleucine-producing host cell is a strain that produces L-isoleucine by substituting alanine for the amino acid at position 323 of the gene of L-threonine dehydratase ilvA.
  • the isoleucine-producing host cells may also be other types of strains capable of producing isoleucine.
  • the host cell is a host cell that produces O-acetyl homoserine.
  • the O-acetylhomoserine-producing host cell is a strain that produces O-acetylhomoserine by inactivating O-acetylhomoserine (thiol)-lyase.
  • the host cell producing O-acetyl homoserine can also be other kinds of bacterial strains with O-acetyl homoserine production capacity.
  • the host cell is a methionine-producing host cell.
  • the methionine-producing host cell is a strain that produces methionine by inactivating transcriptional regulators of methionine and cysteine.
  • the methionine-producing host cells may also be other types of bacterial strains capable of producing methionine.
  • the culture of the host cells of the present disclosure can be carried out according to conventional methods in the art, including but not limited to orifice 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 value of the medium, etc.
  • This disclosure uses the promoter core region sequence of the dapB gene to introduce mutations near the -10 region of the promoter of the dapB gene (the -10 region and the first 16 bp of the -10 region) to obtain the promoter core of the dapB gene including the mutation in the -10 region region mutants.
  • the polynucleotide with promoter activity in the present disclosure by mutating the promoter core region of the dapB gene, specifically introducing a mutation near the -10 region of the promoter core region of the dapB gene (TCTGAACGGGTACGTCTAGAC), and Compared with the wild-type promoter comprising the promoter core region of the dapB gene, the mutant in the present disclosure has significantly improved promoter activity and is a novel strong promoter; when applied to the fermentation of the target compound, the mutant Compared with the wild-type promoter, it shows higher conversion rate and yield of the target compound.
  • the polynucleotides with promoter activity in the present disclosure 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 positions with mutated nucleotides. And compared with the promoter of the wild-type dapB gene of the sequence shown in SEQ ID NO: 1, it has improved promoter activity.
  • the polynucleotide having promoter activity in the present disclosure also includes a polynucleotide reverse complementary to the nucleotide sequence of the mutant of the dapB gene promoter shown in SEQ ID NO:1. And compared with the promoter of the wild-type dapB gene of the sequence shown in SEQ ID NO: 1, the polynucleotide has improved promoter activity.
  • the polynucleotide with promoter activity in the present disclosure further comprises the combination of the dapB gene promoter shown in SEQ ID NO: 1 under high stringency hybridization conditions or very high stringency hybridization conditions
  • the nucleotide sequence of the polynucleotide corresponding to the 75th-95th position of the sequence shown in SEQ ID NO: 1 is not TCTGAACGGGTACGTCTAGAC.
  • the polynucleotide has improved promoter activity.
  • the polynucleotide with promoter activity in the present disclosure is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity (including all ranges and percent) sequence.
  • the nucleotide sequence of the polynucleotide corresponding to the 75th-95th position of the sequence shown in SEQ ID NO: 1 is not TCTGAACGGGTACGTCTAGAC.
  • the polynucleotide has improved promoter activity.
  • the nucleotide sequence corresponding to the 75th-95th position of the sequence shown in SEQ ID NO: 1 of the mutant is selected from the following (P dapB -1)-(P dapB -3) composition Any item in the group:
  • the nucleotide sequence of the mutant is selected from the sequences shown in any one of SEQ ID NO: 2-4.
  • the polynucleotide with promoter activity in the present disclosure compared with the polynucleotide of the sequence shown in SEQ ID NO: 1, has an increased promoter activity of more than 3-12 times. Further, compared with the polynucleotide comprising the sequence shown in SEQ ID NO: 1, it has 3.7, 3.8, 11.2 times increased promoter activity.
  • the present disclosure uses the ATCC13032 genome (Corynebacterium glutamicum ATCC 13032, Gene ID: 2830649) as a template, and uses dapB-1 and dapB-2 as primers to amplify the DNA fragment obtained from the dapB gene promoter, and the dapB gene
  • the N-terminal 180bp fragment of the pEC-XK99E-rfp plasmid is used as a template [2]
  • pEC-1 and pEC-2 are used as primers to amplify the pEC-XK99E plasmid backbone; RFP-1/2 is used as a primer
  • pEC- The XK99E-rfp plasmid was used as a template to amplify the DNA fragment of the red fluorescent protein gene containing the connecting peptide.
  • the present disclosure uses pEC-XK99E-P dapB -rfp as a template, uses PdapB-1 primers and pEC-3 primers to amplify fragments containing mutation regions, and uses pEC-4 primers and pEC-5 primers to amplify
  • the plasmid backbone fragment, the above two fragments were recombined and connected to obtain the recombinant vector pEC-XK99E-P dapB-1- rfp.
  • the present disclosure uses pEC-XK99E-P dapB -rfp as a template, uses PdapB-2 primers and pEC-3 primers to amplify fragments containing mutation regions, and uses pEC-4 primers and pEC-5 primers to amplify
  • the plasmid backbone fragment, the above two fragments were recombined and connected to obtain the recombinant vector pEC-XK99E-P dapB-2- rfp.
  • the present disclosure uses pEC-XK99E-P dapB -rfp as a template, uses PdapB-3 primers and pEC-3 primers to amplify fragments containing mutation regions, and uses pEC-4 primers and pEC-5 primers to amplify
  • the plasmid backbone fragment, the above two fragments were recombined and connected to obtain the recombinant vector pEC-XK99E-P dapB-3- rfp.
  • the present disclosure can also use the promoter mutant shown in any one of (P dapB -1) to (P dapB -3) to construct the required recombinant vector according to specific cloning requirements.
  • the present disclosure is referred to as pEC-XK99E-P dapB -rfp, pEC-XK99E-P dapB-1- rfp, pEC-XK99E-P dapB-2- rfp, pEC-XK99E-P dapB-3- rfp
  • Corynebacterium glutamicum ATCC13032 was transformed respectively to obtain recombinant host cells.
  • the present disclosure uses the promoter mutant library plasmid of the dapB gene as a template, and uses dapB-P1 and dapB-P2 as primers to amplify the promoter mutant fragments of each dapB gene;
  • the genome of Bacillus ATCC13032 was used as a template, and the ppc gene fragment was amplified with ppc-1/ppc-2 primers; the pEC-XK99E plasmid was used as a template, and the plasmid backbone was amplified with PEC-1/PEC-2 primers.
  • Each promoter mutant fragment is recombined with the ppc gene fragment and the plasmid backbone to obtain the promoter mutant plasmid of the dapB gene.
  • the promoter mutant plasmid of dapB gene includes any one of the following: pEC-P dapB-1- ppc, pEC-P dapB-2- ppc, pEC-P dapB-3- ppc.
  • the threonine at position 311 of aspartokinase (encoded by lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 is mutated to isoleucine to construct A strain SCgL30 with certain lysine synthesis ability was obtained.
  • the present disclosure transforms the recombinant expression vector represented by any one of pEC-P dapB-1- ppc to pEC-P dapB-3- ppc into SCgL30 strain to obtain recombinant host cells.
  • the present disclosure can also transform the SCgL30 strain with the recombinant vector containing the promoter mutant shown in any one of (P dapB-1 ) to (P dapB-3 ), to obtain recombinant host cells.
  • a polynucleotide with promoter activity is operably linked to a protein-encoding gene or gene expression regulatory protein-encoding gene related to the synthesis of the target compound to obtain a recombinant protein or gene expression regulatory protein related to the synthesis of the target compound
  • the expression vector is used to transform the host cell with the recombinant expression vector to obtain the recombinant host cell.
  • the transcriptional activity of the gene encoding the protein or gene expression regulation protein related to the synthesis of the target compound is increased, and the protein related to the synthesis of the target compound or The expression level of the gene expression regulation protein is increased, thereby significantly increasing the yield of the target compound.
  • the target compound is an amino acid
  • the protein-coding gene related to the synthesis of the target compound refers to the protein-coding gene related to the synthesis of amino acid.
  • the target compound is L-amino acid
  • the protein coding gene related to the synthesis of amino acid refers to the protein coding gene related to the synthesis of L-amino acid.
  • the protein associated with amino acid synthesis is phosphoenolpyruvate carboxylase
  • increasing the expression of ppc with a polynucleotide with promoter activity can enhance the synthesis of ppc from phosphoenolpyruvate (PEP ) to oxaloacetate, which in turn promotes the production of target compounds that depend on the supply of oxaloacetate precursors, including aspartic acid family amino acids (lysine, threonine, isoleucine, methionine), glutamine Acid family amino acids (glutamic acid, proline, hydroxyproline, arginine, glutamic acid amide), etc.
  • the host cell is Corynebacterium glutamicum, which is an important strain for producing target compounds such as amino acids and organic acids.
  • Corynebacterium glutamicum is an important strain for producing target compounds such as amino acids and organic acids.
  • transcriptional expression cassette or recombinant expression vector with strong constitutive promoter activity is modified to Corynebacterium glutamicum, the expression level of the protein related to the synthesis of the target compound in Corynebacterium glutamicum is significantly increased, thereby making The ability of Corynebacterium glutamicum to accumulate target compounds through long-term fermentation is greatly improved.
  • the host cell is Corynebacterium glutamicum modified as follows: Threonine at position 311 of aspartokinase (encoded by lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 is mutated to isoleucine acid.
  • the culture conditions of the recombinant host cells are as follows: the recombinant host cells are inoculated into TSB liquid medium for culture, the culture is inoculated as a seed into a 24-well plate containing a fermentation medium in each well, and cultured at 30°C for 18h , the rotation speed of the orifice plate shaker was 800rpm, and the L-lysine output was detected after the fermentation was completed.
  • the formula is: glucose, 80g/L; yeast powder, 1g/L; soybean peptone, 1g/L; NaCl, 1g/L; ammonium sulfate, 1g/L; urea, 10g/L; K 2 HPO 4 3H 2 O, 1g/L; MgSO 4 7H 2 O, 0.45g/L; FeSO 4 7H 2 O, 0.05g/L; Biotin, 0.4mg/L; Vitamin B1, 0.1mg /L; MOPS, 40g/L; initial pH7.2. Add 25 ⁇ g/mL kanamycin to the culture medium.
  • the recovery of the target compound from the recombinant host cells or the culture medium of the recombinant cells can be carried out by common methods in the art, including but not limited to: filtration, anion exchange chromatography, crystallization or HPLC.
  • the experimental techniques and experimental methods used in this embodiment are conventional technical methods unless otherwise specified, such as the experimental methods that do not indicate specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: experiment Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through normal commercial channels.
  • Embodiment 1 Construction of Corynebacterium glutamicum dapB gene promoter strength characterization plasmid
  • the disclosure selects the promoter of the Corynebacterium glutamicum dihydrodipicolinate reductase dapB (dihydrodipicolinate reductase) gene for strength characterization, and further introduces specific region mutations to enhance the activity of the promoter, and obtains a promoter mutant with enhanced expression strength.
  • gene expression regulation sequence and N-terminal coding region are the key regions affecting gene expression.
  • the present disclosure adopts the method of connecting the upstream promoter of the dapB gene, the N-terminal 180bp coding region of the dapB gene, a flexible connecting peptide linker and a red fluorescent protein gene rfp sequentially, and characterizes the expression intensity of the target promoter based on the fluorescence intensity.
  • a characterization vector for the promoter of the dapB gene of Corynebacterium glutamicum was firstly constructed. On the basis of the pEC-XK99E plasmid backbone, the N-terminal 60 amino acids of the selected gene, a connecting peptide and the red fluorescent protein gene are expressed from the expression regulatory region including the promoter upstream of the gene.
  • the specific construction is as follows:
  • Amplification primers were designed according to the published genome sequence of Corynebacterium glutamicum ATCC 13032 (Corynebacterium glutamicum ATCC 13032, Gene ID: 2830649) and the annotation information of the dapB gene of Corynebacterium glutamicum. Using dapB-1/2 as primers and the ATCC13032 genome as a template, the wild-type promoter of dapB (sequence shown in SEQ ID NO: 1) and the fragment of the N-terminal 180 bp sequence were amplified.
  • the dapB gene promoter and N-terminal 180bp fragment obtained above were cloned and ligated with the red fluorescent protein gene DNA fragment containing the connecting peptide and the pEC-XK99E plasmid backbone through Novizym's one-step recombination kit to obtain pEC-XK99E-P dapB -rfp characterizes the vector, the plasmid map is shown in Figure 1, and the sequences of the primers used above are shown in Table 1.
  • the core region of the dapB promoter (sequence from position 75 to position 95 of SEQ ID NO: 1) in the pEC-XK99E-P dapB -rfp plasmid is transformed, and the sequence of the core region of the wild-type dapB promoter is as follows , where the bold TAGACT is the main sequence of the -10 region of the promoter:
  • the core region of the wild-type dapB promoter was transformed into the following sequences:
  • the modified polynucleotide sequences with promoter activity are respectively shown in the sequences SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
  • the specific construction is as follows: using the pEC-XK99E-P dapB -rfp plasmid as a template, using PdapB-1/pEC-3, PdapB-2/pEC-3, and PdapB-3/pEC-3 as primers, respectively amplifying three species 3 fragments of the remodeled area.
  • the pEC-XK99E-rfp-2 plasmid as a template and pEC-4/5 as a primer
  • the plasmid backbone was amplified.
  • the components of the assay medium TSB liquid medium are (g/L): glucose, 5g/L; yeast powder, 5g/L; soybean 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. Add 25 ⁇ g/mL kanamycin to the medium.
  • the plate-activated strains were inoculated into 96-well plates containing 200 ⁇ L TSB liquid medium in each well with toothpicks, and each strain was replicated 3 times.
  • the rotation speed of the plate shaker was 800 rpm. After 24 hours of culture at 30°C, it was detected by a microplate reader. Fluorescence intensity of strains.
  • the excitation wavelength of the fluorescence measurement is 560nm, and the emission wavelength is 607nm; at the same time, the OD 600 of the bacterial solution is measured, and the fluorescence intensity of the strain is calculated.
  • Embodiment 3 Corynebacterium glutamicum dapB gene promoter mutant is applied to L-lysine production
  • a T311I point mutation is introduced into the aspartokinase gene lysC of the Corynebacterium glutamicum ATCC13032 strain, and the codon is mutated from ACC to ATC to obtain the SCgL30 strain.
  • the disclosure further uses the dapB gene promoter mutant to overexpress phosphoenolpyruvate carboxylase (PPC, NCBI-GeneID: 1019553, NCBI-ProteinID: NP_600799) to test its effect on L-lysine production.
  • the PdapB -3 promoter mutant was used to overexpress the ppc gene.
  • the construction process of the overexpression plasmid was as follows: the corresponding promoter mutant plasmid screened in Example 2 was used as a template, and the corresponding gene
  • the dapB-P1/dapB-P2 primers amplify the promoter mutant fragment; use the genome of Corynebacterium glutamicum ATCC13032 as a template, and use ppc-1/ppc-2 as primers to amplify the ppc gene fragment; use pEC-XK99E plasmid As a template, the plasmid backbone was amplified with PEC-1/2 primers.
  • the promoter mutant fragments, ppc gene fragments and plasmid backbone fragments obtained above were cloned and connected by Novizym's one-step recombination kit to obtain the pEC-P dapB-3- ppc plasmid.
  • the pEC-XK99E control plasmid and the above plasmids were transformed into the SCgL30 strain, respectively, and the control strain and mutant promoter overexpression strains SCgL30 (pEC-XK99E) and SCgL30 (pEC-P dapB-3- ppc) were obtained.
  • the sequences of the primers used above are shown in Table 4.
  • SCgL30 pEC-XK99E
  • SCgL30 pEC-P dapB-3 - ppc
  • the composition of fermentation medium is: glucose, 80g/L; yeast powder, 1g/L; soybean peptone, 1g/L; NaCl, 1g/L; ammonium sulfate, 1g/L; urea, 10g/L; K 2 HPO 4 ⁇ 3H 2 O, 1g/L; MgSO 4 7H 2 O, 0.45g/L; FeSO 4 7H 2 O, 0.05g/L; Biotin, 0.4mg/L; Vitamin B1, 0.1mg/L; MOPS, 40g/L; initial pH7.2. Add 25 ⁇ g/mL kanamycin to the culture medium.
  • the strain was inoculated into TSB liquid medium and cultured for 8 h, and the culture was inoculated as a seed into a 24-well plate containing 800 ⁇ l of fermentation medium per well, with an inoculum size of 12 ⁇ l, cultured at 30°C for 18 h, and the shaker speed of the well plate was 800 rpm.
  • Three parallels were performed for each strain.
  • the L-lysine production was detected by an SBA biosensor analyzer, and the OD 600 was measured by a microplate reader.
  • the results are shown in Table 5, the L-lysine production of the PdapB -3 promoter mutant overexpression strain increased by 30%. The above results indicate that the promoter mutant of the present disclosure can be used to enhance the expression of PPC gene and be applied to the production of L-lysine.
  • the promoter mutant of the dapB gene of the present disclosure can be used to enhance the expression of PPC in Corynebacterium glutamicum, and then strengthen the synthesis from phosphoenolpyruvate (PEP) to oxaloacetate, which can be applied to Production of target products dependent on the supply of oxaloacetate precursors, including aspartic acid family amino acids (lysine, threonine, isoleucine, methionine), glutamic acid family amino acids (glutamic acid, proline acid, hydroxyproline, arginine, glutamic acid amine), and 5-aminolevulinic acid and other amino acids with oxaloacetate as an important metabolic precursor.
  • enhancing the expression and activity of PPC can be used to increase the yield of target compounds such as 5-aminolevulinic acid [1]
  • the dapB gene promoter mutants of the present disclosure can be used to enhance the expression and activity of PPC, therefore, this The disclosed promoter mutants can also be used for 5-aminolevulinic acid production.
  • the promoter mutant of the present disclosure can express the N-terminal of DapB and RFP fusion protein, and can be used to improve the expression of PPC.
  • the promoter mutant of the dapB gene of the present disclosure can also be used to express other genes and be applied to various production of a product.
  • P dapB -2 (SEQ ID NO: 3):

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Abstract

公开了一种谷氨酸棒杆菌二氢吡啶二羧酸还原酶(dapB)基因启动子的突变体，与野生型dapB基因的启动子相比，突变体的启动子活性显著提高。还公开了包含该启动子突变体的转录表达盒、重组表达载体、重组宿主细胞，以及启动子突变体的构建方法、调控目标基因转录的方法、制备蛋白的方法和生产目标化合物的方法。

Description

基于dapB基因的具有启动子活性的多核苷酸及其用途 技术领域
本公开属于生物技术和基因工程技术领域，具体涉及一种具有启动子活性的多核苷酸，包含具有启动子活性的多核苷酸的转录表达盒、重组表达载体、重组宿主细胞，以及启动子突变体的构建方法、调控目标基因转录的方法、制备蛋白的方法和生产目标化合物的方法。
背景技术
微生物发酵法可以生产多种目标化合物，如氨基酸、有机酸等，这些目标化合物可广泛应用于医药、食品、动物饲料和化妆品等领域，具有巨大的经济价值。近年来，随着对氨基酸、有机酸等市场需求的不断增加，如何提高目标化合物的产量，实现对目标化合物的工业化大规模生产，是当前亟需解决的重要问题。
选育高产的发酵微生物是提高目标化合物工业化产量的重要手段，与传统诱变育种的技术相比，基因工程选育技术由于其强的针对性和高效性获得了广泛性的应用。众多研究表明，目标化合物的合成途径关键基因的高效表达是提高目标化合物产量和转化率的关键。
通过基因工程的方法对微生物代谢途径中的关键基因进行改造，是提高目标化合物的发酵产量的重要方法。启动子是影响基因表达的重要调控元件，启动子的精细调控可以实现目标化合物转化率的最优化。具有不同表达强度的启动子，可以满足不同基因不同表达强度的需求，进而可以提高目标化合物的产量和转化率。
因此，开发更多具有高活性的启动子，以增强目标化合物合成途径关键基因的表达，提高目标化合物的产量，提升生物发酵产业的竞争力，是微生物发酵领域亟需解决的重要问题。
发明内容
发明要解决的问题
鉴于现有技术中存在的技术问题，例如，需要开发更多具有高活性的启动子，以提高目标化合物合成途径中关键基因的表达。为此，本公开提供了一种具有启动子活性的多核苷酸，为包含如SEQ ID NO：1所示序列的多核苷酸的突变体，与野生型启动子相比，本公开提供的突变体的启动子活性显著提高，为目标基因的改造提供了极具应用潜力的表达调控元件。将突变体与目标基因可操作性地连接，可有效提高目标基因的表达，进而可有效提高目标化合物的产量、转化率。
用于解决问题的方案
本公开提供了一种具有启动子活性的多核苷酸，其中，所述多核苷酸选自如下(i)-(iv)组成的组中的任一项：
(i)包含如SEQ ID NO：1所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：1所示序列的第75-95位中的一个或多个位置处具有突变的核苷酸；
(ii)包含与(i)所示的核苷酸序列的反向互补序列的多核苷酸；
(iii)包含在高严格性杂交条件或非常高严格性杂交条件下，能够与(i)或(ii)所示的核苷酸序列杂交的序列的反向互补序列的多核苷酸；
(iv)与(i)或(ii)所示的核苷酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的多核苷酸；
其中，(i)-(iv)任一项所示的多核苷酸在SEQ ID NO：1所示序列的第75-95位的核苷酸序列不为TCTGAACGGGTACGTCTAGAC；并且，与SEQ ID NO：1所示序列的多核苷酸相比，(i)-(iv)任一项所示的多核苷酸具有增强的启动子活性。
在一些实施方式中，根据本公开所述的具有启动子活性的多核苷酸，其中，所述突变体与SEQ ID NO：1所示序列的多核苷酸相比，具有3-12倍以上的提高的启动子活性。
在一些实施方式中，根据本公开所述的具有启动子活性的多核苷酸，其中，所述突变体对应SEQ ID NO：1所示序列的第75-95位的核苷酸序列选自如下(P dapB-1)-(P dapB-3)组成的组中的任一项：
(P dapB-1)CTCTGATGTGATAGTATAATT；
(P dapB-2)ATCATTTGGTGTATACTAAAT；
(P dapB-3)GTCCTGTGGTAAACTTTAGCG。
在一些实施方式中，根据本公开所述的具有启动子活性的多核苷酸，其中，所述突变体的核苷酸序列选自如SEQ ID NO：2-4任一项所示的序列。
本公开还提供了一种转录表达盒，其中，所述转录表达盒包含根据本公开所述的具有启动子活性的多核苷酸；可选地，所述转录表达盒还含有目标基因，所述目标基因与所述具有启动子活性的多核苷酸可操作地连接；优选地，所述目标基因为蛋白编码基因。
本公开还提供了一种重组表达载体，其中，所述重组表达载体包含本公开所述的具有启动子活性的多核苷酸，或本公开所述的转录表达盒。
本公开还提供了一种重组宿主细胞，其中，所述重组宿主细胞包含本公开所述的转录表达盒，或本公开所述的重组表达载体。
在一些实施方式中，根据本公开所述的重组宿主细胞，其中，所述宿主细胞来源于棒状杆菌属、短杆菌属、节杆菌属、微杆菌属或埃希氏菌属；优选地，所述宿主细胞为谷氨酸棒杆菌或大肠杆菌；更优选地，所述宿主细胞为谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869、谷氨酸棒杆菌ATCC 14067或谷氨酸棒杆菌的衍生菌株。
本公开还提供了一种根据本公开所述的具有启动子活性的多核苷酸，根据本公开所述的转录表达盒，根据本公开所述的重组表达载体，根据本公开所述的重组宿主细胞在如下至少一种中的用途：
(a)增强基因的转录水平，或制备用于增强基因的转录水平的试剂或试剂盒；
(b)制备蛋白，或制备用于制备蛋白的试剂或试剂盒；
(c)生产目标化合物，或制备用于生产目标化合物的试剂或试剂盒。
在一些实施方式中，根据本公开所述的用途，其中，所述蛋白选自基因表达调控蛋白、与目标化合物合成相关的蛋白或与膜转运相关的蛋白。
在一些实施方式中，根据本公开所述的用途，其中，所述目标化合物包括氨基酸、有机酸中的至少一种；
可选地，所述氨基酸包括如下的一种或两种以上的组合：脯氨酸、羟脯氨酸、赖氨酸、谷氨酸、苏氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、5-氨基乙酰丙酸或上述任一种的氨基酸的衍生物；
可选地，所述有机酸包括如下的一种或两种以上的组合：柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、草酰乙酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸或上述任一种的有机酸的衍生物。
本公开还提供了一种启动子突变体的构建方法，其中，所述构建方法包括如下步骤：
突变步骤：对SEQ ID NO：1所示序列的多核苷酸进行突变，使SEQ ID NO：1所示序列的第75-95位中的一个或多个位置处具有突变的核苷酸；
筛选步骤：筛选与SEQ ID NO：1所示序列的多核苷酸相比，启动子活性提高的多核苷酸的突变体，得到启动子突变体。
在一些实施方式中，根据本公开所述的构建方法，其中，所述突变步骤包括：对SEQ ID NO：1所示序列的多核苷酸进行突变，使SEQ ID NO：1所示序列的第75-95位的核苷酸突变为如下所示的核苷酸序列：NNNNNNNNNNNNNNNNTANNN；其中，N选自A，T，C或G；
优选地，与SEQ ID NO：1所示序列的多核苷酸相比，启动子突变体具有3-12倍以上的提高的启动子活性。
本公开还提供了一种调控转录的方法，其中，所述方法包括将本公开所述的具有启动子活性的多 核苷酸与目标RNA或目标基因可操作地连接的步骤。可选地，所述目标RNA包括tRNA、sRNA中的至少一种，所述目标基因包括与目标化合物合成相关的蛋白的编码基因、基因表达调控蛋白的编码基因、与膜转运相关的蛋白的编码基因中的至少一种；
可选地，所述目标基因包括如下的至少一种：丙酮酸羧化酶基因、磷酸烯醇式丙酮酸羧化酶基因、γ-谷氨酰激酶基因、谷氨酸半醛脱氢酶基因、吡咯啉-5-羧酸还原酶基因、氨基酸运输蛋白基因、ptsG系统相关基因、丙酮酸脱氢酶基因、高丝氨酸脱氢酶基因、草酰乙酸脱羧酶基因、葡萄糖酸阻遏蛋白基因、葡萄糖脱氢酶基因、天冬氨酸激酶基因、天冬氨酸半醛脱氢酶基因、天冬氨酸氨裂合酶基因、二氢吡啶二羧酸合成酶基因、二氢吡啶甲酸还原酶基因、琥珀酰二氨基庚二酸氨基转移酶基因、四氢吡啶二羧酸酯琥珀酰酶基因、琥珀酰二氨基庚二酸脱酰基酶基因、二氨基庚二酸差向异构酶基因、二氨基庚二酸脱酰基酶基因、甘油醛-3-磷酸脱氢酶基因、转酮酶基因、二氨基庚二酸脱氢酶基因。
本公开还提供了一种制备蛋白的方法，其中，所述方法包括利用本公开所述的转录表达盒，本公开所述的重组表达载体，或本公开所述的重组宿主细胞表达所述蛋白的步骤；可选地，所述蛋白为与目标化合物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白；
任选地，所述方法还包括分离或纯化所述蛋白的步骤。
本公开还提供了一种生产目标化合物的方法，其中，所述方法包括利用本公开所述的转录表达盒，本公开所述的重组表达载体，或本公开所述的重组宿主细胞表达与目标化合物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白，在所述与目标化合物合成相关的蛋白、与膜转运相关的蛋白或所述基因表达调控蛋白存在的环境下生产目标化合物的步骤；
可选地，所述目标化合物包括氨基酸、有机酸中的至少一种；
可选地，所述氨基酸包括如下的一种或两种以上的组合：赖氨酸、谷氨酸、苏氨酸、脯氨酸、羟脯氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、5-氨基乙酰丙酸或上述任一种的氨基酸的衍生物；
可选地，所述有机酸包括如下的一种或两种以上的组合：柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、草酰乙酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸或上述的任一种的有机酸的衍生物；
可选地，所述与目标化合物合成相关的蛋白为与L-氨基酸合成相关的蛋白；可选地，所与L-氨基酸合成相关的蛋白包括丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、γ-谷氨酰激酶、谷氨酸半醛脱氢酶、吡咯啉-5-羧酸还原酶、氨基酸运输蛋白、ptsG系统、丙酮酸脱氢酶、高丝氨酸脱氢酶、草酰乙酸脱羧酶、葡萄糖酸阻遏蛋白、葡萄糖脱氢酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、天冬氨酸氨裂合酶、二氢吡啶二羧酸合成酶、二氢吡啶二羧酸还原酶、二氢吡啶甲酸还原酶、琥珀酰二氨基庚二酸氨基转移酶、四氢吡啶二羧酸酯琥珀酰酶、琥珀酰二氨基庚二酸脱酰基酶、二氨基庚二酸差向异构酶、二氨基庚二酸脱酰基酶、甘油醛-3-磷酸脱氢酶、转酮酶、二氨基庚二酸脱氢酶中的一种或两种以上的组合；
任选地，所述方法还包括分离或纯化所述目标化合物的步骤。
发明的效果
在一些实施方式中，本公开提供的具有启动子活性的多核苷酸，为二氢吡啶二羧酸还原酶基因(dapB基因)启动子的突变体，与野生型dapB基因的启动子相比，突变体的启动子活性显著提高。将突变体与目标基因可操作地连接后，可以显著提高目标基因的表达效率，为目标化合物合成途径中关键基因的改造提供了一种极具应用潜力的表达元件。将突变体应用于目标化合物的生产中，可以显著提高目标化合物的转化率，为氨基酸、有机酸等目标化合物的工业发酵提供了一种极具应用潜力的强启动子。
在一些实施方式中，本公开提供的具有启动子活性的多核苷酸，其启动子活性与野生型dapB基因的启动子相比，具有3-12倍以上提高的启动子活性。
在一些实施方式中，本公开提供了转录表达盒、重组表达载体、重组宿主细胞，包含上述具有启动子活性的多核苷酸。在转录表达盒、重组表达载体、重组宿主细胞中，具有启动子活性的多核苷酸与目标基因可操作地连接，能够实现目标化合物合成途径中关键基因的高效表达。
在一些实施方式中，本公开提供了制备蛋白的方法，能够提高与氨基酸、有机酸等合成相关的蛋白或基因表达调控蛋白的表达量，进而实现目标化合物的高效生产。
在一些实施方式中，本公开提供了生产目标化合物的方法，利用上述具有启动子活性的多核苷酸，能够提高与目标化合物合成相关的蛋白的表达效率，从而有效提高目标化合物的产量和传化率，实现对目标化合物的大规模工业化生产。
附图说明
图1示出了pEC-XK99E-P dapB-rfp的质粒图谱；
图2示出了平板培养基上生长突变体克隆的荧光结果图。
具体实施方式
当在权利要求和/或说明书中与术语“包含”联用时，词语“一(a)”或“一(an)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
在整个申请文件中，术语“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”是指“和/或”。
当用于权利要求书或说明书时，选择/可选/优选的“数值范围”既包括范围两端的数值端点，也包括相对于前述数值端点而言，所述数值端点中间所覆盖的所有自然数。
如本公开所使用的，术语“二氢吡啶二羧酸还原酶”(dihydrodipicolinate reductase)催化二氢吡啶二羧酸的NAD(P)H-依赖的还原性反应，生成六氢吡啶二羧酸。二氢吡啶二羧酸还原酶由dapB基因编码。在一些实施方式中，本公开中的dapB基因来源于谷氨酸棒杆菌(Corynebacterium glutamicum)。
如本公开所使用的，术语“磷酸烯醇式丙酮酸羧化酶”(Phosphopyruvate carboxylase)催化磷酸烯醇式丙酮酸(PEP)到草酰乙酸的转化，由ppc基因编码。
如本公开所使用的，术语“丙酮酸羧化酶”(Pyruvate carboxylase)催化丙酮酸的可逆羧基化，形成草酸乙酰，由pyc基因编码。
如本公开所使用的，术语“多核苷酸”指由核苷酸组成的聚合物。多核苷酸可以是单独片段的形式，也可以是更大的核苷酸序列结构的一个组成部分，其是从至少在数量或浓度上分离一次的核苷酸序列衍生而来的，能够通过标准分子生物学方法(例如，使用克隆载体)识别、操纵以及恢复序列及其组分核苷酸序列。当一个核苷酸序列通过一个DNA序列(即A、T、G、C)表示时，这也包括一个RNA序列(即A、U、G、C)，其中“U”取代“T”。换句话说，“多核苷酸”指从其他核苷酸(单独的片段或整个片段)中去除的核苷酸聚合物，或者可以是一个较大核苷酸结构的组成部分或成分，如表达载体或多顺反子序列。多核苷酸包括DNA、RNA和cDNA序列。
如本公开所使用的，术语“野生型的”指在自然界中可以找到的对象。例如，一种存在于生物体中，可以从自然界的一个来源中分离出来并且在实验室中没有被人类有意修改的多肽或多核苷酸序列是天然存在的。如本公开所用的，“天然存在的”和“野生型的”是同义词。在一些实施方式中，本公开中野生型的启动子是指野生型dapB基因的启动子，也即如SEQ ID NO：1所示序列的多核苷酸。
如本公开所使用的，术语“突变体”是指相对于“野生型”，或者“相比较的”多核苷酸或多肽，在一个或多个(例如，若干个)位置处包含改变(即，取代、插入和/或缺的多核苷酸，其中，取代是指用不同的核苷酸置换占用一个位置的核苷酸。缺失是指去除占据某一位置的核苷酸。插入是指在邻接并且紧随占据位置的核苷酸之后添加核苷酸。
在一些实施方式中，本公开的“突变”为“取代”，是由一个或多个核苷酸中的碱基被另一个不同的碱基取代所引起的突变，也称为碱基置换突变(subsititution)或点突变(point mutation)。
具体来说，SEQ ID NO：1所示的序列是dapB基因的启动子序列，其包含核苷酸序列为“ACGGTCAGTTAGGTATGGATATCAGCACCTTCTGAACGGGTACGTC TAGACTGGTGGGCG”的启动子核心区，其中下划线位置处为-10区序列。本公开中的突变体是在-10区位置附近引入突变的核苷酸，并且发现在上述位置处引入突变后，突变体的启动子活性明显增强，得到了一种新型的强启动子，为目标化合物的高效合成提供丰富的表达调控元件。
在一些实施方式中，具有启动子活性的多核苷酸，是指包含如SEQ ID NO：1所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：1所示序列的第75-95位中的一个或多个位置处具有突变的核苷酸，且不包含SEQ ID NO：1所示序列的第75-95位突变为TCTGAACGGGTACGTCTAGAC的多核苷酸。与包含SEQ ID NO：1所示序列的多核苷酸相比，突变体具有提高的启动子活性。
在一些实施方式中，本公开中包含SEQ ID NO：1所示序列的多核苷酸的突变体，与包含SEQ ID NO：1所示序列的多核苷酸相比，具有3-12倍以上提高的启动子活性。
在一些更为具体的实施方式中，突变体与包含SEQ ID NO：1所示序列的多核苷酸相比，具有3.7、3.8、11.2倍的提高的启动子活性。
如本公开所使用的，术语“启动子”是指一种核酸分子，通常位于目标基因编码序列的上游，为RNA聚合酶提供识别位点，并位于mRNA转录起始位点的5’方向的上游。它是不被翻译的核酸序列，RNA聚合酶与这一核酸序列结合后启动目标基因的转录。在核糖核酸(RNA)的合成中，启动子可以和调控基因转录的转录因子产生相互作用，控制基因表达(转录)的起始时间和表达的程度，包含核心启动子区域和调控区域，就像“开关”，决定基因的活动，继而控制细胞开始生产哪一种蛋白质。
如本公开所使用的，术语“启动子核心区”是指位于原核生物启动子区的一段核酸序列，是发挥启动子功能的核心序列区，主要包括-35区、-10区、-35区和-10区之间的区域以及转录起始位点，-35区是RNA聚合酶的识别位点，-10区是RNA聚合酶的结合位点。在一些实施方式中，本公开的具有启动子活性的多核苷酸，是包含dapB基因的启动子核心区，且在启动子核心区的-10区位置附近引入突变的突变体，以获得相比dapB基因的启动子明显提高的启动子活性。
如本公开所使用的，术语“序列同一性”和“同一性百分比”指两个或更多个多核苷酸或多肽之间相同(即同一)的核苷酸或氨基酸的百分比。两个或更多个多核苷酸或多肽之间的序列同一性可通过以下方法测定：将多核苷酸或多肽的核苷酸或氨基酸序列对准且对经对准的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置数目进行评分，且将其与经对准的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸(即取代或突变)或缺失核苷酸(即一个或两个多核苷酸中的核苷酸插入或核苷酸缺失)而在一个位置处不同。多肽可例如通过含有不同氨基酸(即取代或突变)或缺失氨基酸(即一个或两个多肽中的氨基酸插入或氨基酸缺失)而在一个位置处不同。序列同一性可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中氨基酸残基的总数来计算。举例而言，可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中核苷酸或氨基酸残基的总数且乘以100来计算同一性百分比。
在一些实施方式中，当使用序列比较算法或通过目视检查测量以最大的对应性进行比较和比对时，两个或多个序列或子序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核苷酸的“序列同一性”或“同一性百分比”。在某些实施方式中，所述序列在任一或两个相比较的生物聚合物(例如，多核苷酸)的整个长度上基本相同。
如本公开所使用的，术语“互补的”是指在核苷酸或核苷酸之间的杂交或碱基配对，例如双链DNA分子的两条链之间或者寡核苷酸引物与被测序或扩增的单链核苷酸上的引物结合位点之间等。
如本公开所使用的，术语“高严格条件”是指，对于长度为至少100个核苷酸的探针而言，遵循标准DNA印迹程序，在42℃处在5X SSPE(saline sodium phosphate EDTA)、0.3％SDS、200微克/ml剪切并变性的鲑精DNA和50％甲酰胺中预杂交和杂交12至24小时。最后在65℃处使用2X SSC、 0.2％SDS将载体材料洗涤三次，每次15分钟。
如本公开所使用的，术语“非常高严格条件”是指，对于长度为至少100个核苷酸的探针而言，遵循标准DNA印迹程序，在42℃处在5X SSPE(saline sodium phosphate EDTA)、0.3％SDS、200微克/ml剪切并变性的鲑精DNA和50％甲酰胺中预杂交和杂交12至24小时。最后在70℃处使用2X SSC、0.2％SDS将载体材料洗涤三次，每次15分钟。
在一些具体的实施方式中，本公开中的具有启动子活性的多核苷酸能够用于起始蛋白编码基因的表达。在另外一些实施方式中，本公开中的具有启动子活性的多核苷酸能够用于起始非编码基因的表达。
如本公开所使用的，术语“表达”包括涉及RNA产生及蛋白产生的任何步骤，包括但不限于：转录、转录后修饰、翻译、翻译后修饰和分泌。
如本公开所使用的，术语“转录表达盒”是包含具有启动子活性的多核苷酸的重组表达元件。在一些实施方式中，对目标基因进行调控的转录调控元件除了具有启动子活性的多核苷酸，还可以包含增强子、沉默子、绝缘子，等元件。在一些实施方式中，本公开中目标基因具体为蛋白编码基因。目标基因与具有启动子活性的多核苷酸“可操作地连接”，是指将具有启动子活性的多核苷酸与目标基因功能性连接，以启动和介导目标基因的转录，所述可操作地连接的方式可以采用本领域技术人员所述的任何方式。
如本公开所使用的，术语“载体”指的是DNA构建体，其含有与合适的控制序列可操作地连接的DNA序列，从而在合适的宿主中表达目标基因。“重组表达载体”指用于表达例如编码所需多肽的多核苷酸的DNA结构。重组表达载体可包括，例如包含i)对基因表达具有调控作用的遗传元素的集合，例如启动子和增强子；ii)转录成mRNA并翻译成蛋白质的结构或编码序列；以及iii)适当的转录和翻译起始和终止序列的转录亚单位。重组表达载体以任何合适的方式构建。载体的性质并不重要，并可以使用任何载体，包括质粒、病毒、噬菌体和转座子。用于本公开的可能载体包括但不限于染色体、非染色体和合成DNA序列，例如细菌质粒、噬菌体DNA、酵母质粒以及从质粒和噬菌体DNA的组合中衍生的载体，来自如牛痘、腺病毒、鸡痘、杆状病毒、SV40和伪狂犬病等病毒的DNA。在本公开中，“重组表达载体”与“重组载体”可以互换地使用。
如本公开所使用的，术语“目标RNA”包括在遗传编码、翻译、调控、基因表达等过程中发挥作用的功能性RNA。在本公开中，与具有启动子活性的多核苷酸连接的目标RNA可以是本领域任一种的功能性RNA。
在一些实施方式中，目标RNA为tRNA或sRNA。在另外一些实施方式中，目标RNA还可以是sgRNA、crRNA、tracrRNA、miRNA、siRNA等其他种类的RNA。
如本公开所使用的，术语“目标基因”涉及与本公开中具有启动子活性的多核苷酸连接，以对其转录水平进行调控的任一种的基因。
在一些实施方式中，目标基因为与目标化合物合成相关的蛋白的编码基因。在一些实施方式中，目标基因为基因表达调控蛋白的编码基因。在一些实施方式中，目标基因为与膜转运相关的蛋白的编码基因。
示例性的，目标基因是与目标化合物的生物合成相关的酶的编码基因、与还原力相关的酶的编码基因，与糖酵解或TCA循环相关的酶的编码基因，或与目标化合物的释放相关的酶的编码基因等等。
示例性的，目标基因包括如下的至少一种基因：丙酮酸羧化酶基因、磷酸烯醇式丙酮酸羧化酶基因、γ-谷氨酰激酶基因、谷氨酸半醛脱氢酶基因、吡咯啉-5-羧酸还原酶基因、氨基酸运输蛋白基因、ptsG系统相关基因、丙酮酸脱氢酶基因、高丝氨酸脱氢酶基因、草酰乙酸脱羧酶基因、葡萄糖酸阻遏蛋白基因、葡萄糖脱氢酶基因、天冬氨酸激酶基因、天冬氨酸半醛脱氢酶基因、天冬氨酸氨裂合酶基因、二氢吡啶二羧酸合成酶基因、二氢吡啶甲酸还原酶基因、琥珀酰二氨基庚二酸氨基转移酶基因、四氢吡啶二羧酸酯琥珀酰酶基因、琥珀酰二氨基庚二酸脱酰基酶基因、二氨基庚二酸差向异构酶基因、二氨基庚二酸脱酰基酶基因、甘油醛-3-磷酸脱氢酶基因、转酮酶基因、二氨基庚二酸脱氢酶基因、。如本公开所使用的，术语“目标化合物”可以选自氨基酸、有机酸，也可以选自本领域中可能通过生 物合成得到的其他种类的化合物。
在一些实施方式中，目标化合物为“氨基酸”或“L-氨基酸”。“氨基酸”或“L-氨基酸”通常是指其中氨基和羧基结合至相同碳原子的蛋白质的基本构成单元。示例性的，氨基酸选自如下的一种或多种：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苏氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、谷氨酸、赖氨酸、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、脯氨酸、羟脯氨酸、5-氨基乙酰丙酸或上述的任一种的氨基酸的衍生物。此外，氨基酸也可以是本领域中其他种类的氨基酸。
在一些实施方式中，目标化合物为有机酸。有机酸可以是具有酸性的有机化合物，例如，其中包括羧基和磺酸基的那些化合物。示例性的，有机酸包括如下的一种或多种：乳酸、醋酸、琥珀酸、丁酸、棕榈酸、草酸、草酰乙酸、酒石酸、柠檬酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸或上述的任一种的有机酸的衍生物。此外，有机酸也可以是本领域中其他种类的有机酸。
本公开中的术语“蛋白编码基因”是指能够通过一定的规则指导蛋白的合成DNA分子，蛋白编码基因指导蛋白合成的过程一般包括以双链DNA为模板的转录过程和以mRNA为模板的翻译过程。蛋白编码基因含有CDS序列(Coding Sequence)，能够指导编码蛋白质的mRNA的产生。
示例性的，蛋白编码基因包括但不限于与目标化合物合成相关的蛋白的编码基因，在一些实施方式中，蛋白编码基因涉及与合成L-氨基酸的相关的蛋白的编码基因。示例性的，与合成L-氨基酸的相关的蛋白包括但不限于丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、γ-谷氨酰激酶、谷氨酸半醛脱氢酶、吡咯啉-5-羧酸还原酶、氨基酸运输蛋白、ptsG系统、丙酮酸脱氢酶、高丝氨酸脱氢酶、草酰乙酸脱羧酶、葡萄糖酸阻遏蛋白、葡萄糖脱氢酶中的一种或两种以上的组合。在一些实施方式中，与合成L-氨基酸的相关的蛋白为与合成L-赖氨酸相关的蛋白，对于与合成L-赖氨酸的相关的蛋白，包括天冬氨酸激酶、天冬氨酸半醛脱氢酶、天冬氨酸氨裂合酶、二氢吡啶二羧酸合成酶、二氢吡啶二羧酸还原酶、二氢吡啶甲酸还原酶、琥珀酰二氨基庚二酸氨基转移酶、四氢吡啶二羧酸酯琥珀酰酶、琥珀酰二氨基庚二酸脱酰基酶、二氨基庚二酸差向异构酶、二氨基庚二酸脱酰基酶、甘油醛-3-磷酸脱氢酶、赖氨酸运输蛋白、转酮酶、二氨基庚二酸脱氢酶和丙酮酸羧化酶中的一种或两种以上的组合。
在一些实施方式中，蛋白编码基因涉及与合成有机酸相关的蛋白的编码基因，示例性的，蛋白编码基因用于编码与合成草酰乙酸有关的蛋白，与合成柠檬酸有关的蛋白，或用于编码与合成琥珀酸有关的蛋白。
在一些实施方式中，蛋白编码基因涉及促进草酰乙酸合成的相关酶的编码基因。示例性的，蛋白编码基因为磷酸烯醇式丙酮酸羧化酶的编码基因ppc基因，或丙酮酸羧化酶的编码基因pyc基因。依据现有文献 [1]中报道，促进草酰乙酸合成的相关酶的表达增强后可以提高5-氨基乙酰丙酸产量。
本公开的术语“基因表达调控蛋白”包括不限于外源的基因表达调控工具蛋白，例如CRISPRi调控需要的dCas9蛋白、dCpf1蛋白，sRNA调控需要的Hfq蛋白等，以及内源或外源的转录调控因子，进而调控代谢通路中关键基因的表达。
本公开中的术语“宿主细胞”意指易于用包含本公开的多核苷酸的转录起始元件或表达载体转化、转染、转导等的任何细胞类型。术语“重组宿主细胞”涵盖导入转录起始元件或重组表达载体后不同于亲本细胞的宿主细胞，重组宿主细胞具体通过转化来实现。
本公开中的术语“转化”具有本领域技术人员普遍理解的意思，即将外源性的DNA导入宿主的过程。所述转化的方法包括任何将核酸导入细胞的方法，这些方法包括但不限于电穿孔法、磷酸钙沉淀法、氯化钙(CaCl 2)沉淀法、微注射法、聚乙二醇(PEG)法、DEAE-葡聚糖法、阳离子脂质体法以及乙酸锂-DMSO法。
本公开的宿主细胞可以是原核细胞或真核细胞，只要是能够导入本公开的具有启动子活性的多核苷酸的细胞即可。在一些实施方式中，宿主细胞指原核细胞，具体地，宿主细胞来源于适合发酵生产氨基酸、有机酸的微生物，例如棒状杆菌属、短杆菌属、节杆菌属、微杆菌属或埃希氏菌属。作为优选地，宿主细胞是来源于棒状杆菌属的谷氨酸棒杆菌。其中，谷氨酸棒杆菌可以是谷氨酸棒杆菌ATCC13032、谷氨酸棒杆菌ATCC 13869或谷氨酸棒杆菌ATCC 14067等，以及由上述菌株制备的产生氨基 酸尤其是赖氨酸的突变体菌株或谷氨酸棒杆菌的衍生菌株。在一些实施方式中，本公开中的宿主细胞可以是具有氨基酸生产能力的任意类型的菌株，其包括野生型菌株和重组菌株。
示例地，宿主细胞为生产赖氨酸的宿主细胞。在一些实施方式中，对于生产赖氨酸的宿主细胞，可以是在谷氨酸棒杆菌ATCC 13032基础上表达解除反馈抑制的天冬氨酸激酶的菌株。此外，生产赖氨酸的宿主细胞也可以是具有赖氨酸生产能力的其他种类的菌株。
在一些实施方式中，所述生产赖氨酸的宿主细胞中选自以下的一个或多个基因被弱化或表达降低：
a.编码乙醇脱氢酶的adhE基因；
b.编码乙酸激酶的ackA基因；
c.编码磷酸乙酰转移酶的pta基因；
d.编码乳酸脱氢酶的ldhA基因；
e.编码甲酸转运蛋白的focA基因；
f.编码丙酮酸甲酸裂解酶的pflB基因；
g.编码丙酮酸氧化酶的poxB基因；
h.编码天冬氨酸激酶I/高丝氨酸脱氢酶I双功能酶的thrA基因；
i.编码高丝氨酸激酶的thrB基因；
j.编码赖氨酸脱羧酶的ldcC基因；和
h.编码赖氨酸脱羧酶的cadA基因。
在一些实施方式中，所述生产赖氨酸的宿主细胞中选自以下的一个或多个基因被增强或过表达：
a.编码解除赖氨酸反馈抑制的二氢二吡啶合成酶的dapA基因；
b.编码二氢二吡啶二羧酸还原酶的dapB基因；
c.编码二氨基庚二酸脱氢酶的ddh基因；
d.编码四氢吡啶二羧酸琥珀酰酶的dapD和编码琥珀酰二氨基庚二酸脱酰酶的dapE；
e.编码天冬氨酸-半醛脱氢酶的asd基因；
f.编码磷酸烯醇丙酮酸羧化酶的ppc基因；
g.编码烟酸胺腺嘌呤二核苷酸转氢酶的pntAB基因；
i.编码赖氨酸的运输蛋白lysE基因。
示例地，宿主细胞为生产苏氨酸的宿主细胞。在一些实施方式中，生产苏氨酸的宿主细胞为在谷氨酸棒杆菌ATCC 13032基础上表达解除反馈抑制的天冬氨酸激酶LysC的菌株。在另外一些实施方式中，生产苏氨酸的宿主细胞也可以是具有苏氨酸生产能力的其他种类的菌株。
在一些实施方式中，所述生产苏氨酸的宿主细胞中选自以下的一个或多个基因被增强或过表达：
a.编码苏氨酸操纵子的thrABC基因；
b.编码解除反馈抑制的高丝氨酸脱氢酶的hom基因；
c.编码甘油醛-3-磷酸脱氢酶的gap基因；
d.编码丙酮酸羧化酶的pyc基因；
e.编码苹果酸：醌氧化还原酶的mqo基因；
f.编码转酮酶的tkt基因；
g.编码6-磷酸葡糖酸脱氢酶的gnd基因；
h.编码苏氨酸输出的thrE基因；
i.编码烯醇酶的eno基因。
示例地，宿主细胞为生产异亮氨酸的宿主细胞。在一些实施方式中，生产异亮氨酸的宿主细胞是通过用丙氨酸取代L-苏氨酸脱水酶ilvA基因第323位的氨基酸而产生L-异亮氨酸的菌株。在另外一些实施方式中，生产异亮氨酸的宿主细胞也可以是具有异亮氨酸生产能力的其他种类的菌株。
示例地，宿主细胞为生产O-乙酰高丝氨酸的宿主细胞。在一些实施方式中，生产O-乙酰高丝氨酸的宿主细胞是通过使O-乙酰高丝氨酸(硫醇)-裂解酶失活而产生O-乙酰高丝氨酸的菌株。在另外一些实施方式中，生产O-乙酰高丝氨酸的宿主细胞也可以是具有O-乙酰高丝氨酸生产能力的其他种类 的菌株。
示例地，宿主细胞为生产蛋氨酸的宿主细胞。在一些实施方式中，生产蛋氨酸的宿主细胞是通过使甲硫氨酸和半胱氨酸的转录调节因子失活而产生蛋氨酸的菌株。在另外一些实施方式中，生产蛋氨酸的宿主细胞也可以是具有蛋氨酸生产能力的其他种类的菌株。
本公开的宿主细胞的培养可以根据本领域的常规方法进行，包括但不限于孔板培养、摇瓶培养、批次培养、连续培养和分批补料培养等，并可以根据实际情况适当地调整各种培养条件如温度、时间和培养基的pH值等。
除非在本公开中另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
dapB基因的启动子核心区的突变体
本公开利用dapB基因的启动子核心区序列，在dapB基因的启动子-10区位置附近(-10区及-10区前16bp)引入突变，得到包含-10区突变的dapB基因的启动子核心区的突变体。
本公开中的具有启动子活性的多核苷酸，通过对dapB基因的启动子核心区进行突变，具体地是在dapB基因的启动子核心区的-10区位置附近的(TCTGAACGGGTACGTCTAGAC)引入突变，与包含dapB基因的启动子核心区的野生型启动子相比，本公开中的突变体具有显著提高的启动子活性，是一种新型的强启动子；在应用于目标化合物的发酵时，突变体与野生型启动子相比，表现出更高的目标化合物的转化率和产量。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，在SEQ ID NO：1所示序列的第75-95位的1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个或21个位置处具有突变的核苷酸。且与SEQ ID NO：1所示序列的野生型dapB基因的启动子相比，具有提高的启动子活性。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，还包括与SEQ ID NO：1所示的dapB基因启动子的突变体的核苷酸序列反向互补的多核苷酸。且与SEQ ID NO：1所示序列的野生型dapB基因的启动子相比，多核苷酸具有提高的启动子活性。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，还包含在高严格性杂交条件或非常高严格性杂交条件下，与SEQ ID NO：1所示的dapB基因启动子的突变体的核苷酸序列杂交的序列的反向互补的多核苷酸。并且所述多核苷酸在对应SEQ ID NO：1所示序列的第75-95位中的核苷酸序列不为TCTGAACGGGTACGTCTAGAC。且与SEQ ID NO：1所示序列的野生型dapB基因的启动子相比，多核苷酸具有提高的启动子活性。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，为与上述的多核苷酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性(包括这些数值之间所有范围和百分数)的序列。并且所述多核苷酸在对应SEQ ID NO：1所示序列的第75-95位中的核苷酸序列不为TCTGAACGGGTACGTCTAGAC。且与SEQ ID NO：1所示序列的野生型dapB基因的启动子相比，多核苷酸具有提高的启动子活性。
在一些具体的实施方式中，所述突变体对应SEQ ID NO：1所示序列的第75-95位的核苷酸序列选自如下(P dapB-1)-(P dapB-3)组成的组中的任一项：
(P dapB-1)CTCTGATGTGATAGTATAATT；
(P dapB-2)ATCATTTGGTGTATACTAAAT；
(P dapB-3)GTCCTGTGGTAAACTTTAGCG。
在一些具体的实施方式中，所述突变体的核苷酸序列选自如SEQ ID NO：2-4任一项所示的序列。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，与SEQ ID NO：1所示序列的多核苷酸相比，具有3-12倍以上的提高的启动子活性。进一步的，与包含SEQ ID NO：1所示序列的多核苷酸相比，具有3.7、3.8、11.2倍的提高的启动子活性。
重组表达载体和重组宿主细胞
在一些实施方式中，本公开以ATCC13032基因组(Corynebacterium glutamicum ATCC 13032，Gene ID:2830649)为模板，以dapB-1和dapB-2为引物，扩增获得dapB基因启动子的DNA片段，和dapB基因的N端180bp片段；以pEC-XK99E-rfp质粒为模板 [2]，以pEC-1和pEC-2为引物，扩增pEC-XK99E质粒骨架；以RFP-1/2为引物，以pEC-XK99E-rfp质粒为模板，扩增包含连接肽的红色荧光蛋白基因DNA片段。将上述dapB基因启动子片段和N端180bp片段、包含连接肽的红色荧光蛋白基因DNA片段、pEC-XK99E质粒骨架重组连接，得到重组表达载体pEC-XK99E-P dapB-rfp。
在一些实施方式中，本公开以pEC-XK99E-P dapB-rfp为模板，以PdapB-1引物和pEC-3引物扩增包含突变区的片段，以pEC-4引物和pEC-5引物扩增质粒骨架片段，将上述两种片段重组连接，得到重组载体pEC-XK99E-P dapB-1-rfp。
在一些实施方式中，本公开以pEC-XK99E-P dapB-rfp为模板，以PdapB-2引物和pEC-3引物扩增包含突变区的片段，以pEC-4引物和pEC-5引物扩增质粒骨架片段，将上述两种片段重组连接，得到重组载体pEC-XK99E-P dapB-2-rfp。
在一些实施方式中，本公开以pEC-XK99E-P dapB-rfp为模板，以PdapB-3引物和pEC-3引物扩增包含突变区的片段，以pEC-4引物和pEC-5引物扩增质粒骨架片段，将上述两种片段重组连接，得到重组载体pEC-XK99E-P dapB-3-rfp。
在另外一些实施方式中，本公开还可以根据具体的克隆需要，利用(P dapB-1)～(P dapB-3)任一项所示的启动子突变体构建所需的重组载体。
在一些实施方式中，本公开以pEC-XK99E-P dapB-rfp、pEC-XK99E-P dapB-1-rfp、pEC-XK99E-P dapB-2-rfp、pEC-XK99E-P dapB-3-rfp分别转化谷氨酸棒杆菌ATCC13032，得到重组宿主细胞。
在一些实施方式中，本公开以dapB基因的启动子突变体文库质粒为模板，以dapB-P1和dapB-P2为引物，扩增得到各dapB基因的启动子突变体片段；以谷氨酸棒杆菌ATCC13032的基因组为模板，以ppc-1/ppc-2为引物扩增ppc基因片段；以pEC-XK99E质粒为模板，以PEC-1/PEC-2引物扩增质粒骨架。将各个启动子突变体片段分别与ppc基因片段、质粒骨架重组连接，得到dapB基因的启动子突变体质粒。
在一些具体的实施方式中，dapB基因的启动子突变体质粒包括如下的任一种：pEC-P dapB-1-ppc、pEC-P dapB-2-ppc、pEC-P dapB-3-ppc。
在一些实施方式中，本公开的谷氨酸棒杆菌SCgL30菌株，将谷氨酸棒杆菌ATCC13032基因组上天冬氨酸激酶(lysC基因编码)第311位的苏氨酸突变为异亮氨酸，构建获得一株具有一定赖氨酸合成能力的菌株SCgL30。
在一些具体的实施方式中，本公开将pEC-P dapB-1-ppc～pEC-P dapB-3-ppc中任一项所示的重组表达载体转化SCgL30菌株，获得重组宿主细胞。在另外一些具体的实施方式中，本公开还可以将包含(P dapB-1)～(P dapB-3)任一项所示的启动子突变体的重组载体转化SCgL30菌株，获得重组宿主细胞。
目标化合物的生产过程
(1)将具有启动子活性的多核苷酸，与目标化合物合成相关的蛋白编码基因或基因表达调控蛋白编码基因可操作地连接，得到能够与目标化合物合成相关的蛋白或基因表达调控蛋白的重组表达载体，利用重组表达载体转化宿主细胞，获得重组宿主细胞。
(2)对重组宿主细胞进行发酵培养，从重组宿主细胞或重组宿主细胞的培养液中收集目标化合物，完成目标化合物的生产过程。
上述生产过程中，由于多核苷酸具有改进的启动子活性，在重组宿主细胞中，与目标化合物合成相关的蛋白或基因表达调控蛋白的编码基因的转录活性提高，与目标化合物合成相关的蛋白或基因表达调控蛋白的表达量提高，进而使目标化合物的产量显著提升。
在一些实施方式中，目标化合物为氨基酸，与目标化合物合成相关的蛋白编码基因是指与合成氨基酸相关的蛋白编码基因。在一些实施方式中，目标化合物为L-氨基酸，与合成氨基酸相关的蛋白编 码基因是指与合成L-氨基酸相关的蛋白编码基因。
在一些具体的实施方式中，与氨基酸合成相关的蛋白为磷酸烯醇式丙酮酸羧化酶，以具有启动子活性的多核苷酸增加ppc的表达，可以加强从磷酸烯醇式丙酮酸(PEP)到草酰乙酸的合成，进而促进依赖于草酰乙酸前体供应的目标化合物的生产，包括天冬氨酸家族氨基酸(赖氨酸、苏氨酸、异亮氨酸、蛋氨酸)，谷氨酸家族氨基酸(谷氨酸、脯氨酸、羟脯氨酸、精氨酸、谷氨酸酰胺)等。
在一些具体的实施方式中，宿主细胞为谷氨酸棒杆菌(Corynebacterium glutamicum)，谷氨酸棒杆菌是用于生产氨基酸、有机酸等目标化合物的重要菌株。具有强组成型启动子活性的多核苷酸、转录表达盒或重组表达载体对谷氨酸棒杆菌进行改造后，谷氨酸棒杆菌内与目标化合物合成相关的蛋白的表达量显著提高，进而使谷氨酸棒杆菌长时间发酵积累目标化合物的能力大大提高。
在一些具体的实施方式中，宿主细胞是经过如下改良的谷氨酸棒杆菌：谷氨酸棒杆菌ATCC13032基因组上天冬氨酸激酶(lysC基因编码)第311位的苏氨酸突变为异亮氨酸。
在一些具体的实施方式中，重组宿主细胞的培养条件为：重组宿主细胞接种到TSB液体培养基中培养，培养物作为种子接种到每孔含有发酵培养基的24孔板中，30℃培养18h，孔板摇床转速为800rpm，发酵结束后检测L-赖氨酸产量。
对于赖氨酸发酵培养基，配方为：葡萄糖，80g/L；酵母粉，1g/L；大豆蛋白胨，1g/L；NaCl，1g/L；硫酸铵，1g/L；尿素，10g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.45g/L；FeSO 4·7H 2O，0.05g/L；生物素，0.4mg/L；维生素B1，0.1mg/L；MOPS，40g/L；初始pH7.2。培养基中补加25μg/mL卡那霉素。
在一些具体的实施方式中，对于重组宿主细胞或重组细胞的培养液回收目标化合物，可通过本领域常用方法，包括但不限于：过滤、阴离子交换色谱、结晶或HPLC。
在本领域，用于操纵微生物的方法是已知的，如《分子生物学现代方法》(Online ISBN：9780471142720,John Wiley and Sons,Inc.)、《微生物代谢工程：方法和规程》(Qiong Cheng Ed.,Springer)和《系统代谢工程：方法和规程》(Hal S.Alper Ed.,Springer)等出版物中被解释。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
实施例1.谷氨酸棒杆菌dapB基因启动子强度表征质粒的构建
本公开选择谷氨酸棒杆菌二氢吡啶二羧酸还原酶dapB(dihydrodipicolinate reductase)基因的启动子进行强度表征，并进一步引入特定区域突变增强启动子活性，获得表达强度增强的启动子突变体。在细菌中，基因的表达调控序列及N端编码区是影响基因表达的关键区域。本公开采用dapB基因上游启动子、dapB基因N端180bp编码区、一个柔性连接肽linker和一个红色荧光蛋白基因rfp顺序连接的方法，基于荧光强度表征目标启动子的表达强度。
本实施例首先构建谷氨酸棒杆菌dapB基因启动子的表征载体。在pEC-XK99E质粒骨架基础上，由基因上游包含启动子的表达调控区表达选定基因N端60个氨基酸、一个连接肽和红色荧光蛋白基因。具体构建如下：
(1)扩增启动子和N端序列片段
根据已公开的谷氨酸棒杆菌ATCC13032基因组序列(Corynebacterium glutamicum ATCC 13032，Gene ID:2830649)以及谷氨酸棒杆菌dapB基因的注释信息，设计扩增引物。以dapB-1/2为引物，以ATCC13032基因组为模板，扩增dapB的野生型启动子(序列如SEQ ID NO：1所示)和N端180 bp序列的片段。
(2)扩增质粒骨架和rfp片段
以文献报道的pEC-XK99E-rfp质粒 [2]为模板，分别以pEC-1/2为引物扩增pEC-XK99E质粒骨架，以RFP-1/2为引物扩增包含连接肽的红色荧光蛋白基因DNA片段，其中，DNA序列为：GGCGGTGGCTCTGGAGGTGGTGGGTCCGGCGGTGGCTCT。
以上获得的dapB基因启动子和N端180bp片段，分别与包含连接肽的红色荧光蛋白基因DNA片段、pEC-XK99E质粒骨架通过诺唯赞的一步重组试剂盒克隆连接，获得pEC-XK99E-P dapB-rfp表征载体，质粒图谱如图1所示，以上所用引物序列如表1所示。
表1
Figure PCTCN2022094734-appb-000001
实施例2.改造dapB基因启动子增强表达强度
(1)构建dapB基因启动子的突变体质粒
本实施例对pEC-XK99E-P dapB-rfp质粒中的dapB启动子核心区(SEQ ID NO：1的第75位至95位序列)进行改造，野生型dapB启动子核心区的序列如下所示，其中加粗的TAGACT为该启动子的-10区主要序列：
Figure PCTCN2022094734-appb-000002
野生型dapB启动子核心区分别改造为如下所示序列：
Figure PCTCN2022094734-appb-000003
改造后获得的具有启动子活性的多核苷酸序列分别如序列SEQ ID NO：2、SEQ ID NO：3、和SEQ ID NO：4所示。具体构建如下：以pEC-XK99E-P dapB-rfp质粒为模板，以PdapB-1/pEC-3、PdapB-2/pEC-3、PdapB-3/pEC-3为引物，分别扩增包括3种改造区的3个片段。以pEC-XK99E-rfp-2质粒为模板，以pEC-4/5为引物，扩增质粒骨架。以上3个包括改造区的片段分别与质粒骨架片段通过诺唯赞的一步重组试剂盒克隆连接，分别获得pEC-XK99E-P dapB-1-rfp、pEC-XK99E-P dapB-2-rfp和pEC-XK99E-P dapB-3-rfp表征载体。对获得的质粒进行测序验证，证实pEC-XK99E-P dapB-1-rfp、pEC-XK99E-P dapB-2-rfp和pEC-XK99E-P dapB-3-rfp表征载体中的启动子已分别成功替换为SEQ ID NO：2、SEQ ID NO：3、和SEQ ID NO：4所示序列的启动子。本实施例所用引物序列如表2所示。
表2
Figure PCTCN2022094734-appb-000004
Figure PCTCN2022094734-appb-000005
(2)dapB基因启动子突变体的强度表征
为表征SEQ ID NO：2、SEQ ID NO：3、和SEQ ID NO：4增强表达的强度，将pEC-XK99E-P dapB-1-rfp、pEC-XK99E-P dapB-2-rfp和pEC-XK99E-P dapB-3-rfp质粒及对照质粒pEC-XK99E-P dapB-rfp分别转化至谷氨酸棒杆菌ATCC13032，获得ATCC13032(pEC-XK99E-P dapB-1-rfp)、ATCC13032(pEC-XK99E-P dapB-2-rfp)、ATCC13032(pEC-XK99E-P dapB-3-rfp)和ATCC13032(pEC-XK99E-P dapB-rfp)菌株。以上菌株及对照菌株分别测定荧光强度。
测定培养基TSB液体培养基成份为(g/L)：葡萄糖，5g/L；酵母粉，5g/L；大豆蛋白胨，9g/L；尿素，3g/L；丁二酸，0.5g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。培养基中添加25μg/mL卡那霉素。将平板活化的菌株，分别用牙签接种至每孔含有200μL TSB液体培养基的96孔板中，每个菌株3个平行，孔板摇床转速为800rpm，30℃培养24h后采用酶标仪检测菌株的荧光强度。荧光测定激发波长为560nm，发射波长为607nm；同时测定菌液OD 600，计算菌株的荧光强度。结果如表3所示，ATCC13032(pEC-XK99E-P dapB-1-rfp)、ATCC13032(pEC-XK99E-P dapB-2-rfp)和ATCC13032(pEC-XK99E-P dapB-3-rfp)菌株的荧光强度分别比对照提高了3.7倍、3.8倍、11.2倍，表明启动子核心区改造可以进一步增强dapB基因启动子的活性，可用于增强dapB基因的表达。
表3
Figure PCTCN2022094734-appb-000006
实施例3.谷氨酸棒杆菌dapB基因启动子突变体应用于L-赖氨酸生产
(1)dapB基因启动子突变体应用于L-赖氨酸生产的菌株构建
本公开首先将谷氨酸棒杆菌ATCC13032菌株天冬氨酸激酶基因lysC引入了T311I点突变，密码子由ACC突变为ATC，获得SCgL30菌株。本公开进一步应用dapB基因启动子突变体过表达磷酸烯醇式丙酮酸羧化酶(PPC，NCBI-GeneID:1019553，NCBI-ProteinID:NP_600799)，测试其对L-赖氨酸生产的影响。
在pEC-XK99E质粒骨架基础上采用P dapB-3启动子突变体过表达ppc基因，过表达质粒的构建过程如下：以实施例2中筛选的对应启动子突变体质粒为模板，分别以对应基因的dapB-P1/dapB-P2引物扩增启动子突变体片段；以谷氨酸棒杆菌ATCC13032的基因组为模板，以ppc-1/ppc-2为引物扩增ppc基因片段；以pEC-XK99E质粒为模板，以PEC-1/2引物扩增质粒骨架。以上获得的启动子突变体片段、ppc基因片段和质粒骨架片段通过诺唯赞的一步重组试剂盒克隆连接，获得pEC-P dapB-3-ppc质粒。将pEC-XK99E对照质粒及以上质粒分别转化至SCgL30菌株，获得对照菌株和突变体启动子过表达菌株SCgL30(pEC-XK99E)和SCgL30(pEC-P dapB-3-ppc)。以上所用引物序列如表4所示。
表4
Figure PCTCN2022094734-appb-000007
(2)启动子突变体过表达菌株的L-赖氨酸生产能力评价
为了测试谷氨酸棒杆菌中应用P dapB-3启动子突变体过表达ppc基因对菌株产L-赖氨酸的影响，分别对SCgL30(pEC-XK99E)和SCgL30(pEC-P dapB-3-ppc)进行发酵测试。发酵培养基成份为：葡萄糖，80g/L；酵母粉，1g/L；大豆蛋白胨，1g/L；NaCl，1g/L；硫酸铵，1g/L；尿素，10g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.45g/L；FeSO 4·7H 2O，0.05g/L；生物素，0.4mg/L；维生素B1，0.1mg/L；MOPS，40g/L；初始pH7.2。培养基中补加25μg/mL卡那霉素。首先将菌株接种到TSB液体培养基中培养8h，培养物作为种子接种到每孔含有800μl发酵培养基的24孔板中，接种量为12μl，30℃培养18h，孔板摇床转速为800rpm，每个菌株3个平行，发酵结束后采用SBA生物传感分析仪检测L-赖氨酸产量，并采用酶标仪测定OD 600。结果如表5所示，P dapB-3启动子突变体过表达菌株的L-赖氨酸产量提高达30％。以上结果表明采用本公开的启动子突变体可用于增强PPC基因的表达，并应用于L-赖氨酸生产。
表5
菌株 OD 600 L-赖氨酸产量(g/L)
SCgL30(pEC-XK99E) 17.7±1.2 4.50±0.0
SCgL30(pEC-P dapB-3-ppc) 17.2±1.7 5.83±0.3
以上结果说明：本公开的dapB基因的启动子突变体可用于谷氨酸棒杆菌中增强PPC的表达，进而强化从磷酸烯醇式丙酮酸(PEP)到草酰乙酸的合成，其可应用至依赖于草酰乙酸前体供应的目标产物的生产，包括天冬氨酸家族氨基酸(赖氨酸、苏氨酸、异亮氨酸、蛋氨酸)，谷氨酸家族氨基酸(谷氨酸、脯氨酸、羟脯氨酸、精氨酸、谷氨酸酰胺)，以及5-氨基乙酰丙酸等以草酰乙酸为重要代谢前体的氨基酸的生物法生产。
由于增强PPC的表达和活性可以用于提高5-氨基乙酰丙酸等目标化合物的产量 [1]，而本公开的dapB基因启动子突变体都可以用于增强PPC的表达和活性，因此，本公开的启动子突变体也可以用于5-氨基乙酰丙酸生产。
本公开的启动子突变体已经证实可以DapB的N端与RFP融合蛋白的表达，可以用于提高PPC的表达，本公开的dapB基因的启动子突变体还可以用于表达其他基因，应用于各种产品的生产。
P dapB-WT(SEQ ID NO：1):
Figure PCTCN2022094734-appb-000008
P dapB-1(SEQ ID NO：2):
Figure PCTCN2022094734-appb-000009
P dapB-2(SEQ ID NO：3):
Figure PCTCN2022094734-appb-000010
P dapB-3(SEQ ID NO：4):
Figure PCTCN2022094734-appb-000011
引用文献：
[1]CN103981203A
[2]王迎春等，基于时间序列转录组筛选谷氨酸棒杆菌内源高效组成型启动子[J].生物工程学报，2018，34(11):1760～1771.
本说明书公开的所有技术特征都可以任何组合方式进行组合。本说明所公开的每个特征也可以被其它具有相同、相等或相似作用的特征所替换。因此，除非特殊说明，所公开的每一特征仅仅是一系列相等或相似特征的实例。
此外，从上述描述中，本领域技术人员可从本公开中很容易清楚本公开的关键特征，在不脱离本公开的精神及范围的情况下，可对发明进行很多修改以适应各种不同的使用目的及条件，因此这类修改也旨在落入所附权利要求书的范围内。

Claims (16)

  1. 一种具有启动子活性的多核苷酸，其中，所述多核苷酸选自如下(i)-(iv)组成的组中的任一项：
    (i)包含如SEQ ID NO：1所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：1所示序列的第75-95位中的一个或多个位置处具有突变的核苷酸；
    (ii)包含与(i)所示的核苷酸序列的反向互补序列的多核苷酸；
    (iii)包含在高严格性杂交条件或非常高严格性杂交条件下，能够与(i)或(ii)所示的核苷酸序列杂交的序列的反向互补序列的多核苷酸；
    (iv)与(i)或(ii)所示的核苷酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的多核苷酸；
    其中，(i)-(iv)任一项所示的多核苷酸在SEQ ID NO：1所示序列的第75-95位的核苷酸序列不为TCTGAACGGGTACGTCTAGAC；并且，与SEQ ID NO：1所示序列的多核苷酸相比，(i)-(iv)任一项所示的多核苷酸具有增强的启动子活性。
  2. 根据权利要求1所述的具有启动子活性的多核苷酸，其中，所述突变体与SEQ ID NO：1所示序列的多核苷酸相比，具有3-12倍以上的提高的启动子活性。
  3. 根据权利要求1-2任一项所述具有启动子活性的多核苷酸，其中，所述突变体对应SEQ ID NO：1所示序列的第75-95位的核苷酸序列选自如下(P dapB-1)-(P dapB-3)组成的组中的任一项：
    (P dapB-1)CTCTGATGTGATAGTATAATT；
    (P dapB-2)ATCATTTGGTGTATACTAAAT；
    (P dapB-3)GTCCTGTGGTAAACTTTAGCG。
  4. 根据权利要求1-3任一项所述的具有启动子活性的多核苷酸，其中，所述突变体的核苷酸序列选自如SEQ ID NO：2-4任一项所示的序列。
  5. 一种转录表达盒，其中，所述转录表达盒包含根据权利要求1-4任一项所述的具有启动子活性的多核苷酸；可选地，所述转录表达盒还含有目标基因，所述目标基因与所述具有启动子活性的多核苷酸可操作地连接；优选地，所述目标基因为蛋白编码基因。
  6. 一种重组表达载体，其中，所述重组表达载体包含权利要求1-4任一项所述的具有启动子活性的多核苷酸，或权利要求5所述的转录表达盒。
  7. 一种重组宿主细胞，其中，所述重组宿主细胞包含权利要求5所述的转录表达盒，或权利要求6所述的重组表达载体。
  8. 根据权利要求7所述的重组宿主细胞，其中，所述宿主细胞来源于棒状杆菌属、短杆菌属、节杆菌属、微杆菌属或埃希氏菌属；优选地，所述宿主细胞为谷氨酸棒杆菌或大肠杆菌；更优选地，所述宿主细胞为谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869、谷氨酸棒杆菌ATCC 14067或谷氨酸棒杆菌的衍生菌株。
  9. 一种根据权利要求1-4任一项所述的具有启动子活性的多核苷酸，根据权利要求5所述的转录表达盒，根据权利要求6所述的重组表达载体，根据权利要求7或8所述的重组宿主细胞在如下至少一种中的用途：
    (a)增强基因的转录水平，或制备用于增强基因的转录水平的试剂或试剂盒；
    (b)制备蛋白，或制备用于制备蛋白的试剂或试剂盒；
    (c)生产目标化合物，或制备用于生产目标化合物的试剂或试剂盒。
  10. 根据权利要求9所述的用途，其中，所述蛋白选自基因表达调控蛋白、与目标化合物合成相关的蛋白或与膜转运相关的蛋白。
  11. 根据权利要求9或10所述的用途，其中，所述目标化合物包括氨基酸、有机酸中的至少一种；
    可选地，所述氨基酸包括如下的一种或两种以上的组合：脯氨酸、羟脯氨酸、赖氨酸、谷氨酸、苏氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、5-氨基乙酰丙酸或上述任一种的氨基酸的衍生物；
    可选地，所述有机酸包括如下的一种或两种以上的组合：柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、草酰乙酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸或上述任一种的有机酸的衍生物。
  12. 一种启动子突变体的构建方法，其中，所述构建方法包括如下步骤：
    突变步骤：对SEQ ID NO：1所示序列的多核苷酸进行突变，使SEQ ID NO：1所示序列的第75-95位中的一个或多个位置处具有突变的核苷酸；
    筛选步骤：筛选与SEQ ID NO：1所示序列的多核苷酸相比，启动子活性提高的多核苷酸的突变体，得到启动子突变体。
  13. 根据权利要求12所述的构建方法，其中，所述突变步骤包括：对SEQ ID NO：1所示序列的多核苷酸进行突变，使SEQ ID NO：1所示序列的第75-95位的核苷酸突变为如下所示的核苷酸序列：NNNNNNNNNNNNNNNNTANNN；其中，N选自A，T，C或G；
    优选地，与SEQ ID NO：1所示序列的多核苷酸相比，启动子突变体具有3-12倍以上的提高的启动子活性。
  14. 一种调控转录的方法，其中，所述方法包括将权利要求1-4任一项所述的具有启动子活性的多核苷酸与目标RNA或目标基因可操作地连接的步骤；可选地，所述目标RNA包括tRNA、sRNA中的至少一种，所述目标基因包括与目标化合物合成相关的蛋白的编码基因、基因表达调控蛋白的编码基因、与膜转运相关的蛋白的编码基因中的至少一种；
    可选地，所述目标基因包括如下的至少一种：丙酮酸羧化酶基因、磷酸烯醇式丙酮酸羧化酶基因、γ-谷氨酰激酶基因、谷氨酸半醛脱氢酶基因、吡咯啉-5-羧酸还原酶基因、氨基酸运输蛋白基因、ptsG系统相关基因、丙酮酸脱氢酶基因、高丝氨酸脱氢酶基因、草酰乙酸脱羧酶基因、葡萄糖酸阻遏蛋白基因、葡萄糖脱氢酶基因、天冬氨酸激酶基因、天冬氨酸半醛脱氢酶基因、天冬氨酸氨裂合酶基因、二氢吡啶二羧酸合成酶基因、二氢吡啶甲酸还原酶基因、琥珀酰二氨基庚二酸氨基转移酶基因、四氢吡啶二羧酸酯琥珀酰酶基因、琥珀酰二氨基庚二酸脱酰基酶基因、二氨基庚二酸差向异构酶基因、二氨基庚二酸脱酰基酶基因、甘油醛-3-磷酸脱氢酶基因、转酮酶基因、二氨基庚二酸脱氢酶基因。
  15. 一种制备蛋白的方法，其中，所述方法包括利用权利要求5所述的转录表达盒，权利要求6所述的重组表达载体，或权利要求7-8任一项所述的重组宿主细胞表达所述蛋白的步骤；可选地，所述蛋白为与目标化合物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白；
    任选地，所述方法还包括分离或纯化所述蛋白的步骤。
  16. 一种生产目标化合物的方法，其中，所述方法包括利用权利要求5所述的转录表达盒，权利要求6所述的重组表达载体，或权利要求7-8任一项所述的重组宿主细胞表达与目标化合物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白，在所述与目标化合物合成相关的蛋白、与膜转运相关的蛋白或所述基因表达调控蛋白存在的环境下生产目标化合物的步骤；
    可选地，所述目标化合物包括氨基酸、有机酸中的至少一种；
    可选地，所述氨基酸包括如下的一种或两种以上的组合：赖氨酸、谷氨酸、苏氨酸、脯氨酸、羟脯氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、5-氨基乙酰丙酸或上述任一种的氨基酸的衍生物；
    可选地，所述有机酸包括如下的一种或两种以上的组合：柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、草酰乙酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸或上述的任一种的有机酸的衍生物；
    可选地，所述与目标化合物合成相关的蛋白为与L-氨基酸合成相关的蛋白；可选地，所与L-氨基酸合成相关的蛋白包括丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、γ-谷氨酰激酶、谷氨酸半醛脱氢酶、吡咯啉-5-羧酸还原酶、氨基酸运输蛋白、ptsG系统、丙酮酸脱氢酶、高丝氨酸脱氢酶、草酰乙酸脱羧酶、葡萄糖酸阻遏蛋白、葡萄糖脱氢酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、天冬氨酸氨裂合酶、二氢吡啶二羧酸合成酶、二氢吡啶二羧酸还原酶、二氢吡啶甲酸还原酶、琥珀酰二氨基庚二酸氨 基转移酶、四氢吡啶二羧酸酯琥珀酰酶、琥珀酰二氨基庚二酸脱酰基酶、二氨基庚二酸差向异构酶、二氨基庚二酸脱酰基酶、甘油醛-3-磷酸脱氢酶、转酮酶、二氨基庚二酸脱氢酶中的一种或两种以上的组合；
    任选地，所述方法还包括分离或纯化所述目标化合物的步骤。
PCT/CN2022/094734 2021-06-08 2022-05-24 基于dapB基因的具有启动子活性的多核苷酸及其用途 WO2022257757A1 (zh)

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