WO2022037338A1 - 具有启动子活性的多核苷酸及其在生产氨基酸中的应用 - Google Patents

具有启动子活性的多核苷酸及其在生产氨基酸中的应用 Download PDF

Info

Publication number
WO2022037338A1
WO2022037338A1 PCT/CN2021/106501 CN2021106501W WO2022037338A1 WO 2022037338 A1 WO2022037338 A1 WO 2022037338A1 CN 2021106501 W CN2021106501 W CN 2021106501W WO 2022037338 A1 WO2022037338 A1 WO 2022037338A1
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
gene
promoter activity
amino acids
lysine
Prior art date
Application number
PCT/CN2021/106501
Other languages
English (en)
French (fr)
Inventor
郑平
刘娇
孙际宾
周文娟
石拓
郭轩
马延和
Original Assignee
中国科学院天津工业生物技术研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院天津工业生物技术研究所 filed Critical 中国科学院天津工业生物技术研究所
Priority to EP21857433.3A priority Critical patent/EP4202046A1/en
Priority to US18/020,965 priority patent/US20230399667A1/en
Priority to CA3188476A priority patent/CA3188476A1/en
Publication of WO2022037338A1 publication Critical patent/WO2022037338A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01016—Diaminopimelate dehydrogenase (1.4.1.16)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the present disclosure belongs to the fields of molecular biology and bioengineering, 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 an enhanced target gene Methods of expression, methods of making proteins, and methods of producing amino acids.
  • lysine is 2,6-diaminopimelic acid, which is an essential amino acid for animals and humans. It can promote human development, enhance immunity, and improve the function of central nervous tissue. Lysine has three chemical optical isomers: L-type (left-handed), D-type (right-handed) and DL-type (racemic), of which only the L-type can be used by biology, commonly known as lysine.
  • the acid is L-lysine.
  • L-Lysine is one of the 20 common amino acids that make up proteins, and it belongs to the same basic amino acid as histidine and arginine. Since human and animal bodies cannot synthesize L-lysine by themselves, they can only be obtained from food, which is one of the eight essential amino acids. The content of L-lysine in human staple grain food is relatively low. L-lysine can cause protein metabolism and dysfunction, adversely affect growth, and is easily destroyed during processing, so it is called the first limiting amino acid. It has a very important position in the industries of medicine, health, food, animal feed and cosmetics.
  • the L-lysine biosynthetic pathway can be divided into two completely different pathways, the aminooxalic acid pathway (AAA) and the diamino acid pathway The pimelic acid pathway (DAP).
  • AAA aminooxalic acid pathway
  • DAP diamino acid pathway
  • the diaminopimelic acid pathway (DAP) is a part of the aspartic acid amino acid synthesis pathway.
  • the DAP pathway has a succinylase pathway for synthesizing meso-diaminopimelic acid, The acetylase pathway and the transaminase pathway, and the dehydrogenase pathway for the direct synthesis of racemic diaminopimelate without going through mesodiaminopimelate.
  • Corynebacterium glutamicum is an important industrial microorganism, and its advantage lies in its ability to ferment and produce amino acids on an industrial scale. Corynebacterium glutamicum uses the dehydrogenase pathway to synthesize L-lysine, and there are 6 enzyme-catalyzed reactions, namely aspartate kinase (AK, encoded by the gene lysC), aspartate semialdehyde dehydrogenase (ASADH) , encoded by the gene asd), dihydrodipicolinate synthase (DHDPS, encoded by the gene dapA), dihydrodipicolinate reductase (DHDPR, encoded by the gene dapB), diaminopimelate dehydrogenase ( DAPDH, encoded by the gene ddh) and diaminopimelate decarboxylase (DAPDC, encoded by the gene lysA).
  • AK aspartate kinase
  • ASADH aspartate
  • Citation 1 discloses a coryneform bacterium which, in addition to having at least one copy of an open reading frame (ORF), gene or allele encoding protein or RNA synthesis at its natural site (locus), must also The open reading frame (ORF), gene having a second, alternatively third or fourth copy of the open reading frame (ORF), gene or alleles.
  • ORF open reading frame
  • ORF open reading frame
  • ORF open reading frame
  • ORF open reading frame
  • ORF open reading frame
  • ORF open reading frame
  • ORF open reading frame
  • ORF open reading frame
  • ORF open reading frame
  • gene or alleles gene or alleles.
  • the Corynebacterium of this method improves the gene expression level by increasing the gene copy number, thereby improving the yield of amino acid produced by the Corynebacterium.
  • the increase of gene copy number will reduce the stability of the strain genome, and cannot guarantee the stable and efficient production of L-lysine.
  • Citation 2 discloses a method for producing L-lysine by culturing the following Escherichia coli having L-lysine-producing ability in a medium, and collecting L-lysine from the medium to produce L-Lysine, the E.
  • coli has been modified to reduce one or more enzymes of the meso- ⁇ , ⁇ -diaminopimelate synthesis pathway, such as 2,3,4,5-tetrahydro the activity of pyridine-2,6-dicarboxylic acid N-succinyltransferase, succinyldiaminopimelate transaminase, succinyldiaminopimelate desuccinylase or diaminopimelate epimerase, And a gene encoding diaminopimelate dehydrogenase was introduced therein.
  • enzymes of the meso- ⁇ , ⁇ -diaminopimelate synthesis pathway such as 2,3,4,5-tetrahydro the activity of pyridine-2,6-dicarboxylic acid N-succinyltransferase, succinyldiaminopimelate transaminase, succinyldiaminopimelate desuccinylase or diaminopimelate epimerase, And a
  • This method needs to increase the expression of diaminopimelate dehydrogenase while inhibiting the enzymatic activity of the meso- ⁇ , ⁇ -diaminopimelate synthesis pathway, and has the disadvantages of complicated operation process, high operation cost, and inability to efficiently produce L -Lysine problem.
  • Citation 3 discloses a nucleic acid molecule operably linked to a gene encoding diaminopimelate dehydrogenase, which can increase the enzymatic activity of diaminopimelate dehydrogenase, thereby increasing the L-lysine of the strain Amino acid production.
  • the nucleic acid molecules in this document have higher activity than wild-type promoters, it is not known whether there are nucleic acid molecules with higher promoter activity. It is well known to those skilled in the art that the higher the activity of diaminopimelate dehydrogenase, the better the production of lysine. Therefore, the development of a promoter with higher activity will help to enhance the expression of diaminopimelate dehydrogenase, Thereby more industrial application potential.
  • the Corynebacterium bacterium that improves yield by increasing the number of gene copies has poor genome stability, and the transformation process of the strain with L-lysine production capacity is complicated, and it is impossible to stably and efficiently produce L - Defects in lysine.
  • the present disclosure provides a polynucleotide with promoter activity. Compared with the promoter of the wild-type ddh gene, the promoter activity of the polynucleotide is significantly improved. By operably linking the polynucleotide with promoter activity to the target gene, the expression intensity of the target gene can be improved under the condition of maintaining the stability of the genome.
  • a polynucleotide with promoter activity wherein the polynucleotide is selected from the group shown in any one of the following (i)-(ii):
  • (ii) has at least 90%, alternatively at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence set forth in (i), excluding SEQ ID NO: 9 A polynucleotide of the indicated sequence;
  • the promoter activity of the mutant is higher than that of the polynucleotide of the sequence shown in SEQ ID NO: 9;
  • the nucleotide sequence of the mutant at positions 292-300 of the sequence shown in SEQ ID NO: 9 is not selected from ATGCATTGT.
  • a transcription expression cassette comprising the polynucleotide with promoter activity according to any one of (1)-(4); optionally, the transcription expression cassette further contains a protein-coding gene, and the The protein-coding gene is operably linked to the polynucleotide having promoter activity.
  • a recombinant expression vector comprising the polynucleotide having promoter activity according to any one of (1) to (4), or the transcription expression cassette according to claim 5.
  • a recombinant host cell comprising the transcription expression cassette described in (5), or the recombinant expression vector described in (6).
  • amino acid and its derivatives are selected from the following one or a combination of two or more: proline, hydroxyproline, lysine, glutamic acid, arginine, ornithine, glutamine Acid amide, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, methionine, aspartic acid, asparagine, histidine , phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid or derivatives of any of the above amino acids;
  • the protein is an enzyme involved in synthesizing amino acids; preferably, the enzyme involved in synthesizing amino acids is diaminopimelate dehydrogenase;
  • the amino acid includes L-lysine and derivatives thereof, wherein the derivatives include at least one of pentamethylenediamine, 5-aminovaleric acid and glutaric acid.
  • a method for enhancing the expression of a target gene comprising combining the polynucleotide with promoter activity according to any one of claims (1) to (4) to be operable with a target RNA or a target gene
  • the target RNA includes at least one of tRNA and sRNA
  • the target gene includes the encoding gene of the protein related to the synthesis of the target compound, the encoding gene of the gene expression regulatory protein, and the membrane transporter. At least one of the genes encoding the related proteins.
  • a method for preparing a protein wherein the transcriptional expression cassette described in (5), the recombinant expression vector described in (6), or the recombinant host cell described in any one of (7)-(8) are selected Expressing the protein; alternatively, the protein is a protein related to the synthesis of the target product, a protein related to membrane transport, or a gene expression regulatory protein; alternatively, the protein is an enzyme involved in the synthesis of L-lysine ;
  • the enzymes involved in the synthesis of L-lysine include aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, Dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase , one or
  • a method for producing amino acids and derivatives thereof wherein the transcription expression cassette described in (5), the recombinant expression vector described in (6), or any one of (7) to (8) are selected.
  • the recombinant host cell expresses the enzymes involved in synthesizing amino acids and derivatives thereof, and produces the amino acids and derivatives thereof with the enzymes involved in synthesizing amino acids and derivatives thereof; alternatively, the amino acids and derivatives thereof are selected from the following One or more combinations of: proline, hydroxyproline, lysine, glutamic acid, arginine, ornithine, glutamic acid amide, threonine, glycine, alanine, Valine, Leucine, Isoleucine, Serine, Cysteine, Methionine, Aspartic Acid, Asparagine, Histidine, Phenylalanine, Tyrosine, Tryptophan , 5-aminolevulinic acid or a derivative of any of the above amino acids;
  • the amino acid includes L-lysine and derivatives thereof, wherein the derivatives include at least one of pentamethylenediamine, 5-aminovaleric acid and glutaric acid;
  • the enzyme involved in synthesizing amino acids is diaminopimelate dehydrogenase.
  • the present disclosure provides a polynucleotide having promoter activity that is a mutant of a diaminopimelate dehydrogenase gene (ddh gene) promoter with significantly increased promoter activity.
  • ddh gene diaminopimelate dehydrogenase gene
  • the mutant promoter activity is significantly improved, and operably linking it with the target gene can significantly improve the expression intensity of the target gene without destroying the stability of the genome, enabling the target gene to Stable and high-efficiency expression, and then stable and efficient production of downstream products.
  • the present disclosure provides transcriptional expression cassettes, recombinant expression vectors, and recombinant host cells comprising the above-described polynucleotides having promoter activity.
  • the polynucleotide with promoter activity is operably linked to the protein-coding gene, which can improve the expression intensity of the protein-coding gene.
  • the present disclosure provides a method for producing amino acids, using the above-mentioned polynucleotides with promoter activity to improve the expression of enzymes that synthesize amino acids, thereby producing amino acids stably and efficiently.
  • a method for producing amino acids using the above-mentioned polynucleotides with promoter activity to improve the expression of enzymes that synthesize amino acids, thereby producing amino acids stably and efficiently.
  • Figure 1 shows a schematic diagram of the pEC-XK99E-P ddh -rfp plasmid
  • Figure 2 shows the results of fluorescence display after the mutant clones are grown on the culture plate.
  • the arrows in the figure indicate the position of a single clone with high-intensity red fluorescence.
  • 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.
  • 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.
  • 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 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.
  • mutation in the present disclosure refers to a nucleotide comprising a mutation at one or more (eg, several) positions in a polynucleotide and maintains the promoter activity of the polynucleotide.
  • the mutation in the present disclosure refers specifically to the substitution therein, and the substitution refers to the replacement of a nucleotide occupying a position with a different nucleotide.
  • Deletion refers to the removal of a nucleotide occupying a position.
  • Insertion refers to the addition of a nucleotide adjacent to and immediately following the nucleotide occupying the position.
  • mutants of the present disclosure comprise substituted nucleotides at one or more positions of positions 292-300 of the sequence set forth in SEQ ID NO: 9, and do not comprise SEQ ID NO: : Nucleotides substituted by ATGCATTGT at positions 292-300 of the sequence shown in 9.
  • the mutants in which the nucleotides at the above-mentioned positions are substituted have higher promoter activity than the promoter activity of the polynucleotide of the sequence shown in SEQ ID NO: 9.
  • the "mutations" of the present disclosure comprise 1, 2, 3, 4, 5, 6, 7, 8 of positions 292-300 of the sequence shown in SEQ ID NO: 9 or substituted nucleotides at 9 positions.
  • the "mutations" of the present disclosure are comprised at positions 4, 5, 6, 7, 8, or 9 of positions 292-300 of the sequence set forth in SEQ ID NO:9
  • the nucleotides comprising the mutation preferably comprise mutated nucleotides at positions 6, 7, 8 or 9 of positions 292-300 of the sequence shown in SEQ ID NO:9.
  • the mutants in which the nucleotides at the above positions are substituted have higher promoter activity than those in which the nucleotides are substituted at other positions.
  • 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.
  • the polynucleotide having promoter activity comprises at least 90%, 91%, 92%, 93%, 94%, 90%, 91%, 92%, 93%, 94%, Sequences of 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • having a specific percentage of sequence identity refers to the presence of a mutant sequence in a mutant of a polynucleotide with the sequence shown in SEQ ID NO: 9 that can maintain or increase the transcriptional activity of the mutant.
  • promoter in this 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 gene of interest.
  • RNA ribonucleic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Protein-coding genes contain CDS sequences (Coding Sequences) that can direct the production of protein-coding mRNAs.
  • Protein-coding genes include, but are not limited to, enzymes encoding enzymes involved in the synthesis of amino acids, and in some embodiments, protein-encoding genes involve enzymes encoding enzymes involved in the synthesis of L-lysine.
  • L-lysine For enzymes involved in the synthesis of L-lysine, including aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydropicolinate Reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminoheptane One or a combination of two or more of diacid deacylase, glyceraldehyde-3-phosphate dehydrogenase, lysine transporter, transketolase, diaminopimelate dehydrogenase and pyruvate carboxylase .
  • the polynucleotides with promoter activity disclosed in the present disclosure can be suitable for regulating the expression of target genes, so as to achieve efficient
  • 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.
  • transcriptional regulatory elements include promoters, and on this basis, elements such as enhancers, silencers, and insulators may also be included.
  • 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 with 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.
  • vector in the present disclosure refers to a DNA construct containing 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.
  • vectors are 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 vector involved in the present disclosure is the ddh gene promoter strength characterizing plasmid pEC-XK99E-P ddh -rfp constructed based on the pEC-XK99E-rfp plasmid [1] .
  • the plasmid map of pEC-XK99E-P ddh -rfp is shown in the figure. 1 shown.
  • P ddh is the promoter of the ddh gene; ddh is the wild-type diaminopimelate dehydrogenase gene; linker is the connecting peptide between the ddh gene and the rfp protein; rfp is the red fluorescent protein (Red Fluorescent Protein, RFP); Kan expressed as Kanamycin resistant (Kanamycin resistant).
  • pEC-XK99E-P ddh -rfp can replicate and function independently of the host genome, or in some cases integrate into the genome itself.
  • 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.
  • 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.
  • 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 (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol Alcohol (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.
  • 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.
  • the host cell is Corynebacterium glutamicum derived from the genus Corynebacterium.
  • Corynebacterium glutamicum can be Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 14067 or its derivative strains.
  • 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.
  • the present disclosure utilizes the promoter sequence of the wild-type diaminopimelate dehydrogenase gene (ddh) to mutate the core promoter sequence of the ddh gene to obtain a mutant of the diaminopimelate dehydrogenase gene promoter.
  • the promoter sequence of the diaminopimelate dehydrogenase gene is shown in SEQ ID NO: 9, and the mutant of the promoter of the diaminopimelate dehydrogenase gene is in the 292-300th of the sequence shown in SEQ ID NO: 9
  • the nucleotide sequence of the mutant at position 292-300 of the sequence shown in SEQ ID NO: 9 is not selected from ATGCATTGT.
  • mutant of the diaminopimelate dehydrogenase gene promoter is a polynucleotide with promoter activity; Mutants of the diaminopimelate dehydrogenase gene promoter have improved promoter activity compared to the promoter.
  • the polynucleotide having promoter activity has at least 90%, 91%, 92%, 93%, 94%, compared to the mutant sequence of the diaminopimelate dehydrogenase gene promoter %, 95%, 96%, 97%, 98%, 99%, 100% sequence identity (including all ranges and percentages between these values) and does not include the sequence set forth in SEQ ID NO:9.
  • the polynucleotide having promoter activity is associated with 4, 5, 6, 7, 8 or 9 of the sequence set forth in SEQ ID NO:9 at positions 292-300
  • a nucleotide containing a mutation at the position has a higher promoter activity than that of a polynucleotide of the sequence shown in SEQ ID NO: 9; preferably in the sequence shown in SEQ ID NO: 9 6, 7, 8 or 9 of positions 292-300 comprise mutated nucleotides.
  • the nucleotide sequence at positions 292-300 of the polynucleotide having promoter activity is selected from any of the following groups: (a) ACAAAAGGT; (b) TCTTCATCT; (c) GGAAAGTAT; (d) )TTATTATAT;(e)TAATCCTCT;(f)TCAATTTAT;(g)GCGCAATCT;(h)CAGTTCCGT;(i)AAGTTTTAT;(g)TAAATGTAT;(k)GGATTGTAT;(l)CAAACTCAT;(m)TACAAATCT;(n) )TATCAGGTCT; (o) CGAGGATAT; (p) CCTTGTTAT.
  • a polynucleotide having promoter activity comprises the sequence shown in any of SEQ ID NOs: 10-25.
  • the promoter activity of the mutant is enhanced by more than 18 times, preferably 19 times, preferably 20 times, preferably 22 times, preferably 23 times times, preferably 24 times, preferably 25 times, preferably 26 times, more preferably 27 times, more preferably 28 times, more preferably 30 times, more preferably 31 times or more.
  • a recombinant vector comprising the promoter of the diaminopimelate dehydrogenase gene is constructed first, and then the core promoter region of the ddh gene is mutated to obtain a polynucleotide comprising the promoter activity. recombinant expression vector.
  • primers ddh-F and ddh-R were designed according to the currently published genome information of Corynebacterium glutamicum, and the genome of Corynebacterium glutamicum was amplified by PCR with ddh-F and ddh-R, Obtain the promoter sequence of the ddh gene.
  • plasmid information to design primers pEC-F and pEC-R, using pEC-XK99E-rfp plasmid as template, using primers pEC-F and pEC-R to amplify to obtain pEC-XK99E backbone and connecting peptide and DsRed DNA fragments.
  • the amplified fragments of primers ddh-F and ddh-R are recombined with the amplified fragments of primers pEC-F and pEC-R to obtain a polynucleotide with promoter activity (that is, ddh gene promoter mutation) body sequence) recombinant expression vector.
  • Corynebacterium glutamicum ATCC 13032 Corynebacterium glutamicum ATCC 13032, Gene ID: 2830649.
  • a polynucleotide with promoter activity is operably linked to a gene encoding an enzyme involved in amino acid synthesis to obtain a recombinant expression vector capable of synthesizing an enzyme involved in amino acid synthesis, and the recombinant expression vector is used to transform host cells , to obtain recombinant host cells.
  • 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.
  • the polynucleotide has improved promoter activity
  • the transcriptional activity of the encoding gene of the enzyme involved in synthesizing amino acids is improved, and the expression level of the enzyme involved in amino acid synthesis is improved, thereby increasing the output of amino acids.
  • the amino acids include L-lysine and derivatives thereof.
  • the amino acid is selected from the following one or a combination of two or more: proline, hydroxyproline, lysine, glutamic acid, arginine, ornithine, glutamic acid amide, threonine acid, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, methionine, aspartic acid, asparagine, histidine, phenylalanine , tyrosine, tryptophan, 5-aminolevulinic acid or derivatives of any of the above amino acids.
  • the derivative includes at least one of pentamethylenediamine, 5-aminovaleric acid and glutaric acid.
  • the enzyme involved in the synthesis of amino acids is an enzyme involved in the synthesis of L-lysine; preferably, the enzyme involved in the synthesis of amino acids is diaminopimelate dehydrogenase.
  • the polynucleotide with promoter activity can significantly increase the transcriptional activity of diaminopimelate dehydrogenase, so that the expression level of diaminopimelate dehydrogenase can be significantly increased.
  • the host cells in the present disclosure can be any type of strain with the production capacity of the desired product, including wild-type strains and recombinant strains.
  • the host cells are derived from microorganisms suitable for the fermentative production of target products such as amino acids and derivatives thereof, such as Enterobacter, Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, and the like.
  • the host cell is Enterobacter or Corynebacterium, more preferably Corynebacterium glutamicum, including but not limited to Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum B253, Corynebacterium glutamicum ATCC 14067, and L-amino acid-producing derivative strains prepared from the above strains.
  • the host cell is Corynebacterium glutamicum (Corynebacterium glutamicum), which is an important strain for the production of L-lysine, using polynucleotides with promoter activity, transcription
  • Corynebacterium glutamicum Corynebacterium glutamicum
  • the expression level of the enzyme involved in the synthesis of L-lysine by Corynebacterium glutamicum is significantly increased, specifically, the expression level of diaminopimelate dehydrogenase is significantly increased.
  • the recombinant host cell is Corynebacterium glutamicum modified as follows: 1) the aspartokinase encoding gene in Corynebacterium glutamicum has introduced a T311I mutation coding sequence; 2) glutamic acid The core region from position 279 to position 317 of the pyruvate carboxylase gene promoter in Corynebacterium is CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG; 3) The polynucleotide with promoter activity is operable with the gene encoding diaminopimelate dehydrogenase ligated and then introduced into Corynebacterium glutamicum.
  • the Corynebacterium glutamicum transformed by the above method is a high-producing strain of L-lysine.
  • genes encoding aspartate kinase, pyruvate carboxylase, and diaminopimelate dehydrogenase can be isolated and inserted into expression vectors for transformation production, which can be independent of the host cell
  • the host cell replicates and expresses the enzyme, or in some cases integrates into the host cell's genome.
  • Methods for manipulating microorganisms are known in the art, such as Modern Methods in Molecular Biology (Online ISBN: 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).
  • the host cell can also be other kinds of amino acid producing strains.
  • the "amino acid producing strain” mentioned in the present disclosure refers to a strain that can produce amino acids and accumulate amino acids when bacteria are cultured in a medium, or can secrete amino acids into the medium, that is, can obtain extracellular free amino acids. .
  • it can be a naturally occurring amino acid-producing strain, or an amino acid-producing engineered strain obtained by genetic modification.
  • the host cell is a lysine-producing host cell.
  • the lysine-producing host cell can also include, but is not limited to, one or more genes selected from the group consisting of attenuated or reduced expression:
  • thrA gene encoding aspartate kinase I/homoserine dehydrogenase I bifunctional enzyme
  • the cadA gene encoding lysine decarboxylase.
  • the lysine-producing host cell may also include, but is not limited to, one or more genes selected from the group consisting of enhanced or overexpressed:
  • the dapA gene encoding the dihydrodipyridine synthase that relieves the feedback inhibition of lysine
  • dapB gene encoding dihydrodipicolinate reductase
  • dapD encoding tetrahydrodipicolinate succinylase and dapE encoding succinyldiaminopimelate deacylase
  • pntAB gene encoding niacinamide adenine dinucleotide transhydrogenase
  • lysE gene encoding the lysine transporter protein.
  • the host cell is a threonine-producing host cell.
  • the threonine-producing host cell is a strain expressing the feedback-inhibited aspartokinase LysC based on Corynebacterium glutamicum ATCC 13032.
  • the threonine-producing host cells can also be other strains of threonine-producing ability.
  • one or more genes selected from the following are enhanced or overexpressed in the threonine-producing host cell:
  • thrABC gene encoding the threonine operon
  • 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 L-threonine dehydratase ilvA gene.
  • the isoleucine-producing host cells may also be other species of strains capable of isoleucine production.
  • the host cell is an O-acetylhomoserine-producing host cell.
  • the O-acetylhomoserine-producing host cell is a strain that produces O-acetylhomoserine by inactivating O-acetylhomoserine (thiol)-lyase.
  • the host cell that produces O-acetylhomoserine can also be other species of strains that have the ability to produce O-acetylhomoserine.
  • 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 can also be other strains of methionine-producing ability.
  • the culture conditions of the recombinant host cells are: firstly inoculate Corynebacterium glutamicum into TSB liquid medium for 8 hours, and inoculate the culture as a seed to a 24-well plate of 800 ⁇ l fermentation medium/well , the initial OD was controlled at about 0.1, cultured at 30 °C for 17 h, and the plate shaker speed was 800 rpm.
  • the ingredients are: 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.
  • the ingredients are: glucose, 80g/L; yeast powder, 1g/L; soy peptone, 1g/L; NaCl, 1g/L; ammonium sulfate, 1g/L; urea, 10g/L; K2HPO4 3H2O , 1g/L; MgSO4 7H2O, 0.45g/L; FeSO4 7H2O, 0.05g/L; Biotin, 0.4mg/L; Vitamin B1, 0.1mg/L; MOPS, 40g/L; initial pH 7.2.
  • 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 and HPLC.
  • the experimental techniques and experimental methods used in the present embodiment 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.
  • Example 1 Construction of a plasmid to characterize the strength of the ddh gene promoter of Corynebacterium glutamicum
  • a characterization vector was first constructed, the ddh gene promoter and the ddh gene fragment were connected on the basis of the pEC-XK99E plasmid backbone, and the ddh gene promoter was used to express the ddh gene , linker peptide and red fluorescent protein (rfp) gene. details as follows:
  • the amplified fragment of the genome of Corynebacterium glutamicum ATCC13032 and the amplified fragment of the plasmid of pEC-XK99E-rfp were cloned and connected by Novozan's one-step recombination kit to obtain the pEC-XK99E-P ddh -rfp characterization vector, its plasmid
  • the spectrum is shown in Figure 1.
  • the primer sequences used above are shown in Table 1, and the linker peptide sequence information is shown in Table 2.
  • the core region of the ddh gene promoter of Corynebacterium glutamicum "TGATGAAAGAGATGTCCCTGAA TCATCA TCTAAGTATGCATCTCGG TAAGCT CGACCAGG" is mutated, and the underlined areas are the main sequences of the -35 and -10 regions of the promoter respectively.
  • the mutation "TGATGAAAGAGATGTCCCTGAA TCATCA TCTAAGTNNNNNNNNNGG TAAGCT CGACCAGG” was performed at the corresponding position of the above core region, and two fragments of the plasmid were amplified by primers ddh-M1, ddh-M2, ddh-M3, and ddh-M4, respectively.
  • One-step recombination kit was used for cloning and ligation. All cloned bacteria obtained were collected and plasmids were extracted to obtain ddh gene promoter mutant library.
  • Example 2 The above library, the prior art mutant pEC-XK99E-P ddh-x- rfp and the wild-type control pEC-XK99E-P ddh -rfp obtained in Example 1 were transformed into Corynebacterium glutamicum ATCC13032 respectively, and the TSB plate was coated, The plates with hundreds of clones were photographed by fluorescence imaging system, and mutants with increased expression intensity were initially screened according to the fluorescence brightness of clones.
  • FIG. 2 shows the fluorescent photos after the mutant clones are grown on the culture plate, and the position indicated by the arrow in the figure is a single clone with high-intensity red fluorescence.
  • composition of TSB plate medium is (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, 1g/L; MgSO 4 ⁇ 7H 2 O, 0.1g/L; Biotin, 0.01mg/L; Vitamin B1, 0.1mg/L; MOPS, 20g/L; Agar powder, 15g/L .
  • more than 10,000 clones were preliminarily screened, and about 20 mutants with significantly enhanced fluorescence intensity were obtained.
  • the primer sequences used above are shown in Table 3.
  • TSB liquid medium 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, 1g/L; MgSO 4 ⁇ 7H 2 O, 0.1g/L; Biotin, 0.01mg/ L; Vitamin B1, 0.1mg/L; MOPS, 20g/L.
  • the strains obtained by screening, the wild-type control, and the prior art control were inoculated into a 96-well plate containing 200 ⁇ l of TSB liquid medium in each well with a toothpick, 3 parallel for each strain, and the plate shaker was rotated at 800 rpm and 30° C. After culturing for 24 hours, the fluorescence intensity of the strains was detected, and the strains whose fluorescence intensity was higher than that of the wild-type control were sequenced. Some of the promoter mutants have the same sequence, and finally 16 different promoter mutants with significantly improved expression intensity were successfully obtained. The results are shown in Table 4.
  • the promoters obtained by the present invention are sequentially numbered from P ddh -1 to P ddh - 16. The promoter activity of P ddh -1 to P ddh -16 was increased by about 18-31 times, which provided abundant elements for the expression of genes such as ddh.
  • ATCC13032 aspartate kinase encoding gene introduced T311I (base mutated from ACC to ATC) amino acid mutation, the core region of pyruvate carboxylase gene promoter from 279th to 317th was mutated to CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG strain), painted The transformants of the first recombination were obtained by culturing them on LBHIS solid medium containing 5g/L glucose and 25 ⁇ g/mL kanamycin at 30°C.
  • the correct primary recombinant transformants were respectively inoculated with LB medium containing 5g/L glucose, cultured overnight, diluted and coated with LB solid medium plates added with 100g/L sucrose for screening, and the obtained ddh promoter mutant strains SCgL38, SCgL39 and SCgL40.
  • the fermentation medium components were: glucose, 80g/L; yeast Powder, 1g/L; Soy 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, 0.45 g/L; FeSO4 ⁇ 7H 2 O, 0.05 g/L; biotin, 0.4 mg/L; vitamin B1, 0.1 mg/L; MOPS, 40 g/L; initial pH 7.2.
  • the strains were inoculated into TSB liquid medium for 8 hours, and the culture was inoculated as seeds into a 24-well plate containing 800 ⁇ l of fermentation medium per well. 800 rpm, 3 parallels per strain, L-lysine production and glucose consumption were detected after fermentation, and the conversion rate of sugar and acid from glucose to L-lysine was calculated.
  • Table 6 The results are shown in Table 6. The lysine production and the sugar-acid conversion rate of the strain after the mutation of the ddh promoter are both improved, and the improvement is more significant with the enhancement of the promoter strength. The above results suggest that mutants that enhance the expression strength of the ddh promoter can be applied to L-lysine production.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了具有启动子活性的多核苷酸及其在生产氨基酸中的应用。还公开了含有该多核苷酸的转录表达盒、重组表达载体、重组宿主细胞，以及增强目标基因表达的方法、制备蛋白的方法和生产氨基酸的方法。该具有启动子活性的多核苷酸是SEQ ID NO：9所示序列的多核苷酸的突变体，与SEQ ID NO：9所示序列的多核苷酸相比，突变体的启动子活性显著增强，能够促使目标基因稳定高效表达。

Description

具有启动子活性的多核苷酸及其在生产氨基酸中的应用 技术领域
本公开属于分子生物学和生物工程领域，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，以及增强目标基因表达的方法、制备蛋白的方法和生产氨基酸的方法。
背景技术
赖氨酸(lysine)的化学名称是2,6-二氨基庚二酸，是动物和人体必需氨基酸，能促进人体发育、增强免疫能力，并有提高中枢神经组织功能的作用。赖氨酸有L-型(左旋)、D-型(右旋)和DL型(消旋型)三种化学旋光异构体，其中只有L型才能为生物所利用，通常所说的赖氨酸即为L-赖氨酸。
L-赖氨酸是常见的组成蛋白质的20种氨基酸之一，与组氨酸和精氨酸同属于碱性氨基酸。由于人体和动物体不能靠自身合成L-赖氨酸，只能从食物中获取，属于八大必须氨基酸之一。L-赖氨酸在人类主食谷物食品中的含量较低，缺乏会引起蛋白质代谢及功能障碍，对生长造成不利影响，并且在加工过程中容易被破坏，故被称为第一限制性氨基酸，在医药、健康、食品、动物饲料和化妆品等行业中有着十分重要的地位。
目前为止，工业上生产L-赖氨酸的方法主要有三种：蛋白水解法、化学合成法和微生物发酵法。其中微生物发酵法具有生产成本低、生产强度高、高特异性和对环境污染小等优点而成为当今工业生产L-赖氨酸应用最广泛的方法。棒状细菌和埃希氏菌在L-赖氨酸的工业化生产中获得了广泛应用，常用的埃希氏菌如大肠杆菌(Escherichia coli)，常用的棒状细菌有棒状杆菌属的谷氨酸棒杆菌(Corynebacterium glutamicum)，短杆菌属的黄色短杆菌(Brevibacterium flavum)、乳酸发酵短杆菌(Brevibacterium lactofermentus)，以及节杆菌属的某些种和微杆菌属的某些种。
在已知的具有L-赖氨酸生物合成途径的微生物和植物中，可以将L-赖氨酸生物合成途径划分为两个完全不同的途径，即氨基乙二酸途径(AAA)和二氨基庚二酸途径(DAP)。二氨基庚二酸途径(DAP)是天冬氨酸族氨基酸合成途径中的一部分，以天冬氨酸为底物，DAP途径存在合成内消旋二氨基庚二酸的琥珀酰化酶途径、乙酰酶途径和转氨酶途径，以及不经过内消旋二氨基庚二酸直接合成外消旋二氨基庚二酸的脱氢酶途径。
谷氨酸棒杆菌是一种重要的工业微生物，其优势在于能发酵生产工业化规模的氨基酸。谷氨酸棒杆菌利用脱氢酶途径合成L-赖氨酸有6个酶催化反应，分别是天冬氨酸激酶(AK，由基因lysC编码)、天冬氨酸半醛脱氢酶(ASADH，由基因asd编码)、二氢吡啶二羧酸合酶(DHDPS，由基因dapA编码)、二氢吡啶二羧酸还原酶(DHDPR，由基因dapB编码)、二氨基庚二酸脱氢酶(DAPDH，由基因ddh编码)和二氨基庚二酸脱羧酶(DAPDC，由基因lysA编码)。谷氨酸棒杆菌利用琥珀酰化酶途径合成L-赖氨酸的过程中还涉及合成内消旋二氨基庚二酸的四种酶：琥珀酰二氨基庚二酸氨基转移酶(由基因dapD编码)，四氢吡啶二羧酸酯琥珀酰酶(由基因dapC编码)，琥珀酰二氨基庚二酸脱酰基酶(由基因dapE编码)及二氨基庚二酸差向异构酶(由基因dapF编码)。众所周知，增强L-赖氨酸合成途径中的一个或多个基因的表达，能够有效提高L-赖氨酸的产量。
引用文献1公开了一种棒杆菌细菌，所述细菌除在其天然位点(座位)具有至少一个拷贝的编码蛋白质或RNA合成的可读框(ORF)、基因或等位基因外，一定还在第二个、可选地在第三个或第四个位点以整合入染色体的形式具有第二个、可选地第三个或第四个拷贝的该可读框(ORF)、基因或等位基因。该方法的棒杆菌细菌通过增加基因拷贝数的 方式来提高基因表达水平，进而提高棒杆菌细菌生产氨基酸的产量。但基因拷贝数的增加会降低菌种基因组的稳定性，无法保证L-赖氨酸的稳定高效生产。
引用文献2公开了一种L-赖氨酸的生产方法，通过在培养基中培养下述具有L-赖氨酸生产能力的大肠杆菌，并从该培养基收集L-赖氨酸，来生产L-赖氨酸，所述大肠杆菌经过修饰而降低了内消旋α,ε-二氨基庚二酸合成途径的1种或2种以上的酶，例如2,3,4,5-四氢吡啶-2，6-二羧酸N-琥珀酰转移酶、琥珀酰二氨基庚二酸转氨酶、琥珀酰二氨基庚二酸脱琥珀酰基酶或二氨基庚二酸差向异构酶的活性，并且其中导入了编码二氨基庚二酸脱氢酶的基因。该方法需要在抑制内消旋α,ε-二氨基庚二酸合成途径的酶活性的同时，提高二氨基庚二酸脱氢酶的表达，存在操作过程复杂，操作成本高，无法高效生产L-赖氨酸的问题。
引用文献3公开了一种核酸分子，其被可操作地连接到编码二氨基庚二酸脱氢酶的基因，可以增加二氨基庚二酸脱氢酶的酶活，从而增加菌株的L-赖氨酸产量。虽然该文献中的核酸分子与野生型启动子相比具有更高的活性，但尚不知晓是否存在具备更高启动子活性的核酸分子。本领域技术人员公知二氨基庚二酸脱氢酶的活性越高越利于赖氨酸的生产，因此，开发活性更高的启动子，将有利于增强二氨基庚二酸脱氢酶的表达，从而更具工业应用潜力。
引用文献：
引用文献1：CN1748031A
引用文献2：CN101765659A
引用文献3：CN101939432A
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：通过增加基因拷贝数来提高产量的棒杆菌细菌存在基因组稳定性差，具有L-赖氨酸生产能力的菌株的改造过程复杂，无法稳定、高效生产L-赖氨酸的缺陷。本公开提供了一种具有启动子活性的多核苷酸，与野生型ddh基因的启动子相比，多核苷酸的启动子活性显著提高。将具有启动子活性的多核苷酸与目标基因可操作地连接，可以在保持基因组稳定性的条件下提高目标基因的表达强度。
用于解决问题的方案
(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)-(ii)中任一项所示的组：
(i)如SEQ ID NO：9所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：9所示序列的第292-300位的一个或多个位置处包含突变的核苷酸；
(ii)与(i)所示序列具有至少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，且不包括SEQ ID NO：9所示序列的多核苷酸；
其中，所述突变体的启动子活性与如SEQ ID NO：9所示序列的多核苷酸的启动子活性相比，具有更高的启动子活性；并且，
所述突变体在SEQ ID NO：9所示序列的第292-300位的核苷酸序列不选自ATGCATTGT。
(2)根据(1)所述的具有启动子活性的多核苷酸，其中，所述突变体的启动子活性与如SEQ ID NO：9所示序列的多核苷酸的启动子活性相比，具有18倍以上的增强的启动子活性。
(3)根据(1)或(2)所述的具有启动子活性的多核苷酸，其中，所述突变体在SEQ ID NO：9所示序列的第292-300位的4个、5个、6个、7个、8个或9个位置处包含突变的核苷酸；优选地，所述突变体在SEQ ID NO：9所示序列的第292-300位的6个、7个、8个或9 个位置处包含突变的核苷酸。
(4)根据(1)-(3)任一项所述的具有启动子活性的多核苷酸，其中，所述具有启动子活性的多核苷酸的第292-300位的核苷酸序列选自如下任一组：
(a)ACAAAAGGT；
(b)TCTTCATCT；
(c)GGAAAGTAT；
(d)TTATTATAT；
(e)TAATCCTCT；
(f)TCAATTTAT；
(g)GCGCAATCT；
(h)CAGTTCCGT；
(i)AAGTTTTAT；
(g)TAAATGTAT；
(k)GGATTGTAT；
(l)CAAACTCAT；
(m)TACAAATCT；
(n)TATCAGTCT；
(o)CGAGGATAT；
(p)CCTTGTTAT。
(5)一种转录表达盒，其中，包含(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蛋白编码基因，所述蛋白编码基因与所述具有启动子活性的多核苷酸可操作地连接。
(6)一种重组表达载体，其中，包含(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，包含(5)所述的转录表达盒，或者(6)所述的重组表达载体。
(8)根据(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；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或其衍生菌株。
(9)一种根据(1)-(4)任一项所述的具有启动子活性的多核苷酸、(5)所述的转录表达盒、(6)所述的重组表达载体，或者(7)-(8)任一项所述的重组宿主细胞在制备用于增强基因转录水平的试剂或试剂盒中的应用。
(10)一种根据权利要求(6)所述的重组表达载体，或(7)-(8)任一项所述的重组宿主细胞在制备蛋白或生产氨基酸及其衍生物中的应用；可选地，所述氨基酸及其衍生物选自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、精氨酸、鸟氨酸、谷氨酸酰胺、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、甲硫氨酸、天冬氨酸、天冬酰胺、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；
优选地，所述蛋白为参与合成氨基酸的酶；优选地，所述参与合成氨基酸的酶为二氨基庚二酸脱氢酶；
优选地，所述氨基酸包括L-赖氨酸及其衍生物，其中，所述衍生物包括戊二胺、5-氨基戊酸和戊二酸中的至少一种。
(11)一种增强目标基因表达的方法，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。
(12)一种制备蛋白的方法，其中，选择(5)所述的转录表达盒，(6)所述的重组表达载体，或(7)-(8)任一项所述的重组宿主细胞表达所述蛋白；可选地，所述蛋白为与目标产物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白；可选地，所述蛋白为参与合成L-赖氨酸的酶；可选地，所参与合成L-赖氨酸的酶包括天冬氨酸激酶、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；优选地，所述蛋白为二氨基庚二酸脱氢酶。
(13)一种生产氨基酸及其衍生物的方法，其中，选择(5)所述的转录表达盒，(6)所述的重组表达载体，或(7)-(8)任一项所述的重组宿主细胞表达参与合成氨基酸及其衍生物的酶，以所述参与合成氨基酸及其衍生物的酶生产所述氨基酸及其衍生物；可选地，所述氨基酸及其衍生物选自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、精氨酸、鸟氨酸、谷氨酸酰胺、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、甲硫氨酸、天冬氨酸、天冬酰胺、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；
优选地，所述氨基酸包括L-赖氨酸及其衍生物，其中，所述衍生物包括戊二胺、5-氨基戊酸和戊二酸中的至少一种；
优选地，所述参与合成氨基酸的酶为二氨基庚二酸脱氢酶。
发明的效果
在一个实施方案中，本公开提供了具有启动子活性的多核苷酸，是启动子活性显著提高的二氨基庚二酸脱氢酶基因(ddh基因)启动子的突变体。与野生型ddh基因启动子相比，突变体启动子活性显著提高，将其与目标基因可操作地连接，可以显著提高目标基因的表达强度，且不会破坏基因组的稳定性，使目标基因能够稳定高效表达，进而稳定、高效的生产下游产物。
在另一个实施方案中，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蛋白编码基因可操作地连接，可以提高蛋白编码基因的表达强度。
在另一个实施方案中，本公开提供了生产氨基酸的方法，利用上述具有启动子活性的多核苷酸，能够提高合成氨基酸的酶的表达，进而稳定、高效的生产氨基酸。当用于L-赖氨酸生产时，能够获得稳定高产的L-赖氨酸。
附图说明
图1示出了pEC-XK99E-P ddh-rfp质粒的示意图；
图2示出了培养平板上生长突变体克隆菌后的荧光显示结果，图中箭头标识位置为具有高强度红色荧光显示的单个克隆菌。
具体实施方式
定义
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。
本公开中的术语“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。
本公开中的术语“突变”是指在多核苷酸的一个或多个(例如，若干个)位置处包含突变的核苷酸，并且保持多核苷酸的启动子活性。其中，本公开中的突变(包含，取代、插入和/或缺失)特指其中的取代，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。
在一些具体的实施方案中，本公开的“突变”包含在SEQ ID NO：9所示序列的第292-300位的一个或多个位置处的取代的核苷酸，且不包含SEQ ID NO：9所示序列的第292-300位被ATGCATTGT取代的核苷酸。上述位置处的核苷酸被取代后的突变体，与如SEQ ID NO：9所示序列的多核苷酸的启动子活性相比，具有更高的启动子活性。
示例性地，本公开的“突变”包含在SEQ ID NO：9所示序列的第292-300位的1个、2个、3个、4个、5个、6个、7个、8个或9个位置处的取代的核苷酸。在一些具体的实施方案中，本公开的“突变”包含在SEQ ID NO：9所示序列的第292-300位的4个、5个、6个、7个、8个或9个位置处包含突变的核苷酸，优选在SEQ ID NO：9所示序列的第292-300位的6个、7个、8个或9个位置处包含突变的核苷酸。上述位置处的核苷酸被取代后的突变体，与其他位置处被取代的突变比相比，具有更高的启动子活性。
本公开中的术语“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。
在一些具体的实施方案中，具有启动子活性的多核苷酸包含与SEQ ID NO：9所示序列的多核苷酸的突变体具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％的序列同一性的序列。在本公开中，具有特定百分数的序列同一性指的是， 与SEQ ID NO：9所示序列的多核苷酸的突变体中存在可使突变体的转录活性保持或提高的突变序列。
本公开中的术语“启动子”是指一种核酸分子，通常位于目的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的基因的转录。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。
本公开中的术语“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。
在一些具体的实施方案中，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。在另外一些实施方案中，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。
本公开中的术语“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。
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。蛋白编码基因包括但不限于用于编码参与合成氨基酸的酶，在一些实施方案中，蛋白编码基因涉及用于编码参与合成L-赖氨酸的酶。对于参与合成L-赖氨酸的酶，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。本公开的具有启动子活性的多核苷酸，可适于调控目标基因的表达，实现目标产物的高效生产。
本公开中的术语“转录表达盒”指的包含转录调控元件与目标基因，利用转录调控元件对目标基因的表达进行调控的一类表达元件。在本公开中，转录调控元件包含启动子，在此基础上，还可以包含增强子、沉默子、绝缘子等元件。在本公开中，目标基因具体为蛋白编码基因。目标基因与多核苷酸“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，所述可操作地链接的方式可以采用本领域技术人员所述的任何方式。
本公开中的术语“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的基因。“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。
示例性的，本公开涉及的载体为基于pEC-XK99E-rfp质粒 [1]构建的ddh基因启动子强度表征质粒pEC-XK99E-P ddh-rfp，pEC-XK99E-P ddh-rfp质粒图谱如图1所示。图1中P ddh表示 为ddh基因的启动子；ddh表示野生型二氨基庚二酸脱氢酶基因；linker表示为位于ddh基因与rfp蛋白之间的连接肽；rfp表示为红色荧光蛋白(Red Fluorescent Protein，RFP)；Kan表示为卡那霉素抗性(Kanamycin resistant)。pEC-XK99E-P ddh-rfp转化入合适的宿主之后，可以复制并独立于宿主基因组发挥功能，或者在某些情况下整合入基因组本身。
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(CaPO 4)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。在一个实施方案中，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ATCC 13032、谷氨酸棒杆菌ATCC 13869、谷氨酸棒杆菌ATCC 14067或其衍生菌株。
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。
二氨基庚二酸脱氢酶基因启动子的突变体
本公开利用野生型二氨基庚二酸脱氢酶基因(ddh)的启动子序列，对ddh基因的核心启动子区序列进行突变，得到二氨基庚二酸脱氢酶基因启动子的突变体。二氨基庚二酸脱氢酶基因的启动子序列如SEQ ID NO：9所示，二氨基庚二酸脱氢酶基因启动子的突变体在SEQ ID NO：9所示序列的第292-300位的一个或多个位置处包含突变的核苷酸，且所述突变体在SEQ ID NO：9所示序列的第292-300位的核苷酸序列不选自ATGCATTGT。
经研究发现，二氨基庚二酸脱氢酶基因启动子的突变体，是具有启动子活性的多核苷酸；且与SEQ ID NO：9所示序列的二氨基庚二酸脱氢酶基因启动子相比，二氨基庚二酸脱氢酶基因启动子的突变体具有改进的启动子活性。
在一些具体的实施方案中，具有启动子活性的多核苷酸与二氨基庚二酸脱氢酶基因启动子的突变体序列相比，具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％的序列同一性(包括这些数值之间所有范围和百分数)，且不包括SEQ ID NO：9所示序列。
在一些具体的实施方案中，具有启动子活性的多核苷酸与在SEQ ID NO：9所示序列的第292-300位的4个、5个、6个、7个、8个或9个位置处包含突变的核苷酸，且与如SEQ ID NO：9所示序列的多核苷酸的启动子活性相比，具有更高的启动子活性；优选在SEQ ID NO：9所示序列的第292-300位的6个、7个、8个或9个位置处包含突变的核苷酸。
在一些具体的实施方案中，具有启动子活性的多核苷酸第292-300位的核苷酸序列选自如下任一组：(a)ACAAAAGGT；(b)TCTTCATCT；(c)GGAAAGTAT；(d)TTATTATAT；(e)TAATCCTCT；(f)TCAATTTAT；(g)GCGCAATCT；(h)CAGTTCCGT；(i)AAGTTTTAT；(g)TAAATGTAT；(k)GGATTGTAT；(l)CAAACTCAT；(m)TACAAATCT；(n)TATCAGTCT；(o)CGAGGATAT；(p) CCTTGTTAT。具有启动子活性的多核苷酸包含如SEQ ID NO：10-25任一所示的序列。与SEQ ID NO：9所示序列的二氨基庚二酸脱氢酶基因启动子相比，突变体的启动子活性得到增强18倍以上，优选19倍，优选20倍，优选22倍，优选23倍，优选24倍，优选25倍，优选26倍，更优选27倍，更优选28倍，更优选30倍，更优选31倍以上。
重组表达载体的构建
在一些具体的实施方案中，首先构建包含二氨基庚二酸脱氢酶基因启动子的重组载体，然后对ddh基因的核心启动子区进行突变，获得包含有具有启动子活性的多核苷酸的重组表达载体。
对于重组载体的构建，根据目前已公开的谷氨酸棒杆菌的基因组信息设计引物ddh-F和ddh-R，以ddh-F和ddh-R对谷氨酸棒杆菌的基因组进行PCR扩增，获得ddh基因的启动子序列。
利用pEC-XK99E-rfp [1]质粒信息设计引物pEC-F和pEC-R，以pEC-XK99E-rfp质粒为模板，利用引物pEC-F和pEC-R扩增得到pEC-XK99E骨架、连接肽和红色荧光蛋白的DNA片段。
将引物ddh-F和ddh-R的扩增片段与引物pEC-F和pEC-R的扩增片段进行重组连接，获得连接有具有启动子活性的多核苷酸(也即，ddh基因启动子突变体序列)的重组表达载体。
谷氨酸棒杆菌的具体来源为谷氨酸棒杆菌ATCC 13032(Corynebacterium glutamicum ATCC 13032，Gene ID:2830649)。
氨基酸的生产过程
(1)本公开中将具有启动子活性的多核苷酸，与参与合成氨基酸的酶的编码基因可操作的连接，得到能够合成参与合成氨基酸的酶的重组表达载体，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氨基酸，完成氨基酸的生产过程。
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，参与合成氨基酸的酶的编码基因的转录活性提高，参与氨基酸合成的酶的表达量提高，进而使氨基酸的产量显著提升。
对于生产的氨基酸，所述氨基酸包括L-赖氨酸及其衍生物。可选地，氨基酸选自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、精氨酸、鸟氨酸、谷氨酸酰胺、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、甲硫氨酸、天冬氨酸、天冬酰胺、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。优选地，所述衍生物包括戊二胺、5-氨基戊酸和戊二酸中的至少一种。
对于参与合成氨基酸的酶，所述参与合成氨基酸的酶是参与合成L-赖氨酸的酶；优选地，参与合成氨基酸的酶为二氨基庚二酸脱氢酶。具有启动子活性的多核苷酸可以显著提高二氨基庚二酸脱氢酶的转录活性，使二氨基庚二酸脱氢酶的表达量显著提高。
在一些实施方式中，本公开中的宿主细胞可以是具有目标产物的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。示例性的，宿主细胞来源于适合发酵生产氨基酸及其衍生物等目标产物的微生物，例如肠杆菌、棒杆菌、短杆菌、节杆菌、微杆菌等。
在一些优选地实施方式中，宿主细胞为肠杆菌或棒杆菌，更优选谷氨酸棒杆菌，包括但不限于谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067，以及由上述菌株制备的产生L-氨基酸的衍生菌株。
在一些具体的实施方案中，宿主细胞为谷氨酸棒杆菌(Corynebacterium glutamicum)， 谷氨酸棒杆菌是用于生产L-赖氨酸的重要菌株，利用具有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，谷氨酸棒杆菌参与合成L-赖氨酸的酶的表达量显著提高，具体为二氨基庚二酸脱氢酶的表达量显著提高，使谷氨酸棒杆菌发酵生产L-赖氨酸的能力大大提高。
在一些具体的实施方案中，重组宿主细胞是经过如下改良的谷氨酸棒杆菌：1)谷氨酸棒杆菌中的天冬氨酸激酶编码基因引入了T311I突变编码序列；2)谷氨酸棒杆菌中丙酮酸羧化酶基因启动子的第279位至第317位的核心区为CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG；3)将具有启动子活性的多核苷酸与编码二氨基庚二酸脱氢酶的基因可操作地连接，然后导入谷氨酸棒杆菌。以上述方法改造的谷氨酸棒杆菌，是L-赖氨酸的高产菌株。
具体地，天冬氨酸激酶、丙酮酸羧化酶以及二氨基庚二酸脱氢酶的编码基因可被分离，插入到用于转化生产的表达载体中，表达载体在宿主细胞中可独立于宿主细胞复制并表达所述酶，或在某些情况下整合进入宿主细胞的基因组中。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。
在另外一些实施方式中，宿主细胞还可以是其他种类的氨基酸生产菌株。本公开所说的“氨基酸生产菌株”是指，当细菌在培养基中培养时可以生产氨基酸并且能够积累氨基酸，或者能够将氨基酸分泌到培养基中，也就是能够得到胞外的游离氨基酸的菌株。例如，可以是天然存在的氨基酸生产菌株，也可以是经过遗传改造获得的氨基酸生产工程菌株。
示例性地，宿主细胞为生产赖氨酸的宿主细胞。在一些实施方式中，所述生产赖氨酸的宿主细胞中还可以包括但不限于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，所述生产赖氨酸宿主细胞中还可以包括但不限于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。
示例性地，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。
示例性地，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。
示例性地，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。
示例性地，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。
在一些具体的实施方案中，重组宿主细胞的培养条件为：首先将谷氨酸棒杆菌接种到TSB液体培养基中培养8h，培养物作为种子接种到的800μl发酵培养基/孔的24孔板中，初始OD控制约为0.1，30℃培养17h，孔板摇床转速为800rpm。
对于TSB液体培养基，成分为：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。
对于发酵培养基，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；K2HPO4·3H2O，1g/L；MgSO4·7H2O，0.45g/L；FeSO4·7H2O，0.05g/L；生物素，0.4mg/L；维生素B1，0.1mg/L；MOPS，40g/L；初始pH7.2。
在一些具体的实施方案中，对于重组宿主细胞或重组细胞的培养液回收氨基酸，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、结晶和HPLC。
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ddh基因启动子强度表征质粒的构建
为了表征谷氨酸棒杆菌ddh基因启动子的强度，本实施例首先构建一个表征载体，将ddh基因启动子和ddh基因片段连接在pEC-XK99E质粒骨架基础上，利用ddh基因启动子表达ddh基因、连接肽和红色荧光蛋白(rfp)基因。具体如下：
(1)根据已公开的谷氨酸棒杆菌ATCC13032(Corynebacterium glutamicum ATCC 13032，Gene ID:2830649)基因组序列及ddh基因注释信息，设计引物ddh-F和ddh-R，以ATCC13032基因组为模板，通过PCR扩增获得ddh基因启动子和ddh基因片段，扩增片段的核苷酸序列如SEQ ID NO：34所示。
(2)以pEC-XK99E-rfp质粒为模板，以pEC-F和pEC-R引物进行扩增，扩增pEC-XK99E质粒骨架、连接肽和红色荧光蛋白基因的DNA片段，扩增片段的核苷酸序列如SEQ ID NO：35所示。
将谷氨酸棒杆菌ATCC13032的基因组扩增片段，以及pEC-XK99E-rfp的质粒扩增片段通过诺唯赞的一步重组试剂盒克隆连接，获得pEC-XK99E-P ddh-rfp表征载体，其质粒图谱如图1所示。以上所用引物序列如表1所示，连接肽序列信息如表2所示。
表1
Figure PCTCN2021106501-appb-000001
表2
Figure PCTCN2021106501-appb-000002
实施例2.谷氨酸棒杆菌ddh基因启动子突变体筛选及强度表征
(1)谷氨酸棒杆菌ddh基因启动子突变体文库的构建
本实施例对谷氨酸棒杆菌ddh基因启动子的核心区“TGATGAAAGAGATGTCCCTGAA TCATCATCTAAGTATGCATCTCGG TAAGCTCGACCAGG”进行突变，其中下划线处分别为该启动子的-35区和-10区主要序列。本实施例在以上核心区对应位置进行突变“TGATGAAAGAGATGTCCCTGAA TCATCATCTAAGTNNNNNNNNNGG TAAGCTCGACCAGG”，分别采用ddh-M1、ddh-M2和ddh-M3、ddh-M4引物扩增质粒的两个片段，通过诺唯赞的一步重组试剂盒克隆连接，对获得的所有克隆菌进行收集并提取质粒，获得ddh基因启动子突变体文库。为了对比现有技术中已经公开的ddh启动子突变体，我们同时在pEC-XK99E-Pddh-rfp质粒基础上，构建引用文献3(CN101939432A)中相同 的ddh启动子突变体，具体为启动子核心区序列为“TGATGAAAGAGATGTCCCTGAATCATCATCTAAGTATGCAT tgtGGTAAGCTCGACCAGG”，横线标出的是引用文献3公开的突变位点。将以上文库、现有技术突变体pEC-XK99E-P ddh-x-rfp和实施例1中获得野生型对照pEC-XK99E-P ddh-rfp分别转化谷氨酸棒杆菌ATCC13032，涂布TSB平板，通过荧光成像系统对长有数百个克隆的平板进行荧光拍照，根据克隆的荧光亮度初步筛选表达强度提高的突变体。图2示出了培养平板上生长突变体克隆菌后的荧光照片，图中箭头标识位置为具有高强度红色荧光显示的单个克隆菌。TSB平板培养基成份为(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；琼脂粉，15g/L。本实施例对大于1万个克隆进行初步筛选，获得约20个荧光强度显著增强的突变体。以上所用引物序列如表3所示。
表3
Figure PCTCN2021106501-appb-000003
(2)谷氨酸棒杆菌ddh基因启动子突变体文库的强度表征
对以上平板观察荧光强度增强的所有突变体进行96孔板培养表征启动子的强度，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。将筛选获得的菌株和野生型对照、现有技术对照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后检测菌株的荧光强度，并对荧光强度较野生型对照提高的菌株进行测序。部分启动子突变体序列相同，最终成功获得16个表达强度显著提高的不同启动子突变体，结果如表4所示，按顺序将本发明获得的启动子编号为P ddh-1至P ddh-16。P ddh-1至P ddh-16启动子活性提高范围为18-31倍左右，为改造ddh等基因的表达提供丰富元件。
以上述方法对引用文献3的ddh启动子突变体(P ddh-x)进行荧光强度的检测，结果如表4所示，P ddh-x相对野生型启动子活性仅提高15倍，本发明的启动子相对于现有技术公开的启动子表达强度提高了15-101％，具有实质性的进步。
表4
Figure PCTCN2021106501-appb-000004
Figure PCTCN2021106501-appb-000005
实施例3.谷氨酸棒杆菌ddh基因启动子突变体应用于赖氨酸生产
(1)谷氨酸棒杆菌ddh基因启动子突变体的重组载体构建
根据已报道的谷氨酸棒杆菌ATCC13032基因组序列，分别以ATCC13032基因组为模板，以ddh-UF/ddh-UR和ddh-DF1/ddh-DR，ddh-UF/ddh-UR和ddh-DF10/ddh-DR，ddh-UF/ddh-UR和ddh-DF16/ddh-DR为引物，PCR扩增P ddh-1、P ddh-10和P ddh-16启动子突变的上下游同源臂；同时以pK18-1/2引物扩增pK18mobsacB(GenBank:FJ437239.1)的骨架。上述两种PCR片段回收后，通过诺唯赞的一步重组试剂盒克隆连接，分别获得启动子突变的重组载体pK18-P ddh-1、pK18-P ddh-10、和pK18-P ddh-16。以上所用引物序列如表5所示。
表5
Figure PCTCN2021106501-appb-000006
(2)谷氨酸棒杆菌赖氨酸生产菌的pyc基因启动子突变体构建
将上述构建的重组载体pK18-P ddh-1、pK18-P ddh-10、和pK18-P ddh-16分别转化谷氨酸棒杆菌赖氨酸生产菌SCgL37(该菌株是将谷氨酸棒杆菌ATCC13032天冬氨酸激酶编码基因引入了T311I(碱基由ACC突变为ATC)氨基酸突变，丙酮酸羧化酶基因启动子的第279位至第317位的核心区突变为CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG的菌株)，涂布含有5g/L葡萄糖和25μg/mL卡那霉素的LBHIS固体培养基上，30℃培养获得第一次重组的转化子。正确的一次重组转化子分别接种含有5g/L葡萄糖的LB培养基，过夜培养，分别稀释涂布添加100g/L蔗糖的LB固体培养基平板进行筛选，分别获得的ddh启动子突变的菌株 SCgL38、SCgL39和SCgL40。
(3)谷氨酸棒杆菌赖氨酸生产菌ddh基因启动子突变体的L-赖氨酸生产能力评价
为了测试谷氨酸棒杆菌中ddh启动子突变对菌株产L-赖氨酸的影响，分别对SCgL37、SCgL38、SCgL39和SCgL40菌株进行发酵测试，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；FeSO4·7H 2O，0.05g/L；生物素，0.4mg/L；维生素B1，0.1mg/L；MOPS，40g/L；初始pH7.2。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，初始OD 600控制约为0.1，30℃培养17h，孔板摇床转速为800rpm，每个菌株3个平行，发酵结束后检测L-赖氨酸产量和葡萄糖消耗量，并计算从葡萄糖到L-赖氨酸的糖酸转化率。结果如表6所示，ddh启动子突变后菌株的赖氨酸产量和糖酸转化率均有提高，而且随着启动子强度的增强而提高更加显著。以上结果表明增强ddh启动子表达强度的突变体可应用于L-赖氨酸生产。
表6
菌株 赖氨酸产量(g/L) 转化率(％)
SCgL37 2.97±0.06 5.41±0.04
SCgL38 3.47±0.21 6.47±0.57
SCgL39 3.53±0.21 7.05±1.09
SCgL40 3.60±0.36 7.49±1.26
由于L-赖氨酸下游产物的合成均依赖于二氨基庚二酸脱氢酶催化的反应步骤，通过本公开的ddh基因启动子的突变体增强ddh基因的表达也可以提高下游产物的产量。因此，本公开提供的技术方案也可用于L-赖氨酸下游产物的生产，如戊二胺、5-氨基戊酸、戊二酸等的生产。
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。
参考文献：
[1]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

Claims (13)

  1. 一种具有启动子活性的多核苷酸，其特征在于，所述多核苷酸选自如下(i)-(ii)中任一项所示的组：
    (i)如SEQ ID NO：9所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：9所示序列的第292-300位的一个或多个位置处包含突变的核苷酸；
    (ii)与(i)所示序列具有至少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，且不包括SEQ ID NO：9所示序列的多核苷酸；
    其中，所述突变体的启动子活性与如SEQ ID NO：9所示序列的多核苷酸的启动子活性相比，具有更高的启动子活性；并且，
    所述突变体在SEQ ID NO：9所示序列的第292-300位的核苷酸序列不选自ATGCATTGT。
  2. 根据权利要求1所述的具有启动子活性的多核苷酸，其特征在于，所述突变体的启动子活性与如SEQ ID NO：9所示序列的多核苷酸的启动子活性相比，具有18倍以上的增强的启动子活性。
  3. 根据权利要求1或2所述的具有启动子活性的多核苷酸，其特征在于，所述突变体在SEQ ID NO：9所示序列的第292-300位的4个、5个、6个、7个、8个或9个位置处包含突变的核苷酸；优选地，所述突变体在SEQ ID NO：9所示序列的第292-300位的6个、7个、8个或9个位置处包含突变的核苷酸。
  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，其特征在于，所述具有启动子活性的多核苷酸的第292-300位的核苷酸序列选自如下任一组：
    (a)ACAAAAGGT；
    (b)TCTTCATCT；
    (c)GGAAAGTAT；
    (d)TTATTATAT；
    (e)TAATCCTCT；
    (f)TCAATTTAT；
    (g)GCGCAATCT；
    (h)CAGTTCCGT；
    (i)AAGTTTTAT；
    (g)TAAATGTAT；
    (k)GGATTGTAT；
    (l)CAAACTCAT；
    (m)TACAAATCT；
    (n)TATCAGTCT；
    (o)CGAGGATAT；
    (p)CCTTGTTAT。
  5. 一种转录表达盒，其特征在于，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；可选地，所述转录表达盒还含有蛋白编码基因，所述蛋白编码基因与所述具有启动子活性的多核苷酸可操作地连接。
  6. 一种重组表达载体，其特征在于，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，其特征在于，包含权利要求5所述的转录表达盒，或者权利要求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，其特征在于，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；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或其衍生菌株。
  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、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，或者权利要求7-8任一项所述的重组宿主细胞在制备用于增强基因转录水平的试剂或试剂盒中的应用。
  10. 一种根据权利要求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在制备蛋白或生产氨基酸及其衍生物中的应用；
    优选地，所述蛋白为参与合成氨基酸及其衍生物的酶；优选地，所述参与合成氨基酸的酶为二氨基庚二酸脱氢酶；可选地，所述氨基酸及其衍生物选自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、精氨酸、鸟氨酸、谷氨酸酰胺、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、甲硫氨酸、天冬氨酸、天冬酰胺、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；
    优选地，所述氨基酸包括L-赖氨酸及其衍生物，其中，所述衍生物包括戊二胺、5-氨基戊酸和戊二酸中的至少一种。
  11. 一种增强目标基因表达的方法，其特征在于，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。
  12. 一种制备蛋白的方法，其特征在于，选择权利要求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；可选地，所述蛋白为与目标产物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白；可选地，所述蛋白为参与合成L-赖氨酸的酶；可选地，所参与合成L-赖氨酸的酶包括天冬氨酸激酶、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；优选地，所述蛋白为二氨基庚二酸脱氢酶。
  13. 一种生产氨基酸及其衍生物的方法，其特征在于，选择权利要求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参与合成氨基酸及其衍生物的酶，以所述参与合成氨基酸及其衍生物的酶生产所述氨基酸及其衍生物；可选地，所述氨基酸及其衍生物选自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、精氨酸、鸟氨酸、谷氨酸酰胺、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、甲硫氨酸、天冬氨酸、天冬酰胺、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；
    优选地，所述氨基酸包括L-赖氨酸及其衍生物，其中，所述衍生物包括戊二胺、5-氨基戊酸和戊二酸中的至少一种；
    优选地，所述参与合成氨基酸的酶为二氨基庚二酸脱氢酶。
PCT/CN2021/106501 2020-08-19 2021-07-15 具有启动子活性的多核苷酸及其在生产氨基酸中的应用 WO2022037338A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP21857433.3A EP4202046A1 (en) 2020-08-19 2021-07-15 Polynucleotide having promoter activity and application of polynucleotide in producing amino acid
US18/020,965 US20230399667A1 (en) 2020-08-19 2021-07-15 Polynucleotide Having Promoter Activity and Application of Polynucleotide in Producing Amino Acid
CA3188476A CA3188476A1 (en) 2020-08-19 2021-07-15 Polynucleotide having promoter activity and application of polynucleotide in producing amino acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010838604.9 2020-08-19
CN202010838604.9A CN113201536B (zh) 2020-08-19 2020-08-19 具有启动子活性的多核苷酸及其在生产氨基酸中的应用

Publications (1)

Publication Number Publication Date
WO2022037338A1 true WO2022037338A1 (zh) 2022-02-24

Family

ID=77024998

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/106501 WO2022037338A1 (zh) 2020-08-19 2021-07-15 具有启动子活性的多核苷酸及其在生产氨基酸中的应用

Country Status (5)

Country Link
US (1) US20230399667A1 (zh)
EP (1) EP4202046A1 (zh)
CN (1) CN113201536B (zh)
CA (1) CA3188476A1 (zh)
WO (1) WO2022037338A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114107141A (zh) * 2021-08-19 2022-03-01 中国科学院天津工业生物技术研究所 高产l-脯氨酸的谷氨酸棒杆菌以及高产l-脯氨酸的方法
WO2023236634A1 (zh) * 2022-06-10 2023-12-14 宁夏伊品生物科技股份有限公司 一种ep6启动子与其相关生物材料及应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113994003B (zh) * 2021-05-07 2022-10-14 Cj第一制糖株式会社 新型启动子及其用途
CN115490761B (zh) 2021-11-01 2023-06-09 中国科学院天津工业生物技术研究所 基于赖氨酸外排蛋白构建的重组微生物及生产赖氨酸的方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1748031A (zh) 2003-02-05 2006-03-15 底古萨股份公司 细菌和用所述细菌生产化合物的方法
CN101765659A (zh) 2007-07-23 2010-06-30 味之素株式会社 L-赖氨酸的生产方法
CN101939432A (zh) 2008-01-31 2011-01-05 Cj第一制糖株式会社 改进的启动子和用其产生l-赖氨酸的方法
US20190194647A1 (en) * 2016-06-30 2019-06-27 Zymergen Inc. Methods for generating a glucose permease library and uses thereof
CN111197021A (zh) * 2020-01-13 2020-05-26 江南大学 一种l-赖氨酸产量提高的重组谷氨酸棒杆菌及其构建方法
CN112111469A (zh) * 2020-11-23 2020-12-22 中国科学院天津工业生物技术研究所 γ-谷氨酰激酶突变体及其应用
CN112695036A (zh) * 2021-03-23 2021-04-23 中国科学院天津工业生物技术研究所 一种天冬氨酸激酶基因表达调控序列及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011118019A1 (de) * 2011-06-28 2013-01-03 Evonik Degussa Gmbh Varianten des Promotors des für die Glyzerinaldehyd-3-phosphat-Dehydrogenase kodierenden gap-Gens
ES2856755T3 (es) * 2015-12-07 2021-09-28 Zymergen Inc Promotores de Corynebacterium glutamicum
CN109536428B (zh) * 2018-12-07 2022-08-30 武汉远大弘元股份有限公司 一种产l-异亮氨酸的基因工程菌及其构建方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1748031A (zh) 2003-02-05 2006-03-15 底古萨股份公司 细菌和用所述细菌生产化合物的方法
CN101765659A (zh) 2007-07-23 2010-06-30 味之素株式会社 L-赖氨酸的生产方法
CN101939432A (zh) 2008-01-31 2011-01-05 Cj第一制糖株式会社 改进的启动子和用其产生l-赖氨酸的方法
US20190194647A1 (en) * 2016-06-30 2019-06-27 Zymergen Inc. Methods for generating a glucose permease library and uses thereof
CN111197021A (zh) * 2020-01-13 2020-05-26 江南大学 一种l-赖氨酸产量提高的重组谷氨酸棒杆菌及其构建方法
CN112111469A (zh) * 2020-11-23 2020-12-22 中国科学院天津工业生物技术研究所 γ-谷氨酰激酶突变体及其应用
CN112695036A (zh) * 2021-03-23 2021-04-23 中国科学院天津工业生物技术研究所 一种天冬氨酸激酶基因表达调控序列及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Microbial Metabolic Engineering: Methods and Protocols", 1989, COLD SPRING HARBOR LABORATORY PRESS
ACORD JOHN, MILLICENT MASTERS: "Expression from the Escherichia coli dapA promoter is regulated by intracellular levels of diaminopimelic acid", FEMS MICROBIOLOGY LETTERS AUG 2009, vol. 235, no. 1, 23 April 2004 (2004-04-23), pages 131 - 137, XP055901946, ISSN: 1574-6968, DOI: 10.1016/j.femsle.2004.04.022 *
WANG YINGCHUN, LIU JIAO, NI XIAOMENG, LEI YU, ZHENG PING, DIAO AIPO: "Screening efficient constitutive promoters in Corynebacterium glutamicum based on time-series transcriptome analysis", CHINESE JOURNAL OF BIOTECHNOLOGY, ZHONGGUO KEXUEYUAN WEISHENGWU YANJIUSUO, CHINESE ACADEMY OF SCIENCES, INSTITUTE OF MICROBIOLOGY, CN, vol. 34, no. 11, 25 November 2018 (2018-11-25), CN , pages 1760 - 1771, XP055901944, ISSN: 1000-3061, DOI: 10.13345/j.cjb.180041 *
WANG, YC ET AL.: "Screening efficient constitutive promoters in Corynebacterium glutamicum based on time-series transcriptome analysis", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 34, no. 11, 2018, pages 1760 - 1771, XP055901944, DOI: 10.13345/j.cjb.180041

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114107141A (zh) * 2021-08-19 2022-03-01 中国科学院天津工业生物技术研究所 高产l-脯氨酸的谷氨酸棒杆菌以及高产l-脯氨酸的方法
WO2023236634A1 (zh) * 2022-06-10 2023-12-14 宁夏伊品生物科技股份有限公司 一种ep6启动子与其相关生物材料及应用

Also Published As

Publication number Publication date
CN113201536A (zh) 2021-08-03
CA3188476A1 (en) 2022-02-24
US20230399667A1 (en) 2023-12-14
EP4202046A1 (en) 2023-06-28
CN113201536B (zh) 2022-09-13

Similar Documents

Publication Publication Date Title
WO2022037338A1 (zh) 具有启动子活性的多核苷酸及其在生产氨基酸中的应用
WO2022199460A1 (zh) 一种天冬氨酸激酶基因表达调控序列及其应用
WO2022017221A1 (zh) 谷氨酸脱氢酶基因启动子的突变体及其应用
WO2023284419A1 (zh) 丙酮酸脱氢酶的突变体及其用于生产l-氨基酸的方法
WO2022017223A1 (zh) 丙酮酸羧化酶基因启动子的突变体及其应用
CN101273138A (zh) 产生l-氨基酸的细菌和用于产生l-氨基酸的方法
CN113278620B (zh) 一种突变的高渗诱导型启动子PproP及其应用
CN113201539B (zh) 具有启动子活性的多核苷酸及其在生产目标化合物中的用途
WO2022078127A1 (zh) 具有天冬氨酸激酶活性的多肽及其在生产氨基酸中的应用
RU2812048C9 (ru) Мутант промотора гена пируваткарбоксилазы и его применение
RU2812048C1 (ru) Мутант промотора гена пируваткарбоксилазы и его применение
WO2022257758A1 (zh) 基于mdh基因的具有启动子活性的多核苷酸及其用途
WO2022257757A1 (zh) 基于dapB基因的具有启动子活性的多核苷酸及其用途
RU2825450C2 (ru) Полинуклеотид на основе гена mdh, обладающий промоторной активностью, и его применение
WO2023071580A1 (zh) 基于赖氨酸外排蛋白构建的重组微生物及生产赖氨酸的方法
JPWO2013154182A1 (ja) アミノ酸の製造法
WO2022152036A1 (zh) 具有启动子活性的多核苷酸及其在生产目标化合物中的用途
CN115322990B (zh) 具有启动子活性的多核苷酸及其在生产目标化合物中的用途
CN115806962A (zh) 磷酸烯醇式丙酮酸羧化酶突变体及其应用
CN115506035A (zh) 启动子突变体文库的构建方法及启动子突变体文库
CN115927325A (zh) 柠檬酸合酶启动子突变体及其应用
CN116891868A (zh) 一种渗透压耐受性功能基因鉴定方法及功能基因的应用
CN115948396A (zh) 谷氨酸脱氢酶启动子突变体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21857433

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3188476

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021857433

Country of ref document: EP

Effective date: 20230320