WO2022199460A1 - 一种天冬氨酸激酶基因表达调控序列及其应用 - Google Patents

一种天冬氨酸激酶基因表达调控序列及其应用 Download PDF

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WO2022199460A1
WO2022199460A1 PCT/CN2022/081527 CN2022081527W WO2022199460A1 WO 2022199460 A1 WO2022199460 A1 WO 2022199460A1 CN 2022081527 W CN2022081527 W CN 2022081527W WO 2022199460 A1 WO2022199460 A1 WO 2022199460A1
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host cell
expression
polynucleotide
corynebacterium glutamicum
sequence
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French (fr)
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孙际宾
刘娇
郑平
石拓
周文娟
马延和
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Tianjin Institute of Industrial Biotechnology of CAS
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Tianjin Institute of Industrial Biotechnology of CAS
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Priority to EP22774121.2A priority patent/EP4282965A4/en
Priority to CA3209940A priority patent/CA3209940A1/en
Publication of WO2022199460A1 publication Critical patent/WO2022199460A1/zh
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    • C12N2830/30—Vector systems having a special element relevant for transcription being an enhancer not forming part of the promoter region

Definitions

  • the invention belongs to the fields of molecular biology and bioengineering, and in particular relates to a polynucleotide sequence for regulating the transcription and expression of aspartate kinase gene, and the use of the polynucleotide sequence to transcribe and express aspartate kinase to produce L-lysine sour method.
  • L-Lysine referred to as lysine
  • lysine is a basic amino acid and is the most important essential amino acid in human and animal nutrition. Because the content of lysine in cereal food is very low, and it is easily destroyed during processing, it is deficient. , so called the first limiting amino acid, is widely used in medicine, health, food, animal feed and other industries. Lysine is mainly produced by microbial fermentation. At present, the main production strains include microorganisms such as Escherichia coli and Corynebacterium glutamicum.
  • the synthetic pathway of L-lysine starts from aspartic acid in many microorganisms, including two steps shared by methionine and threonine, and finally produces L after nine steps of enzymatic catalysis.
  • -Lysine Aspartate Kinase (ASK or AK, encoding gene lysC) is the first enzyme that initiates aspartate in the L-lysine synthesis pathway, and its activity determines the flow of metabolic flow to L-lysine. The ratio of the lysine synthesis pathway, the rate-limiting step in lysine production. Aspartokinase activity is complexly regulated in microorganisms.
  • one of the objects of the present invention is to provide a polynucleotide sequence that enhances the regulation of lysC gene expression.
  • RBS ribosome binding site
  • the inventors of the present invention have found that by adjusting the ribosome binding site (ribosome binding site, hereinafter referred to as RBS) of the artificially synthesized expression control sequence (the nucleotide sequence of which is shown in SEQ ID NO: 1) and the The spacer partial sequence TTACTCTA (that is, the 366th to 373rd position of the polynucleotide sequence shown in SEQ ID NO: 1) between the initiation codons is modified, and the sequence is formed by connecting the sequence with the initiation codon GTG or TTG.
  • RBS ribosome binding site
  • the polynucleotide sequence has transcriptional expression regulation activity, and these polynucleotide sequences with transcriptional expression regulation activity can increase the expression of the aspartokinase encoding gene lysC, thereby achieving a large accumulation of L-lysine, and on this basis the present invention.
  • Another object of the present invention is to provide a vector containing a polynucleotide sequence that exhibits improved promoter activity.
  • the third object of the present invention is to provide a host cell containing a lysC gene expression control sequence.
  • a fourth object of the present invention is to provide a method for producing L-lysine by fermenting host cells.
  • the present invention provides a polynucleotide for gene expression regulation, the polynucleotide is:
  • the polynucleotide has the activity of enhancing the expression of the lysC gene compared to the unmodified SEQ ID NO: 1.
  • nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the present invention provides a vector comprising the above-mentioned polynucleotide.
  • the vector uses pK18mobsacB as the backbone, and the GenBank accession number of the pK18mobsacB is FJ437239.1.
  • the present invention provides an expression cassette for lysC gene expression regulation, wherein the expression cassette is a polynucleotide formed by operably linking the above-mentioned polynucleotide with an lysC gene without an initiation codon.
  • nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the present invention provides a host cell comprising the above-mentioned polynucleotide, or the above-mentioned expression cassette.
  • nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the host cell is derived from Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia; preferably, the host cell is Corynebacterium glutamicum or Escherichia coli; more Preferably, the host cell is Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067 and derivative strains thereof.
  • the present invention provides a method for enhancing the expression of an aspartokinase-encoding gene lysC, which comprises operably linking the above-mentioned polynucleotide to a lysC gene without an initiation codon.
  • nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the operably linked is to introduce the vector containing the above-mentioned polynucleotide into a host cell and integrate into the genome of the host cell by homologous recombination.
  • the host cell is derived from Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia; preferably, the host cell is Corynebacterium glutamicum or Escherichia coli; more Preferably, the host cell is Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067.
  • the host cell is Corynebacterium glutamicum modified as follows: 1) the aspartokinase coding gene lysC in Corynebacterium glutamicum ATCC13032 has introduced the T311I mutation coding sequence; 2) glutamic acid The core region from position 279 to position 317 of the pyc promoter of the pyruvate carboxylase gene in Corynebacterium is CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG; 3) The 279th position of the ddh promoter of the diaminopimelate dehydrogenase gene in Corynebacterium glutamicum Nucleotides to position 317 were mutated from wild-type ATGCATCTC to CCTTGTTAT.
  • the present invention provides the use of the polynucleotide of the first aspect, the vector of the second aspect, the expression cassette of the third aspect, and the host cell of the fourth aspect in preparing L-lysine.
  • the present invention provides a method for producing L-lysine, the method comprising the step of culturing the host cell of the fourth aspect to produce L-lysine.
  • a step of isolating L-lysine from the fermentation broth is included.
  • the present invention provides applications of the polynucleotide of the first aspect, the vector of the second aspect, the expression cassette of the third aspect, and the host cell of the fourth aspect in the preparation of aspartic acid family amino acids and derivatives thereof
  • the aspartic acid family amino acids and their derivatives include L-threonine, L-isoleucine, L-homoserine, L-methionine and L-lysine downstream products such as pentamethylenediamine, 5-aminovaleric acid, glutaric acid.
  • the present invention provides a polynucleotide with the activity of enhancing the expression of lysC gene, which can be operably linked with the target gene lysC, which can significantly improve the expression intensity of lysC, thereby stably and efficiently producing downstream products.
  • the present invention provides a method for producing L-lysine, which can improve the expression of aspartokinase by utilizing the above-mentioned polynucleotides with gene transcription and expression regulation activity, thereby stably and efficiently producing L-lysine, L-
  • the lysine yield can reach 1.9 times that of the starting strain.
  • Figure 1 shows the pEC-XK99E-rfp-1 plasmid map
  • Figure 2 shows the pEC-XK99E-rfp-2 plasmid map.
  • the starting materials and reagents used in the following examples are commercially available or can be prepared by known methods.
  • the experimental method of unreceipted specific conditions in the following examples usually according to conventional conditions such as Sambrook et al., molecular cloning: conditions described in laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer the proposed conditions.
  • Selected/optional/preferred "numerical ranges" as used herein include 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 the present invention 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 invention refers to a nucleotide comprising a mutation at one or more (eg, several) positions of a polynucleotide and maintains the promoter activity of the polynucleotide.
  • the mutation including, substitution, insertion and/or deletion
  • the substitution refers to replacing a nucleotide occupying a position with a different nucleotide.
  • Deletion refers to the removal of a nucleotide occupying a position.
  • the "mutations" of the invention comprise the partial sequence TTACTCTA of the RBS spacer of the sequence shown in SEQ ID NO: 1 (ie, positions 366 to 373 of the sequence shown in SEQ ID NO: 1)
  • the substitution and/or deletion of nucleotides at one or more positions is performed, and the polynucleotide sequence formed after the sequence is connected with the initiation codon GTG or TTG, compared with the unmodified SEQ ID NO: 1, has Activity to enhance lysC expression.
  • the "mutations" of the present invention comprise 1, 2, 3, 4, 5, 6, 7, 8 at positions 366 to 373 of the sequence shown in SEQ ID NO: 1 Substitutions and/or deletions at positions.
  • the “mutations” of the present invention comprise deletions of 6 nucleotides from positions 363 to 373 of the sequence shown in SEQ ID NO: 1, and/or substitutions of 0-2 nucleotides in the deleted sequence , and linked to the start codon GTG or TTG.
  • SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 formed after the above mutation have higher gene transcription expression regulation activity compared with unsubstituted SEQ ID NO: 1.
  • promoter in the present invention refers to a nucleic acid molecule generally located upstream of the coding sequence of the gene of interest, providing a recognition site for RNA polymerase, and 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.
  • RBS spacer in the present invention refers to a nucleic acid sequence located in the prokaryotic promoter region, which is a nucleotide sequence between the RBS and the initiation codon.
  • initiation codon in the present invention has a definition well known to those skilled in the art, and refers to the codon at the initiation site of protein synthesis, usually including ATG, GTG, TTG and ATA.
  • initiation codon included in SEQ ID NO: 2 and SEQ ID NO: 3 of the present invention is GTG
  • initiation codon included in SEQ ID NO: 4 is TTG.
  • 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 in the present invention refers to a type of expression element that includes transcriptional regulatory elements and target genes, and utilizes transcriptional regulatory elements to regulate the expression of target genes.
  • 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.
  • “Operably linked" with a target gene and a polynucleotide means that a polynucleotide with promoter activity is functionally linked to the target gene to initiate and mediate the transcription of the target gene, and the operably linked manner may be Use any means described by those skilled in the art.
  • the target gene and polynucleotide of the present invention are operably linked to a polynucleotide sequence obtained by operably linking SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 to the lysC gene.
  • the "lysC gene” in the present invention refers to the gene encoding aspartokinase (EC: 2.7.2.4) derived from Corynebacterium glutamicum, including but not limited to the naturally occurring lysC gene derived from Corynebacterium glutamicum , lysC gene mutants (such as T311I, D274A, Q298G, N299L, N374A, E382A, R384L, etc.) that have released the feedback inhibition of lysine, and artificially engineered ones derived from Corynebacterium glutamicum that still have aspartate kinase Active lysC gene mutants.
  • the lysC gene of the present invention is the aspartate kinase encoding gene of Corynebacterium glutamicum ATCC13032, the gene number is NCgl0247 or Cgl0251, the NCBI database Gene ID: 1021294, the encoded protein Protein ID: NP_599504 or WP_003855724. 1.
  • the "lysC gene without initiation codon" of the present invention refers to a gene encoding an aspartokinase (EC: 2.7.2.4) in which the initiation codon GTG is deleted.
  • vector in the present invention refers to a DNA construct comprising DNA sequences operably linked to suitable control sequences for expressing 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 invention 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 vectors involved in the present invention are characterization plasmids pEC-XK99E-rfp-1 and pEC-XK99E-rfp-2, pEC-XK99E-rfp-1 and The pEC-XK99E-rfp-2 plasmid maps are shown in Figures 1 and 2, respectively.
  • PlysC is the wild-type promoter of lysC gene
  • lysC180 is the 180 bp coding region at the N-terminal of the wild-type aspartate kinase LysC encoding gene
  • linker is the connecting peptide between the lysC gene and the rfp protein
  • rfp is the For red fluorescent protein (Red Fluorescent Protein, RFP); Kan expressed as kanamycin resistance (Kanamycin resistant).
  • SEQ ID NO: 1 represents the promoter shown in SEQ ID NO: 1; lysC180 represents the 180 bp coding region at the N-terminus of the wild-type aspartate kinase LysC encoding gene; linker represents the position between the lysC gene and the rfp protein
  • the connecting peptide of ; rfp is expressed as red fluorescent protein (Red Fluorescent Protein, RFP); Kan is expressed as kanamycin resistant (Kanamycin resistant).
  • host cell in the present invention means any cell type that is readily transformed, transfected, transduced, etc. with a transcription initiation element or expression vector comprising a polynucleotide of the present invention.
  • 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 invention 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 invention is a prokaryotic cell, as long as it is a cell into which the polynucleotide having transcriptional expression regulation activity of the present invention can be introduced.
  • a host cell refers to a microorganism 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.
  • the Corynebacterium glutamicum can be Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067, etc. and derivative strains thereof.
  • the derivative strain may be any strain as long as the strain has the ability to produce L-amino acids.
  • the host cell is a lysine-producing host cell.
  • the host cell for lysine production may be a derivative strain expressing feedback-relieved aspartokinase based on Corynebacterium glutamicum ATCC 13032.
  • the lysine-producing host cells may also be other kinds of strains having lysine-producing ability.
  • the host cell for producing lysine may also include, but is not limited to, one or more genes selected from the group consisting of weakened 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-relieved 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 group consisting of the threonine-producing host cells are enhanced or overexpressed:
  • 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 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 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 of the host cells of the present invention can be carried out 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 invention adopts the pEC-XK99E-rfp-2 plasmid in the (the underline is the sequence of positions 366 to 373 of SEQ ID NO: 1, which is a partial sequence of the RBS spacer; the bold is the start codon of the lysC gene) to be transformed into a sequence
  • the sequences containing the above mutations were cloned and connected with the pEC-XK99E plasmid backbone fragment by Novozan's one-step recombination kit to obtain pEC-XK99E-rfp-3, pEC-XK99E-rfp-4 and pEC-XK99E-rfp- 5 Characterize the vector.
  • ATCC13032 pEC-XK99E-rfp-3
  • ATCC13032 pEC-XK99E-rfp-4
  • ATCC13032 pEC-XK99E-rfp-5 strains.
  • ATCC13032 pEC-XK99E-rfp-2
  • it has higher lysC gene transcriptional expression regulation activity.
  • the polynucleotide with enhanced lysC gene expression activity is operably connected with the encoding gene of the enzyme involved in synthesizing amino acids to obtain a recombinant expression vector capable of synthesizing the enzyme involved in synthesizing amino acids, and the recombinant expression vector is utilized to transform host cells to obtain recombinant host cells.
  • the host cell is Corynebacterium glutamicum.
  • the recombinant host cell is Corynebacterium glutamicum modified as follows: 1) the aspartokinase encoding gene lysC in Corynebacterium glutamicum ATCC13032 has introduced a T311I mutation coding sequence; The core region from position 279 to position 317 of the pyc promoter of the pyruvate carboxylase gene in Corynebacterium glutamicum is CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG; 3) The first ddh promoter of the diaminopimelate dehydrogenase gene in Corynebacterium glutamicum Nucleotides from positions 279 to 317 are mutated from wild-type ATGCATCTC to CCTTGTTAT; 4) Recombination of polynucleotides containing SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 with transcription
  • the host cells according to the present invention have improved aspartokinase gene expression compared to wild type. Because aspartokinase is the most important enzyme in the lysine biosynthetic pathway, fermentation of the host cell results in the production of lysine in higher yields.
  • the fermentation of the transformant can be carried out by a known method, and the fermentation conditions including temperature, time, pH and the like can be appropriately controlled.
  • the following documents provide a detailed description of the fermentation [Chmiel; Bioreatechnik 1. Einbowung in die Biovonstechnik (Gustav Fischer Verlag, Stuttgart, 1991), and Storhas; Bioreaktoren undperiphere River Junior (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)]. Fermentation can be achieved by batch culture, continuous culture or fed-batch culture.
  • the medium may contain, for example, saccharides and carbohydrates (eg, glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), lipids and fats (eg, soybean oil, sunflower oil, peanut oil) and coconut oil), fatty acids (such as palmitic acid, stearic acid, rinoleic acid), alcohols (such as glycerol and ethanol), and organic acids (such as acetic acid).
  • saccharides and carbohydrates eg, glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose
  • lipids and fats eg, soybean oil, sunflower oil, peanut oil
  • coconut oil fatty acids
  • fatty acids such as palmitic acid, stearic acid, rinoleic acid
  • alcohols such as glycerol and ethanol
  • organic acids such as acetic acid
  • nitrogen-containing organic compounds such as peptone, yeast extract, broth, wort, corn steep liquor, soybean meal, and urea
  • nitrogen-containing inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and nitric acid
  • examples of phosphorus sources for the medium include dipotassium hydrogen phosphate, potassium dihydrogen phosphate and the corresponding sodium salts.
  • the medium may contain metal salts necessary for cell growth (eg, magnesium sulfate or ferric sulfate), and may be supplemented with essential nutrients such as amino acids and vitamins to stimulate growth.
  • metal salts necessary for cell growth eg, magnesium sulfate or ferric sulfate
  • essential nutrients such as amino acids and vitamins to stimulate growth.
  • appropriate precursors can be added to the medium. Nutrients and additives can be added all at once or separately during fermentation.
  • the pH of the medium can be adjusted with basic compounds such as sodium hydroxide, potassium hydroxide or ammonia, or acidic compounds such as phosphoric acid or sulfuric acid.
  • the formation of foam in the medium can be inhibited with antifoaming agents such as polyethylene glycol fatty acid esters.
  • the medium can be maintained under aerobic conditions by introducing oxygen or oxygen-containing gas mixtures therein. For the incubation temperature, it is usually between 20°C and 45°C, preferably between 25°C and 40°C. Fermentation continued until the maximum amount of L-lysine was produced. In this regard, it can be done within 10 to 160 hours. After L-lysine is produced, L-lysine can be exported to the medium or can be maintained within the cell.
  • the culture conditions of the recombinant host cells are: firstly inoculate the strain into TSB liquid medium for 8 hours, and inoculate the culture as a seed into a 24-well plate containing 800 ⁇ l of fermentation medium per well, and the initial OD 600 control was about 0.1, cultured at 30 °C for 19 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, 8g/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, 40 g/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.
  • Embodiment 1 Synthetic expression control sequences enhance the expression of the lysC gene of Corynebacterium glutamicum
  • gene expression control sequences and N-terminal coding regions are key regions that affect gene expression.
  • the invention adopts the method of sequentially connecting the expression control region of the gene, the 180bp coding region at the N end of the gene, a flexible linker and a red fluorescent protein gene rfp, and characterizes the expression intensity of the expression control sequence of the lysC gene based on the fluorescence intensity.
  • a vector is first constructed to characterize the expression strength of the expression control sequence of the lysC gene of Corynebacterium glutamicum.
  • the specific construction is as follows: On the basis of the pEC-XK99E plasmid backbone, the lysC gene's own promoter and RBS express the 60 amino acids at the N-terminal of the lysC gene, a linker peptide and the red fluorescent protein gene.
  • Plasmid was used as template, and pEC-1/2 primers were used to amplify the DNA fragment of pEC-XK99E plasmid backbone, connecting peptide (DNA sequence: GGCGGTGGCTCTGGAGGTGGTGGGTCCGGCGGTGGCTCT) and red fluorescent protein gene.
  • the above two fragments were cloned and connected by Novozan's one-step recombination kit to obtain pEC-XK99E-rfp-1 characterization vector.
  • the plasmid map is shown in Figure 1.
  • the present embodiment artificially synthesized the expression control sequence shown in SEQ ID NO: 1, and inserted it upstream of lysC gene.
  • a characterisation vector was further constructed based on the pEC-XK99E-rfp-1 characterisation vector. The specific construction is as follows: using the nucleotide sequence synthesized by DNA Synthesis Company as the expression control sequence shown in SEQ ID NO: 1 as a template, and using pyc-F/R as a primer to amplify an artificial sequence with a 20bp homology arm.
  • the plasmid backbone was amplified with pEC-XK99E-rfp-1 plasmid as template and pEC-3/4 primers.
  • the above two fragments were cloned and connected by Novozan's one-step recombination kit to obtain pEC-XK99E-rfp-2 characterization vector.
  • the plasmid map is shown in Figure 2.
  • the primer sequences used in this example are shown in Table 1.
  • the constructed pEC-XK99E-rfp-1 and pEC-XK99E-rfp-2 were respectively transformed into Corynebacterium glutamicum ATCC13032 to obtain ATCC13032 (pEC-XK99E-rfp-1) and ATCC13032 (pEC-XK99E-rfp-2) strains.
  • 96-well plate culture was used to characterize the intensity of lysC expressed by the synthetic expression control sequence (SEQ ID NO: 1).
  • composition of TSB liquid 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, 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.
  • TSB solid medium was supplemented with 15g/L agar powder.
  • the strains obtained from the TSB plate were inoculated into a 96-well plate containing 200 ⁇ l of TSB liquid medium in each well with a toothpick, and each strain was 3 parallel.
  • the fluorescence intensity (excitation wavelength: 560 nm, emission wavelength: 607 nm).
  • the measurement results are shown in Table 2.
  • the fluorescence intensity of the artificially synthesized expression control sequence (SEQ ID NO: 1) was increased by 4.8 times, indicating that the insertion of the above artificial expression control sequence can enhance the expression intensity of the lysC gene.
  • Embodiment 2 Synthetic expression control sequence and lysC gene start codon modification to further enhance the expression intensity of lysC gene
  • the modification of the base sequence of the RBS spacer region and the start codon can be used to further enhance the expression strength. (underlined is the sequence of positions 366 to 373 of SEQ ID NO: 1, which is a partial sequence of the RBS spacer; bold is the start codon of the lysC gene) to transform into a sequence
  • SEQ ID NO: 2 SEQ ID NO: 3
  • SEQ ID NO: 4 SEQ ID NO: 4
  • the specific construction is as follows: using plasmid pEC-XK99E-rfp-2 as template and RBS1/pEC-5, RBS2/pEC-5 and RBS3/pEC-5 as primers, respectively amplify three fragments including three modified regions.
  • the plasmid backbone was amplified with pEC-XK99E-rfp-2 plasmid as template and pEC-6/7 as primers.
  • the above three fragments including the modified region were cloned and connected to the plasmid backbone fragment by Novozan's one-step recombination kit to obtain pEC-XK99E-rfp-3, pEC-XK99E-rfp-4 and pEC-XK99E-rfp-5 respectively Characterization vector.
  • the primer sequences used in this example are shown in Table 3.
  • the pEC-XK99E-rfp-3, pEC-XK99E-rfp-4 and pEC-XK99E-rfp-5 plasmids were respectively transformed into Corynebacterium glutamicum ATCC13032 to obtain ATCC13032 (pEC-XK99E-rfp-3), ATCC13032 (pEC-XK99E-rfp-4) and ATCC13032 (pEC-XK99E-rfp-5) strains.
  • the above strains and the control strain ATCC13032 used the same strategy in Example 1 to measure the fluorescence intensity again.
  • the results are shown in Table 4, the fluorescence intensity of ATCC13032 (pEC-XK99E-rfp-3), ATCC13032 (pEC-XK99E-rfp-4) and ATCC13032 (pEC-XK99E-rfp-5) strains were increased by 1.3 times compared with the control. , 2.7 times, and 5.0 times, indicating that the modification of the start codon and upstream RBS spacer sequence can further enhance the expression of lysC gene.
  • SEQ ID NO: 2 SEQ ID NO: 3
  • SEQ ID NO: 4 were inserted upstream of the lysC gene on the genome of Corynebacterium glutamicum ATCC13032 in the same manner as before to enhance Expression of the lysC gene.
  • the genetic modification of L-lysine-producing bacteria SCgL40 of Corynebacterium glutamicum includes: introducing the amino acid mutation T311I (base mutation from AC C to ATC) of the aspartokinase gene lysC of Corynebacterium glutamicum ATCC13032, pyruvate
  • the nucleotides from 279 to 317 of the carboxylase gene pyc promoter were mutated from wild-type CGATGTTTGATTGGGGGAATCGGGGTTACGATACTAGG to CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG, and the 279 to 317 nucleotides of the diaminopimelate dehydrogenase gene ddh promoter Acid was mutated from wild-type ATGCATCTC to CCTTGTTAT.
  • the recombinant vectors pK18-2, pK18-3 and pK18-4 constructed in the above (1) were transformed into Corynebacterium glutamicum lysine-producing bacteria SCgL40 respectively, and the coating containing 5g/L Glucose and 25 ⁇ g/mL kanamycin on LBHIS solid medium, cultured at 30°C to obtain the first recombination transformants.
  • the correct primary recombinant transformants were respectively inoculated into LB medium containing 5g/L glucose, cultured overnight, diluted and coated with LB solid medium plates supplemented with 100g/L sucrose respectively, and cultured on resistant and non-resistant plates at the same time.
  • the obtained clones were screened for kanamycin sensitivity.
  • the clones sensitive to kanamycin were amplified and sequenced by PCR using primers lysC-C1 (sequence: CCCAGTTCAAGATGAGTCCC) and lysC-C2 (sequence: CCGGGATCATTACTATAAGACG), and the clones with correct sequencing were obtained after the lysC gene expression sequence was transformed.
  • Strains SCgL42, SCgL43 and SCgL44 were obtained after the lysC gene expression sequence was transformed.
  • 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. The results are shown in Table 6. After the lysC gene expression element was modified, the lysine yield and the sugar-acid conversion rate of the strain were improved, and the yield of the SCgL42 strain was increased by 89%. The above results indicate that expression sequences that enhance the expression intensity of the lysC gene can be applied to L-lysine production.
  • the method for enhancing the expression of the lysC gene disclosed in the present invention can also be used to increase the yield of the downstream products of the reactions catalyzed by LysC. Therefore, the technical solution provided by the present invention can also be used for L-threonine, L-isoleucine, L-homoserine, L-methionine and L-lysine downstream products such as pentamethylenediamine, 5- Production of aminovaleric acid, glutaric acid, etc.

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Abstract

本发明公开了一种天冬氨酸激酶基因的表达调控序列及其应用。通过对SEQ ID NO：1所示序列的第366位至373位进行改造，将该序列与起始密码子GTG或TTG连接后形成的多核苷酸，具有转录表达调控活性，这些多核苷酸能够增加天冬氨酸激酶编码基因lysC的表达，从而实现L-赖氨酸的大量积累。

Description

一种天冬氨酸激酶基因表达调控序列及其应用
本发明要求2021年03月23日向中国国家知识产权局提交的专利申请号为202110305010.6，发明名称为“一种天冬氨酸激酶基因表达调控序列及其应用”的在先申请的优先权。该件在先申请的全文通过引用的方式结合于本发明中。
技术领域
本发明属于分子生物学和生物工程领域，具体涉及用于调控天冬氨酸激酶基因转录表达的多核苷酸序列，以及利用该多核苷酸序列转录表达天冬氨酸激酶进而生产L-赖氨酸的方法。
背景技术
L-赖氨酸，简称赖氨酸，为碱性氨基酸，是人类和动物营养中最重要的必需氨基酸，由于谷物食品中的赖氨酸含量甚低，且在加工过程中易被破坏而缺乏，故称为第一限制性氨基酸，被广泛应用于医药、健康、食品、动物饲料等行业中。赖氨酸主要采用微生物发酵法来生产，目前，主要的生产菌株包括大肠杆菌、谷氨酸棒杆菌等的微生物。
L-赖氨酸的合成途径在许多微生物中都是从天冬氨酸起始的，包括两步和甲硫氨酸和苏氨酸共用的步骤，经过九步的酶催化过程，最终产生L-赖氨酸。其中，天冬氨酸激酶(Aspartate Kinase，ASK或AK，编码基因lysC)是L-赖氨酸合成途径中天冬氨酸起始的第一个酶，它的活力决定着代谢流流向L-赖氨酸合成途径的比例，是赖氨酸生产的限速步骤。天冬氨酸激酶的活力在微生物体内受复杂的调控。在许多L-赖氨酸的生产菌 中，天冬氨酸激酶的活力受到终产物L-赖氨酸的负反馈抑制。目前已报道了大量的解除反馈抑制的天冬氨酸激酶突变体，如EP2374873A1。而如何进一步提高解除了反馈抑制的天冬氨酸激酶的表达，是微生物高产赖氨酸的必需途径。
发明内容
为了解决现有技术中存在的问题，本发明的目的之一在于提供增强lysC基因表达调控的多核苷酸序列。本发明的发明人经过大量的工作发现，通过对人工合成的表达调控序列(其核苷酸序列如SEQ ID NO：1所示)的核糖体结合位点(ribosomebinding site，以下简称RBS)和起始密码子之间的间隔区部分序列TTACTCTA(即SEQ ID NO：1所示多核苷酸序列的第366位至373位)进行改造，将该序列与起始密码子GTG或TTG连接后形成的多核苷酸序列具有转录表达调控活性，这些具有转录表达调控活性的多核苷酸序列能够增加天冬氨酸激酶编码基因lysC的表达，从而实现L-赖氨酸的大量积累，在此基础上完成了本发明。
本发明的目的之二是提供含有多核苷酸序列的载体，该多核苷酸序列显示改进的启动子活性。
本发明的目的之三是提供含有lysC基因表达调控序列的宿主细胞。
本发明的目的之四是提供通过发酵宿主细胞产生L-赖氨酸的方法。
为了实现上述目的，本发明采用如下技术方案：
在第一方面，本发明提供一种用于基因表达调控的多核苷酸，所述多核苷酸为：
(1)在SEQ ID NO：1所示序列的第366位至373位进行一个或多个位置的核苷酸的取代和/或缺失，将所示经核苷酸取代和/或缺失后所得的序列与起始密码子GTG连接后形成的多核苷酸序列；
或者，
(2)在SEQ ID NO：1所示序列的第366位至373位进行一个或多个位置的核苷酸的取代和/或缺失，将所示经核苷酸取代和/或缺失后所得的序列与起始密码子TTG连接形成的多核苷酸序列；
与未改造的SEQ ID NO：1相比，所述多核苷酸具有增强lysC基因表达的活性。
优选地，所述多核苷酸的核苷酸序列如SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示。
在第二方面，本发明提供一种包含上述多核苷酸的载体。
优选地，所述载体是以pK18mobsacB为骨架，所述pK18mobsacB的GenBank登记号为FJ437239.1。
在第三方面，本发明提供一种用于lysC基因表达调控的表达盒，所述表达盒为将上述多核苷酸与无起始密码子的lysC基因可操作地连接形成的多核苷酸。
优选地，所述多核苷酸的核苷酸序列如SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示。
在第四方面，本发明提供一种包含上述多核苷酸，或上述表达盒的宿主细胞。
优选地，所述多核苷酸的核苷酸序列如SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示。
任选地，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或谷氨酸棒杆菌ATCC 14067及其衍生菌株。
在第五方面，本发明提供一种增强天冬氨酸激酶编码基因lysC表达的方法，所述方法为将上述多核苷酸与无起始密码子的lysC基因可操作地连接。
优选地，所述多核苷酸的核苷酸序列如SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示。
优选地，所述可操作地连接为将含有上述多核苷酸的载体导入宿主细胞中，通过同源重组整合到宿主细胞的基因组中。
进一步优选地，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或谷氨酸棒杆菌ATCC 14067。
更进一步优选地，所述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ATCC13032中的天冬氨酸激酶编码基因lysC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pyc启动子的第279位至第317位的核心区为CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG；3)谷氨酸棒杆菌中二氨基庚二酸脱氢酶基因ddh启动子的第279位至第317位的核苷酸由野生型ATGCATCTC突变为CCTTGTTAT。
第六方面，本发明提供第一方面的多核苷酸、第二方面的载体、第三方面的表达盒、第四方面的宿主细胞在制备L-赖氨酸中的应用。
第七方面，本发明提供了一种生产L-赖氨酸的方法，所述方法包括培养第四方面的宿主细胞，使之生产L-赖氨酸的步骤。
任选地，包括从发酵液中分离L-赖氨酸的步骤。
第八方面，本发明提供第一方面的多核苷酸、第二方面的载体、第三方面的表达盒、第四方面的宿主细胞在制备天冬氨酸家族氨基酸及其衍生物中的应用，所述天冬氨酸家族氨基酸及其衍生物包括L-苏氨酸、L-异亮氨酸、L-高丝氨酸、L-甲硫氨酸和L-赖氨酸 下游产物如戊二胺、5-氨基戊酸、戊二酸。
本发明的有益效果
本发明提供了具有增强lysC基因表达活性的多核苷酸，将其与目标基因lysC可操作地连接，可以显著提高lysC的表达强度，进而稳定、高效的生产下游产物。
本发明提供了生产L-赖氨酸的方法，利用上述具有基因转录表达调控活性的多核苷酸，能够提高天冬氨酸激酶的表达，进而稳定、高效的生产L-赖氨酸，L-赖氨酸产量可达到起始菌株的1.9倍。
附图说明
图1示出了pEC-XK99E-rfp-1质粒图谱；
图2示出了pEC-XK99E-rfp-2质粒图谱。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。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。
定义与说明：
本发明所用的术语″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。
本发明所用的″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。
本发明所用的″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″。
本发明所用的选择/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。
本发明中的术语″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。
本发明中的术语″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，本发明中的突变(包含，取代、插入和/或缺失)特指其中的取代和缺失，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。
在一些具体的实施方案中，本发明的″突变″包含在SEQ ID NO：1所示序列的RBS间隔区的部分序列TTACTCTA(即SEQ ID NO：1所示序列的第366位至373位)进行一个或多个位置的核苷酸的取代和/或缺失，将该序列与起始密码子GTG或TTG连 接后形成的多核苷酸序列，与未改造的SEQ ID NO：1相比，具有增强lysC表达的活性。
示例性地，本发明的″突变″包含在SEQ ID NO：1所示序列的第366位至373位进行1个、2个、3个、4个、5个、6个、7个、8个位置的取代和/或缺失。本发明的″突变″包含在SEQ ID NO：1所示序列的第363位至373位进行6个核苷酸的缺失，和/或对缺失后的序列进行0-2个核苷酸的取代，并与起始密码子GTG或TTG连接。上述突变后形成的SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4，与未被取代的SEQ ID NO：1相比，具有更高的基因转录表达调控活性。
本发明中的术语″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。
本发明中的术语″RBS间隔区″是指位于原核生物启动子区的一段核酸序列，是RBS与起始密码子之间的一段核苷酸序列。
本发明中的术语″起始密码子″具有本领域技术人员公知的定义，是指蛋白质合成起始位点的密码子，通常包括ATG、GTG、TTG和ATA。示例性的，本发明SEQ ID NO：2和SEQ ID NO：3中包含的起始密码子为GTG，SEQ ID NO：4包含的起始密码子为TTG。
本发明中的术语″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。
本发明中的术语″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。在本发明中，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。在本发明中，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，所述可操作地连接的方式可以采用本领域技术人员所述的任何方式。示例性的，本发明的目标基因与多核苷酸可操作地连接为将SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4与lysC基因可操作连接后的多核苷酸序列。
本发明中的″lysC基因″指的是谷氨酸棒杆菌来源的天冬氨酸激酶(EC：2.7.2.4)的编码基因，包括但不限于天然存在的谷氨酸棒杆菌来源的lysC基因、解除了赖氨酸反馈抑制的lysC基因突变体(如T311I、D274A、Q298G、N299L、N374A、E382A、R384L等)，以及谷氨酸棒杆菌来源的经过人工改造的仍然具有天冬氨酸激酶活性的lysC基因突变体。示例性的，本发明的lysC基因是谷氨酸棒杆菌ATCC13032的天冬氨酸激酶编码基因，基因编号为NCgl0247或Cgl0251，NCBI数据库Gene ID：1021294，所编码的蛋白Protein ID：NP_599504或WP_003855724.1。
本发明的″无起始密码子的lysC基因″是指缺失了起始密码子GTG的天冬氨酸激酶(EC：2.7.2.4)的编码基因。
本发明中的术语″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。用于本发明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，本发明涉及的载体为基于pEC-XK99E-rfp质粒构建的具有表达调控序列的表征质粒pEC-XK99E-rfp-1和pEC-XK99E-rfp-2，pEC-XK99E-rfp-1和pEC-XK99E-rfp-2质粒图谱分别如图1和2所示。图1中P lysC表示为lysC基因的野生型启动子；lysC180表示野生型天冬氨酸激酶LysC编码基因N端的180bp编码区；linker表示为位于lysC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)。图2中SEQ ID NO：1表示为SEQ ID NO：1所示的启动子；lysC180表示野生型天冬氨酸激酶LysC编码基因N端的180bp编码区；linker表示为位于lysC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-rfp-1和pEC-XK99E-rfp-2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。
本发明中的术语″宿主细胞″意指易于用包含本发明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。
本发明中的术语″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。
本发明的宿主细胞是原核细胞，只要是能够导入本发明的具有转录表达调控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或谷氨酸棒杆菌ATCC 14067等及其衍生菌株等。示例地，所述衍生菌株可以是任何菌株，只要该菌株具有生产L-氨基酸的能力。
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衍生菌株。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。
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。
本发明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，否则在本发明中的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。
具有增强lysC基因的表达活性的多核苷酸
本发明通过对pEC-XK99E-rfp-2质粒中的
Figure PCTCN2022081527-appb-000001
(下划线为SEQ ID NO：1的第366位至373位序列，是RBS间隔区的部分序列；粗体为lysC基因的起始密码子)进行改造，改造为序列
Figure PCTCN2022081527-appb-000002
并将含有以上突变的序列与pEC-XK99E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-rfp-3、pEC-XK99E-rfp-4和pEC-XK99E-rfp-5表征载体。将以上质粒转化谷氨酸棒杆菌ATCC13032，获得ATCC13032(pEC-XK99E-rfp-3)、ATCC13032(pEC-XK99E-rfp-4)和ATCC13032(pEC-XK99E-rfp-5)菌株。与对照菌株ATCC13032(pEC-XK99E-rfp-2)相比，具有更高的lysC基因转录表达调控活性。
赖氨酸的生产过程
(1)本发明中将具有增强lysC基因表达活性的多核苷酸，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赖氨酸，完成赖氨酸的生产过程。
在一些实施方案中，宿主细胞为谷氨酸棒杆菌(Corynebacterium glutamicum)。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ATCC13032中的天冬氨酸激酶编码基因lysC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pyc启动子的第279位至第317位的核心区为CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG；3)谷氨酸棒杆菌中二氨基庚二酸脱氢酶基因ddh启动子的第279位至第317位的核苷酸由野生型ATGCATCTC突变为CCTTGTTAT；4)将含有具有转录表达调控活性的多核苷酸SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4的重组载体pK18-1、pK18-2、pK18-3或pK18-4转化上述谷氨酸棒杆菌，获得重组的谷氨酸棒杆菌。以上述方法改造的谷氨酸棒杆菌，是L-赖氨酸的高产菌株。
与野生型相比，根据本发明所述的宿主细胞在天冬氨酸激酶基因表达上有所改良。因为天冬氨酸激酶是赖氨酸生物合成途径中最重要的酶，所以宿主细胞的发酵引起赖氨酸较高产率的产生。在本发明中，可利用公知方法进行转化体的发酵，并可适当地控制发酵条件，包括温度、时间、pH等。以下文件提供了发酵的详细描述[Chmiel；Bioprozesstechnik 1.Einfuhrung in die Bioverfahrenstechnik(Gustav Fischer Verlag，Stuttgart，1991)，以及Storhas；Bioreaktoren undperiphere Einrichtungen(Vieweg Verlag，Braunschweig/Wiesbaden，1994)]。发酵可通过分批培养、连续培养或补料分批培养实现。
用于发酵，培养基必须满足所用菌株的需要。适用于培养各种微生物的培养基是本领域公知的(如，来自American Society forBacteriology(Washington D.C.，USA，1981)的″Manual of Methods forGeneral Bacteriology″)。培养基可包含如碳源糖(saccharides)和碳水化合物(carbohydrates)(如葡萄糖、蔗糖、乳糖、果糖、麦芽糖、糖蜜、淀粉和纤维素)，脂质和脂肪(如豆油、葵花籽油、花生油和椰子油)，脂肪酸(如 棕榈酸、硬脂酸、蓖麻酸(rinoleic acid))，醇(如甘油和乙醇)和有机酸(如醋酸)。这些物质可分别使用或组合使用。作为氮源，含氮有机化合物(如蛋白胨、酵母提取物、肉汤、麦芽汁、玉米浆、豆粕和尿素)或含氮无机化合物(如硫酸铵、氯化铵、磷酸铵、碳酸铵和硝酸铵)可分别使用或组合使用。用于培养基的磷源的实例包括磷酸氢二钾、磷酸二氢钾和相应的钠盐。
另外，培养基可包含细胞生长必需的金属盐(如硫酸镁或硫酸铁)，并可补充必需营养物如氨基酸和维生素以刺激生长。此外，可向培养基添加适当的前体。可一次一起或在发酵过程中分别添加营养物和添加剂。
培养基的pH可用碱性化合物(如氢氧化钠、氢氧化钾或氨水)或酸性化合物(如磷酸或硫酸)调节。培养基中泡沫的生成可用消泡剂如聚乙二醇脂肪酸酯抑制。可通过在其中引入氧气或含氧气体混合物将培养基保持在有氧条件下。对于培养温度，通常在20℃到45℃之间，优选地在25℃到40℃之间。发酵连续进行直到产生L-赖氨酸的最大量。在这方面，可在10到160小时内完成。L-赖氨酸产生后，L-赖氨酸可输出至培养基或可保持在细胞内。
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19h，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，8g/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。
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1.人工合成表达调控序列增强谷氨酸棒杆菌lysC基因的表达
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。本发明采用基因的表达调控区、基因N端的180bp编码区、一个柔性linker和一个红色荧光蛋白基因rfp顺序连接的方法，基于荧光强度对lysC基因的表达调控序列进行表达强度表征。
本实施例首先构建1个表征谷氨酸棒杆菌lysC基因自身表达调控序列表达的表达强度的载体。具体构建如下：在pEC-XK99E质粒骨架基础上，由lysC基因自身启动子和RBS表达lysC基因N端60个氨基酸、一个连接肽和红色荧光蛋白基因。根据已公开的谷氨酸棒杆菌ATCC13032基因组序列(Gene ID：2830649)及lysC基因注释信息，设计引物lysC-F/R，以ATCC13032基因组为模板，通过PCR扩增获得lysC基因启动子、RBS和N端180bp的DNA片段。以文献报道的pEC-XK99E-rfp(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)质粒为模板，以pEC-1/2引物，扩增pEC-XK99E质粒骨架、连接肽(DNA序列为：GGCGGTGGCTCTGGAGGTGGTGGGTCCGGCGGTGGCTCT)和红色荧光蛋白基因的DNA片段。以上两个片段通过诺唯赞的一步重组试剂盒克隆连接，获得pEC-XK99E-rfp-1表征载体，质粒图谱如图1所示。
为了增强lysC基因的表达强度，本实施例人工合成了SEQ ID NO：1所示的表达 调控序列，并将其插入lysC基因上游。为表征插入人工合成表达调控序列增强表达强度的效果，在pEC-XK99E-rfp-1表征载体基础上进一步构建表征载体。具体构建如下：以DNA合成公司合成的核苷酸序列如SEQ ID NO：1所示的表达调控序列为模板，以pyc-F/R为引物扩增带有20bp同源臂的人工序列。以pEC-XK99E-rfp-1质粒为模板，以pEC-3/4引物，扩增质粒骨架。以上两个片段通过诺唯赞的一步重组试剂盒克隆连接，获得pEC-XK99E-rfp-2表征载体，质粒图谱如图2所示。本实施例所用引物序列如表1所示。
表1
Figure PCTCN2022081527-appb-000003
为表征插入人工合成表达调控序列(SEQ ID NO：1)增强表达的强度，将构建的pEC-XK99E-rfp-1和pEC-XK99E-rfp-2分别转化至谷氨酸棒杆菌ATCC13032，获得ATCC13032(pEC-XK99E-rfp-1)和ATCC13032(pEC-XK99E-rfp-2)菌株。采用96孔板培养表征人工合成表达调控序列(SEQ ID NO：1)表达lysC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。TSB固体培养基再补充15g/L琼脂粉。将TSB平板获得的菌株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(激发波长：560nm，发射波长：607nm)。测定结果如表2所示，插入人工合成表达调控序列(SEQ ID NO：1)荧光强度提高了4.8倍，说明插入以上人工表达调控序列可以增强lysC基因的表达强度。
表2
菌株 荧光强度(RFU/OD 600)
ATCC13032(pEC-XK99E-rfp-1) 277±3
ATCC13032(pEC-XK99E-rfp-2) 1613±38
实施例2.人工合成表达调控序列及lysC基因起始密码子改造进一步增强lysC基因的表达强度
RBS间隔区碱基序列和起始密码子的改造可用于进一步增强表达强度，本实施例对pEC-XK99E-rfp-2质粒中的
Figure PCTCN2022081527-appb-000004
(下划线为SEQ ID NO：1的第366位至 373位序列，是RBS间隔区的部分序列；粗体为lysC基因的起始密码子)进行改造，改造为序列
Figure PCTCN2022081527-appb-000005
改造后获得的具有表达调控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-rfp-2质粒为模板，以RBS1/pEC-5、RBS2/pEC-5、RBS3/pEC-5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-6/7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-rfp-3、pEC-XK99E-rfp-4和pEC-XK99E-rfp-5表征载体。本实施例所用引物序列如表3所示。
表3
Figure PCTCN2022081527-appb-000006
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-rfp-3、pEC-XK99E-rfp-4和pEC-XK99E-rfp-5质粒分别转化至谷氨酸棒杆菌ATCC13032，获得ATCC13032(pEC-XK99E-rfp-3)、ATCC13032 (pEC-XK99E-rfp-4)和ATCC13032(pEC-XK99E-rfp-5)菌株。以上菌株及对照菌株ATCC13032(pEC-XK99E-rfp-2)再次采用实施例1相同策略测定荧光强度。结果如表4所示，ATCC13032(pEC-XK99E-rfp-3)、ATCC13032(pEC-XK99E-rfp-4)和ATCC13032(pEC-XK99E-rfp-5)菌株的荧光强度分别比对照提高了1.3倍、2.7倍、5.0倍，表明起始密码子及上游RBS间隔区序列改造可以进一步增强lysC基因的表达。
表4
菌株 荧光强度(RFU/OD600)
ATCC13032(pEC-XK99E-rfp-2) 1711±66
ATCC13032(pEC-XK99E-rfp-3) 3928±963
ATCC13032(pEC-XK99E-rfp-4) 6280±1324
ATCC13032(pEC-XK99E-rfp-5) 8509±144
实施例3.改造后的表达调控序列应用于L-赖氨酸生产
(1)谷氨酸棒杆菌lysC基因表达调控序列改造的重组载体构建
根据实施例1和2表征的表达强度，分别将SEQ ID NO：2、SEQ ID NO：3、和SEQ ID NO：4按照前面相同方式插入谷氨酸棒杆菌ATCC13032基因组上lysC基因上游，以增强lysC基因的表达。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lysC-UF/lysC-UR为引物，PCR扩增部分上游同源臂；分别以pEC-XK99E-rfp-3、pEC-XK99E-rfp-4和pEC-XK99E-rfp-5质粒为模板，以Pyc-RBS1/Pyc-RBS2为引物，分别扩增对应的表达调控序列和部分上下游同源臂；以lysC-DF/lysC-DR为引物，PCR扩增另一部分下游同源臂；以pK18-1F/2R 引物扩增pK18mobsacB(GenBank：FJ437239.1)的骨架。上述4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lysC基因表达调控序列改造的重组载体pK18-2、pK18-3和pK18-4。以上所用引物序列如表5所示。
表5
Figure PCTCN2022081527-appb-000007
(2)将lysC基因表达调控序列改造的重组载体引入谷氨酸棒杆菌赖氨酸生产菌
为了验证lysC基因表达序列改造对L-赖氨酸生产的影响，本发明首先构建一个可以生产L-赖氨酸的基础菌株SCgL40。谷氨酸棒杆菌L-赖氨酸生产菌SCgL40的遗传改造包括：将谷氨酸棒杆菌ATCC13032天冬氨酸激酶基因lysC引入了T311I(碱基由AC C突变为ATC)氨基酸突变，丙酮酸羧化酶基因pyc启动子的第279位至第317位的核苷酸由野生型CGATGTTTGATTGGGGGAATCGGGGGTTACGATACTAGG突变为CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG，二氨基庚二酸脱氢酶基因ddh启动子的第279位至第317位的核苷酸由野生型ATGCATCTC突变为CCTTGTTAT。为了进一步引入lysC基因表达调控序列改造，将上述(1)构建的重组载体pK18-2、pK18-3和pK18-4分别转化谷氨酸棒杆菌赖氨酸生产菌SCgL40，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，通过抗性平板和无抗性平板同时点板培养对获得的克隆进行卡那霉素敏感性筛选。对卡那霉素敏感的克隆分别采用引物lysC-C1(序列：CCCAGTTCAAGATGAGTCCC)和lysC-C2(序列：CCGGGATCATTACTATAAGACG)进行PCR扩增及测序，测序正确的克隆即为获得的lysC基因表达序列改造后的菌株SCgL42、SCgL43和SCgL44。
(3)谷氨酸棒杆菌赖氨酸生产菌lysC基因表达元件改造突变体的L-赖氨酸生产能力评价
为了测试谷氨酸棒杆菌中lysC基因表达元件改造对菌株产L-赖氨酸的影响，分别对SCgL40、SCgL42、SCgL43和SCgL44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，8g/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19h，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，lysC基因表达元件改造后菌株的赖氨酸产量和糖酸转化率均有提高，SCgL42菌株产量提高达89％。以上结果表明增强lysC基因表达强度的表达序列可应用于L-赖氨酸生产。
表6
菌株 OD 600 L-赖氨酸产量(g/L) 转化率(％)
SCgL40 15.7±0.4 3.7±0.3 6.9±0.5
SCgL42 15.9±0.7 7.0±1.0 14.0±2.0
SCgL43 17.3±0.3 5.0±0.0 8.9±0.2
SCgL44 10.9±0.3 4.7±0.6 14.1±1.0
由于天冬氨酸下游产物的合成均依赖于天冬氨酸激酶LysC催化的反应步骤，通过本发明公开的lysC基因表达增强的方法也可以用于提高依赖LysC催化反应的下游产物的产量。因此，本发明提供的技术方案也可用于L-苏氨酸、L-异亮氨酸、L-高丝氨酸、L-甲硫氨酸和L-赖氨酸下游产物如戊二胺、5-氨基戊酸、戊二酸等的生产。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (20)

  1. 一种用于天冬氨酸激酶基因lysC表达调控的多核苷酸，其特征在于，所述多核苷酸的多核苷酸序列为：
    (1)在SEQ ID NO：1所示序列的第366位至373位进行一个或多个位置的核苷酸的取代和/或缺失，将所示经核苷酸取代和/或缺失后所得的序列与起始密码子GTG连接形成的多核苷酸序列。
    或者，
    (2)在SEQ ID NO：1所示序列的第366位至373位进行一个或多个位置的核苷酸的取代和/或缺失，将所示经核苷酸取代和/或缺失后所得的序列与起始密码子TTG连接形成的多核苷酸序列。
  2. 根据权利要求1所述的多核苷酸序列，其特征在于，所述多核苷酸序列的核苷酸序列如SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示。
  3. 一种包含权利要求1或2所述多核苷酸的载体。
  4. 根据权利要求3所述的载体，其特征在于，所述载体是以pK18mobsacB为骨架，所述pK18mobsacB的GenBank登记号为FJ437239.1。
  5. 一种用于天冬氨酸激酶基因lysC表达调控的表达盒，其特征在于，所述表达盒为将权利要求1或2所述的多核苷酸与无起始密码子的天冬氨酸激酶基因lysC可操作地连接形成的多核苷酸。
  6. 一种包含权利要求1或2所述的多核苷酸或权利要求5所述的表达盒的宿主细胞。
  7. 根据权利要求6所述的宿主细胞，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任一种。
  8. 根据权利要求7所述的宿主细胞，其特征在于，所述宿主细胞为谷氨酸棒杆菌或大肠杆菌。
  9. 根据权利要求8所述的宿主细胞，其特征在于，所述宿主细胞为谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869或谷氨酸棒杆菌ATCC 14067及其衍生菌株。
  10. 根据权利要求9所述的宿主细胞，其特征在于，所述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ATCC 13032中的天冬氨酸激酶编码基因lysC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pyc启动子的第279位至第317位的核心区由野生型CGATGTTTGATTGGGGGAATCGGGGGTTACGATACTAGG突变为CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG；3)谷氨酸棒杆菌中二氨基庚二酸脱氢酶基因ddh启动子的第279位至第317位的核苷酸序列由野生型ATGCATCTC突变为CCTTGTTAT。
  11. 一种增强天冬氨酸激酶编码基因lysC表达的方法，其特征在于，所述方法为将权利要求1或2所述的多核苷酸与无起始密码子的lysC基因可操作地连接。
  12. 根据权利要求11所述的增强天冬氨酸激酶编码基因lysC表达的方法，其特征在于，所述可操作地连接为将含有上述多核苷酸的载体导入宿主细胞中，通过同源重组整合到宿主细胞的基因组中。
  13. 根据权利要求12所述的增强天冬氨酸激酶编码基因lysC表达的方法，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。
  14. 根据权利要求13所述的增强天冬氨酸激酶编码基因lysC表达的方法，其特征在于，所述宿主细胞为谷氨酸棒杆菌或大肠杆菌。
  15. 根据权利要求14所述的增强天冬氨酸激酶编码基因lysC表达的方法，其特征在于，所述宿主细胞为谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869或谷氨酸棒杆菌ATCC 14067及其衍生菌株。
  16. 根据权利要求15所述的增强天冬氨酸激酶编码基因lysC表达的方法，其特征在于，所述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ATCC 13032中的天冬氨酸激酶编码基因lysC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pyc启动子的第279位至第317位的核心区由野生型CGATGTTTGATTGGGGGAATCGGGGGTTACGATACTAGG突变为CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG；3)谷氨酸棒杆菌中二氨基庚二酸脱氢酶基因ddh启动子的第279位至第317位的核苷酸序列由野生型ATGCATCTC突变为CCTTGTTAT。
  17. 权利要求1-2所述的多核苷酸序列、权利要求3或4所述的载体、权利要求5所述的表达盒、权利要求6-10任一项所述的宿主细胞在制备L-赖氨酸中的应用。
  18. 一种生产L-赖氨酸的方法，所述方法包括培养权利要求6-10任一项所述的宿主细胞，使之生产L-赖氨酸，从发酵液中分离L-赖氨酸。
  19. 权利要求1-2所述的多核苷酸、权利要求3或4所述的载体、权利要求5所述的表达盒、权利要求6-10任一项所述的宿主细胞在制备天冬氨酸家族氨基酸及其衍生物中的应用。
  20. 根据权利要求19所述的应用，其特征在于，所述天冬氨酸家族氨基酸及其衍生物为L-苏氨酸、L-异亮氨酸、L-高丝氨酸、L-甲硫氨酸和L-赖氨酸下游产物，所述L-赖氨酸下游产物为戊二胺、5-氨基戊酸、戊二酸。
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Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113755492B (zh) * 2020-07-20 2023-05-30 中国科学院天津工业生物技术研究所 丙酮酸羧化酶基因启动子的突变体及其应用
CN113201536B (zh) * 2020-08-19 2022-09-13 中国科学院天津工业生物技术研究所 具有启动子活性的多核苷酸及其在生产氨基酸中的应用
CN112695036B (zh) * 2021-03-23 2021-07-06 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
CN115322990B (zh) * 2021-05-10 2024-09-20 中国科学院天津工业生物技术研究所 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
CN113278571A (zh) * 2021-05-27 2021-08-20 齐鲁工业大学 一种棒状杆菌工程菌构建方法及应用
CN115449518B (zh) * 2021-06-08 2024-01-26 中国科学院天津工业生物技术研究所 基于mdh基因的具有启动子活性的多核苷酸及其用途
CN114107141B (zh) * 2021-08-19 2022-07-12 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
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
US20250051785A1 (en) * 2021-12-13 2025-02-13 Kao Corporation Novel promoter
CN115896133B (zh) * 2022-08-25 2023-09-05 中国科学院天津工业生物技术研究所 一种谷氨酰激酶的表达调控序列及其应用
CN117327677B (zh) * 2023-09-26 2024-09-20 江南大学 天冬氨酸激酶突变体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1289368A (zh) * 1998-09-25 2001-03-28 味之素株式会社 构建产生氨基酸的细菌的方法,及通过发酵该经构建的产生氨基酸的细菌以制备氨基酸的方法
EP2374873A1 (en) 2010-04-08 2011-10-12 Technische Universität Hamburg-Harburg Modified aspartate kinase from corynebacterium and its application for amino acid production
CN105849249A (zh) * 2013-10-28 2016-08-10 Cj第制糖株式会社 具有增强的l-赖氨酸生产能力的棒杆菌属物种的微生物及使用其生产l-赖氨酸的方法
WO2017100376A2 (en) * 2015-12-07 2017-06-15 Zymergen, Inc. Promoters from corynebacterium glutamicum
CN110951662A (zh) * 2019-12-26 2020-04-03 新疆梅花氨基酸有限责任公司 一种高产赖氨酸的棒状细菌及其构建方法与应用
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 (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0613660A2 (pt) * 2005-07-18 2012-11-06 Basf Ag microorganismo, cassete de expressão de metl, vetor, métodos para produzir metionina, licopeno, nìveis incrementados de um carotenóide desejado, pelo menos dois compostos em um processo de fermentação, um composto carotenóide desejado, pelo menos dois compostos em um processo de fermentação, um composto carotenóide, e um produto quìmico fino contendo enxofre, e para incrementar a capacidade de produção de metionina em um microorganismo, e, sequência de dna
WO2009031565A1 (ja) * 2007-09-04 2009-03-12 Ajinomoto Co., Inc. アミノ酸生産微生物及びアミノ酸の製造法
CN108486082A (zh) * 2012-10-18 2018-09-04 中粮生物化学（安徽）股份有限公司 天冬氨酸激酶iii突变体及其宿主细胞和应用
CN103215286B (zh) * 2012-11-12 2015-11-25 江南大学 用于发酵生产l-赖氨酸的重组dna、菌株及其应用
CN104099308B (zh) * 2013-04-03 2019-05-31 上海凯赛生物技术研发中心有限公司 一种具有天冬氨酸激酶活性的多肽及其应用
CN104845923B (zh) * 2014-02-14 2018-03-23 中国科学院微生物研究所 生产l‑组氨酸的方法及其专用重组菌
US10428359B2 (en) * 2016-10-03 2019-10-01 Ajinomoto Co, Inc. Method for producing L-amino acid
JP7066977B2 (ja) * 2017-04-03 2022-05-16 味の素株式会社 Ｌ－アミノ酸の製造法
CN109750069B (zh) * 2017-11-01 2025-08-01 宁夏伊品生物科技股份有限公司 生产l-赖氨酸的重组菌、其构建方法以及l-赖氨酸的生产方法
CN111471638B (zh) * 2020-05-22 2021-11-23 江南大学 一株产l-高丝氨酸的谷氨酸棒杆菌突变株的构建与应用
CN113755492B (zh) * 2020-07-20 2023-05-30 中国科学院天津工业生物技术研究所 丙酮酸羧化酶基因启动子的突变体及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1289368A (zh) * 1998-09-25 2001-03-28 味之素株式会社 构建产生氨基酸的细菌的方法,及通过发酵该经构建的产生氨基酸的细菌以制备氨基酸的方法
EP2374873A1 (en) 2010-04-08 2011-10-12 Technische Universität Hamburg-Harburg Modified aspartate kinase from corynebacterium and its application for amino acid production
CN105849249A (zh) * 2013-10-28 2016-08-10 Cj第制糖株式会社 具有增强的l-赖氨酸生产能力的棒杆菌属物种的微生物及使用其生产l-赖氨酸的方法
WO2017100376A2 (en) * 2015-12-07 2017-06-15 Zymergen, Inc. Promoters from corynebacterium glutamicum
CN110951662A (zh) * 2019-12-26 2020-04-03 新疆梅花氨基酸有限责任公司 一种高产赖氨酸的棒状细菌及其构建方法与应用
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 (7)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. FJ437239.1
"Manual of Methods for General Bacteriology", 1981, AMERICAN SOCIETY FOR BACTERIOLOGY
JUDITH BECKER, OSKAR ZELDER, STEFAN HäFNER, HARTWIG SCHRöDER, CHRISTOPH WITTMANN: "From zero to hero—Design-based systems metabolic engineering of Corynebacterium glutamicum for l-lysine production", METABOLIC ENGINEERING, ACADEMIC PRESS, AMSTERDAM, NL, vol. 13, no. 2, 15 January 2011 (2011-01-15), AMSTERDAM, NL, pages 159 - 168, XP055523642, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2011.01.003 *
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP4282965A4
WANG Y C 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
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 *

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