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WO2025058109A1 - Molécule d'acide aminé recombiné, cellules hôtes pour la production de l-arginine, et procédés de production de l-arginine les utilisant - Google Patents

Molécule d'acide aminé recombiné, cellules hôtes pour la production de l-arginine, et procédés de production de l-arginine les utilisant Download PDF

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WO2025058109A1
WO2025058109A1 PCT/KR2023/013895 KR2023013895W WO2025058109A1 WO 2025058109 A1 WO2025058109 A1 WO 2025058109A1 KR 2023013895 W KR2023013895 W KR 2023013895W WO 2025058109 A1 WO2025058109 A1 WO 2025058109A1
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seq
sequence
amino acid
acid molecule
host cell
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Ye-Eun Kim
Zeewon LEE
Hanhyoung LEE
Ju Eun Kim
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CJ CheilJedang Corp
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CJ CheilJedang Corp
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Priority to AU2023465726A priority Critical patent/AU2023465726A1/en
Priority to PCT/KR2023/013895 priority patent/WO2025058109A1/fr
Publication of WO2025058109A1 publication Critical patent/WO2025058109A1/fr
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    • 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
    • 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
    • C12N9/93—Ligases (6)
    • 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
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/04—Other carbon-nitrogen ligases (6.3.4)
    • C12Y603/04005—Argininosuccinate synthase (6.3.4.5)

Definitions

  • the present disclosure relates to improved amino acid molecules, host cells expressing the amino acid molecules and use thereof to produce L-arginine, nucleic acid molecules with sequences encoding the amino acid molecule, vectors comprising nucleic acid molecules with sequences encoding the amino acid molecule, and methods of producing L-arginine and for increasing L-arginine production by a host cell expressing the amino acid molecule.
  • L-arginine is an amino acid with extensive application for medical, food, animal feed and industrial applications. L-Arginine is produced by the body and, for example, has effects as a vasodilator. L-Arginine may also be used as a feed additive. Currently, various studies are being conducted to develop host cells and fermentation process technology that produce L-arginine at a high efficiency.
  • An object of the present disclosure provides an amino acid molecule such as, for example, an amino acid molecule protein disclosure herein.
  • Another object of the present disclosure provides a host cell or microorganism expressing the amino acid molecule disclosure herein.
  • Another object of the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid molecule disclosed herein.
  • Another object of the present disclosure provides a vector comprising a nucleic acid molecule disclosed herein.
  • Another object of the present disclosure provides an engineered host cell expressing a recombinant amino acid molecule, including a recombinant protein that is an argininosuccinate synthase.
  • Another object of the present disclosure provides use of the engineered host cell or microorganism disclosed herein for producing L-arginine.
  • Another object of the present disclosure provides a method for producing L-arginine disclosed herein.
  • Figure 1 depicts a protein sequence alignment comparing alignment of exemplary argininosuccinate synthase proteins between E. coli and Corynebacterium glutamicum to determine amino acid residues essential for binding to substrate of argininosuccinate synthase.
  • Figures 2a to 2c depict a sequence alignment carried out on exemplary argininosuccinate synthase proteins of different heterogeneous model microorganism species to determine the non-conserved structures of the substrate binding site.
  • Figure 3 depicts an exemplary tertiary structure of an argininosuccinate synthase protein without the amino acid extension (derived from C. glutamicum ) overlayed with a tertiary structure of an argininosuccinate synthase protein with the amino acid extension (derived from E. coli ) to compare the structures and to predict the role of the amino acid extension in terms of protein function.
  • Figures 4a to 4c list identified exemplary motifs amongst the argininosuccinate synthase proteins of the heterogeneous model microorganisms for which sequence alignment was carried out, along with generalized versions of the motif sequences.
  • Figures 5a to 5f depict the exemplary DNA sequences for the argG gene encoding the argininosuccinate synthase proteins of different heterogeneous model microorganism species.
  • Figure 6 depicts the exemplary DNA sequences for the argR deletion mutation and the argB (M54V) mutation of the argG gene encoding argininosuccinate synthase protein.
  • amino acid molecules including argininosuccinate synthase proteins, host cells, nucleic acid molecules, vectors, and methods described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and microarray and sequencing technology, which are within the skill of those who practice in the art.
  • conventional techniques include polymerase chain reaction (PCR), protein sequence alignment, and sequencing of proteins and oligonucleotides.
  • the present disclosure relates to an improved amino acid molecule including, for example, an improved argininosuccinate synthase protein, which is an enzyme responsible for the rate-limiting reaction step in the process of producing L-arginine.
  • Argininosuccinate synthase catalyzes synthesis of argininosuccinate from substrates citrulline and aspartate. Argininosuccinate is then cleaved by the action of argininosuccinate lyase into fumaric acid and L-arginine.
  • L-arginine is an amino acid and is necessary for the production for nitric oxide, which regulates vasodilation, vascular tone, and blood flow.
  • L-arginine is recognized for its effects as a vasodilator, and has been studied as an option for treating high blood pressure, angina, and erectile dysfunction.
  • the present disclosure further relates to recombinant and non-naturally occurring amino acid molecules including argininosuccinate synthase proteins, engineered host cells capable of expressing the amino acid molecules and producing L-arginine, recombinant nucleic acid molecules and vectors for expressing the amino acid molecules, and use and methods for employing these proteins, host cells, vectors, and nucleic acid molecules to produce L-arginine or to increase production of L-arginine.
  • nucleic acid or protein refers to one, more than one, or mixtures of such regions
  • an assay may include reference to equivalent steps and methods known to those skilled in the art, and so forth.
  • heterologous when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • conservative amino acid substitutions means amino acid sequence modifications which do not abrogate the function or a binding cite of an enzyme.
  • Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix.
  • the term "variant" encompasses but is not limited to enzymes which comprise an amino acid sequence which differs from the amino acid sequence of a reference protein by way of one or more substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the reference protein.
  • the variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.
  • the variant retains the ability, for example, to specifically bind to a substrate of the reference protein.
  • the term variant also includes pegylated proteins.
  • Nucleic acid sequences implicitly encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. Batzer, et al., Nucleic Acid Res. 1991, 19, 5081; Ohtsuka, et al., J. Biol. Chem. 1985, 260, 2605-2608; Rossolini, et al., Mol. Cell. Probes 1994, 8, 91-98. The term nucleic acid is used interchangeably with cDNA, mRNA, oligonucleotide, and polynucleotide.
  • homology refers to the degree of similarity between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.
  • the terms “correspond(s) to” and “corresponding to,” as they relate to sequence alignment, are intended to mean enumerated positions within the reference protein, and those positions in the sequence of interest that align with the positions on the reference protein.
  • the amino acids in the subject sequence that "correspond to" certain enumerated positions of the reference sequence are those that align with these positions of the reference sequence, but are not necessarily in these exact numerical positions of the reference sequence.
  • amino acid molecule generally refer to a chain of amino acids that are held together by peptide bonds (also called amide bonds).
  • amino acid refers to an organic molecule that contains both an amino group (i.e., -NH 2 ) and a carboxylic acid group (i.e., -COOH).
  • nucleotide molecule refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain shape by covalent bonds, more specifically a polynucleotide fragment encoding the protein.
  • the term "enhancement" of polypeptide activity means that the activity of a polypeptide is increased compared to the intrinsic activity.
  • operably linked in the context of a promoter sequence, which initiates and mediates transcription of the polynucleotide encoding the target polypeptide of the present application, means that the promoter sequence and the polynucleotide sequence are functionally linked to each other.
  • the present disclosure provides an amino acid molecule such as, for example, an amino acid molecule protein.
  • the amino acid molecule may comprise a peptide sequence having SEQ ID NO:1, YKPWLDX 1 X 2 FX 3 X 4 EL, in which each of X 1 , X 2 , and X 4 is an amino acid; and X 3 is I or V, is provided herein.
  • X 1 of SEQ ID NO:1 is S. In some embodiments, X 1 of SEQ ID NO:1 is Q. In some embodiments, X 1 of SEQ ID NO:1 is T.
  • X 2 of SEQ ID NO:1 is D. In some embodiments, X 2 of SEQ ID NO:1 is A. In some embodiments, X 2 of SEQ ID NO:1 is T. In some embodiments, X 2 of SEQ ID NO:1 is Q.
  • X 3 of SEQ ID NO:1 is V. In some embodiments, X 3 of SEQ ID NO:1 is I.
  • X 4 of SEQ ID NO:1 is D.
  • SEQ ID NO:1 consists of a sequence selected from the group consisting of YKPWLDSAFIDEL (SEQ ID NO: 2), YKPWLDQTFIDEL (SEQ ID NO: 3), YKPWLDQQFIDEL (SEQ ID NO: 4), and YKPWLDTDFIDEL (SEQ ID NO: 5).
  • SEQ ID NO: 1 consists of the sequence YKPWLDSAFIDEL (SEQ ID NO: 2). In some embodiments, SEQ ID NO: 1 consists of the sequence YKPWLDQTFIDEL (SEQ ID NO: 3). In some embodiments, SEQ ID NO: 1 consists of the sequence YKPWLDQQFIDEL (SEQ ID NO: 4). In some embodiments, SEQ ID NO: 1 consists of the sequence YKPWLDTDFIDEL (SEQ ID NO: 5).
  • amino acid molecules e.g., the amino acid molecule proteins
  • the amino acid molecules may be "enhanced" in function and may yield higher quantities of L-arginine than their naturally occurring enzyme counterparts; e.g. , they may have enhanced catalytic activity in comparison to the naturally occurring counterparts.
  • the amino acid molecules may be derived from a host cell or a microorganism.
  • the amino acid molecule may be derived from a microorganism that is modified (e.g., artificially and/or specifically genetically modified) from its naturally occurring variant or strain.
  • the host cell or the microorganism described herein may be Corynebacterium, Escherichia, Bacillus, Streptomyces, Penicillum, Klebsiella, Erwinia, or Pantoea .
  • the host cell or the microorganism may be Acidobacterium capsulatum, Alcaligenes faecalis, Bacillus amyloliquefaciens, Burkholderia pyrrocinia, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Cupriavidus necator, Escherichia coli, Mycobacterium smegmatis, or Neisseria weaveri.
  • amino acid molecules described herein also may be specifically derived from a microorganism of the genus Corynebacterium , more specifically derived from Corynebacterium glutamicum, Corynebacterium deserti, Corynebacterium crenatum, Corynebacterium efficiens, Corynebacterium suranareeae , and the like, but is not limited thereto.
  • activation, enhancement, up-regulation, overexpression, and increase may include both exhibiting activity that is not originally possessed and exhibiting an improved activity compared to the intrinsic activity or activity before modification or alteration of the amino acid sequence of the polypeptide.
  • intrinsic activity refers to the activity of a specific polypeptide originally possessed by the parent strain before transformation or the unmodified host cell or microorganism when the trait is changed by genetic mutation due to natural or artificial factors.
  • introduction activity may be used interchangeably with the term "activity before modification”.
  • the activity of a polypeptide being "enhanced, up-regulated, overexpressed, or increased” compared to the intrinsic activity means that the activity of a polypeptide is improved compared to the activity and/or concentration (expression level) of a specific polypeptide originally possessed by the parent strain before transformation or unmodified host cell or microorganism.
  • the enhancement may be achieved by introducing an exogenous polypeptide or by enhancing the activity and/or concentration (expression level) of the endogenous polypeptide. Whether or not the activity of a polypeptide is enhanced may be confirmed from an increase in the activity degree or expression level of the polypeptide or the amount of product generated from the activity of polypeptide.
  • the claimed peptide and/or the enhancement in activity are not limited as long as the activity of a target polypeptide has been enhanced compared to that in the host cell or microorganism before modification.
  • the procedures used for enhancing the activity of the peptide may be modifications using genetic engineering and/or protein engineering well known to those skilled in the art, which are routinely used in molecular biology, but are not limited thereto (for example, Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012).
  • the enhancement of the polypeptide activity of the present disclosure may be achieved by:
  • the increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introduction into a host cell of a vector capable of replicating and functioning independently of a host, to which a polynucleotide encoding the polypeptide is operably linked.
  • the increase may be achieved by introduction of one or two or more copies of a polynucleotide encoding the polypeptide into a chromosome in a host cell.
  • the introduction into a chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome in a host cell into the host cell, but is not limited thereto.
  • the replacement of the gene expression control region (or expression control sequence) on the chromosome encoding a polypeptide with a sequence having strong activity may be, for example, occurrence of mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof to further enhance the activity of the expression control region; or replacement with a sequence having stronger activity.
  • the expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating the termination of transcription and translation.
  • the replacement may be to replace the original promoter with a strong promoter, but is not limited thereto.
  • strong promoters include, but are not limited to, CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, and yccA promoter.
  • modification of the nucleotide sequence encoding the start codon or 5'-UTR region of a gene transcript encoding a polypeptide may include, for example, substitution with a nucleotide sequence encoding another (i.e., a different) start codon having a higher polypeptide expression rate compared to the endogenous start codon.
  • the modification of the amino acid sequence or polynucleotide sequence may be, but is not limited to, effectuated by a mutation in the amino acid sequence of a polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof so that the activity of the polypeptide is enhanced, or replacement of the sequence with an amino acid sequence or polynucleotide sequence modified to have stronger activity or an amino acid sequence or polynucleotide sequence modified to increase activity.
  • the replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto.
  • the vector used at this time may further include a selection marker for confirming chromosome insertion.
  • introduction of an exogenous polypeptide exhibiting the activity of a polypeptide or an exogenous polynucleotide encoding the same may be introduction of an exogenous polynucleotide encoding a polypeptide exhibiting activity the same as/similar to that of the polypeptide into a host cell.
  • the exogenous polynucleotide is not limited in origin or sequence as long as it exhibits activity the same as/similar to that of the polypeptide.
  • a known transformation method may be appropriately selected by those skilled in the art.
  • As the introduced polynucleotide is expressed in the host cell, a polypeptide may be produced and its activity may be increased.
  • codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of the endogenous polynucleotide so that the transcription or translation is increased in a host cell, or codon optimization of the exogenous polynucleotide so that the optimized transcription and translation are performed in a host cell.
  • modification or chemical modification of an exposed site selected through analysis of the tertiary structure of a polypeptide may be, for example, determining a template protein candidate according to the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, confirming the structure based on this, selecting an exposed site to be modified or chemically modified, selecting the exposed site to be modified or chemically modified, and modifying or chemically modifying the same.
  • Such enhancement of the amino acid molecule activity may be an increase in the activity or concentration expression level of the corresponding amino acid molecule compared to the activity or concentration of the amino acid molecule expressed in the wild-type host cell or host cell before modification, or an increase in the amount of product produced with the amino acid molecule, but is not limited thereto.
  • the amino acid molecule is a recombinant protein such as, for example, a recombinant argininosuccinate synthase.
  • the amino acid molecule comprises a sequence having SEQ ID NO:6, X 5 PX 6 X 7 X 8 X 9 GX 10 AFSGGLDTSX 11 AX 12 , in which X 5 is I, L or V; X 6 is an amino acid; X 7 is A, E or Q; X 8 is K or R; X 9 is I or V; X 10 is I or L; X 11 is A, T or V; and X 12 is I, L or V.
  • X 5 of SEQ ID NO:6 is I. In some embodiments, X 5 of SEQ ID NO:6 is L. In some embodiments, X 5 of SEQ ID NO:6 is V.
  • X 7 of SEQ ID NO:6 is A. In some embodiments, X 7 of SEQ ID NO:6 is E. In some embodiments, X 7 of SEQ ID NO:6 is Q.
  • X 8 of SEQ ID NO:6 is K. In some embodiments, X 8 of SEQ ID NO:6 is R.
  • X 9 of SEQ ID NO:6 is I. In some embodiments, X 9 of SEQ ID NO:6 is V.
  • X 10 of SEQ ID NO:6 is I. In some embodiments, X 10 of SEQ ID NO:6 is L.
  • X 11 of SEQ ID NO:6 is A. In some embodiments, X 11 of SEQ ID NO:6is T. In some embodiments, X 11 of SEQ ID NO:6 is V.
  • X 12 of SEQ ID NO:6 is I. In some embodiments, X 12 of SEQ ID NO:6 is L. In some embodiments, X 12 of SEQ ID NO:6 is V.
  • the amino acid molecule comprises a sequence having SEQ ID NO:7, X 13 GAX a X 14 X 15 X 16 YTA X 17 X 18 GQ X 19 DE, in which X 13 is K or N; X a is an amino acid; X 14 is C or P; X 15 is C or Y; X 16 is A, S or T; X 17 is D or N; X 18 is I or L; and X 19 is A, P or Y.
  • X 13 of SEQ ID NO:7 is K. In some embodiments, X 13 of SEQ ID NO:7 is N.
  • X 14 of SEQ ID NO:7 is C. In some embodiments, X 14 of SEQ ID NO:7 is P.
  • X 15 of SEQ ID NO:7 is C. In some embodiments, X 15 of SEQ ID NO:7 is Y.
  • X 16 of SEQ ID NO:7 is A. In some embodiments, X 16 of SEQ ID NO:7 is S. In some embodiments, X 16 of SEQ ID NO:7 is T.
  • X 17 of SEQ ID NO:7 is D. In some embodiments, X 17 of SEQ ID NO:7 is N.
  • X 18 of SEQ ID NO:7 is I. In some embodiments, X 18 of SEQ ID NO:7 is L.
  • X 19 of SEQ ID NO:7 is A. In some embodiments, X 19 of SEQ ID NO:7 is P. In some embodiments, X 19 of SEQ ID NO:7 is Y.
  • the amino acid molecule comprises a sequence having SEQ ID NO:8, X 20 X 21 X 22 X 23 X 24 X 25 X 26 X a LX 27 , in which X 20 is A or S; X 21 is R or V; X 22 is I or L; X 23 is I or V; X 24 is E or D; X 25 is C or G; X 26 is K or R; X a is an amino acid; and X 27 is A or V.
  • X 20 of SEQ ID NO:8 is A. In some embodiments, X 20 of SEQ ID NO:8 is S.
  • X 21 of SEQ ID NO:8 is R. In some embodiments, X 21 of SEQ ID NO:8 is V.
  • X 22 of SEQ ID NO:8 is I. In some embodiments, X 22 of SEQ ID NO:8 is L.
  • X 23 of SEQ ID NO:8 is I. In some embodiments, X 23 of SEQ ID NO:8 is V.
  • X 24 of SEQ ID NO:8 is E. In some embodiments, X 24 of SEQ ID NO:8 is D.
  • X 25 of SEQ ID NO:8 is C. In some embodiments, X 25 of SEQ ID NO:8 is G.
  • X 26 of SEQ ID NO:8 is K. In some embodiments, X 26 of SEQ ID NO:8 is R.
  • X 27 of SEQ ID NO:8 is A. In some embodiments, X 27 of SEQ ID NO:8 is V.
  • the amino acid molecule comprises a sequence having SEQ ID NO:9, X 28 AFX 29 X a X a X 30 X 31 G, in in which X 28 is G or N; X 29 is H or N; X a is an amino acid; X 30 is S or T; and X 31 is A or G.
  • X 28 of SEQ ID NO:9 is G. In some embodiments, X 28 of SEQ ID NO:9 is N.
  • X 29 of SEQ ID NO:9 is H. In some embodiments, X 29 of SEQ ID NO:9 is N.
  • X 30 of SEQ ID NO:9 is S. In some embodiments, X 30 of SEQ ID NO:9 is T.
  • X 31 of SEQ ID NO:9 is A. In some embodiments, X 31 of SEQ ID NO:9 is G.
  • the amino acid molecule comprises a sequence having SEQ ID NO:10, YFNTTPX 32 GRAVX 33 X 34 TX 35 LV, in which X 32 is I or L; X 33 is A or T; X 34 is A or G; and X 35 is L or M.
  • X 32 of SEQ ID NO:10 is I. In some embodiments, X 32 of SEQ ID NO:10 is L.
  • X 33 of SEQ ID NO:10 is A. In some embodiments, X 33 of SEQ ID NO:10 is T.
  • X 34 of SEQ ID NO:10 is A. In some embodiments, X 34 of SEQ ID NO:10 is G.
  • X 35 of SEQ ID NO:10 is L. In some embodiments, X 35 of SEQ ID NO:10 is M.
  • the amino acid molecule comprises a sequence having SEQ ID NO:11, TX 36 KGNDIERF, in which X 36 is F or Y.
  • X 36 of SEQ ID NO:11 is F. In some embodiments, X 36 of SEQ ID NO:11 is Y.
  • X 37 of SEQ ID NO:12 is L. In some embodiments, X 37 of SEQ ID NO:12 is V.
  • X 38 of SEQ ID NO:12 is A. In some embodiments, X 38 of SEQ ID NO:12 is T. In some embodiments, X 38 of SEQ ID NO:12 is V.
  • the amino acid molecule comprises a sequence having SEQ ID NO:13, GGRX a EMX 39 X 40 X 41 X 42 , in which X a is an amino acid; X 39 is A or S; X 40 is A, E or Q; X 41 is F, W or Y; and X 42 is L or M.
  • X 39 of SEQ ID NO:13 is A. In some embodiments, X 39 of SEQ ID NO:13 is S.
  • X 40 of SEQ ID NO:13 is A. In some embodiments, X 40 of SEQ ID NO:13 is E. In some embodiments, X 40 of SEQ ID NO:13 is Q.
  • X 41 of SEQ ID NO:13 is F. In some embodiments, X 41 of SEQ ID NO:13 is W. In some embodiments, X 41 of SEQ ID NO:13 is Y.
  • X 42 of SEQ ID NO:13 is L. In some embodiments, X 42 of SEQ ID NO:13 is M.
  • the amino acid molecule comprises a sequence having SEQ ID NO:14, EKAYSTDX 43 NX 44 X 45 GATHE, in which X 43 is A or S; X 44 is I, L or M; and X 45 is L or W.
  • X 43 of SEQ ID NO:14 is A. In some embodiments, X 43 of SEQ ID NO:14 is S.
  • X 44 of SEQ ID NO:14 is I. In some embodiments, X 44 of SEQ ID NO:14 is L. In some embodiments, X 44 of SEQ ID NO:14 is M.
  • X 45 of SEQ ID NO:14 is L. In some embodiments, X 45 of SEQ ID NO:14 is W.
  • the amino acid molecule comprises a sequence having SEQ ID NO:15, VX a PIMGVX a X 46 W, in which X a is an amino acid and X 46 is F, H or S.
  • X 46 of SEQ ID NO:15 is F. In some embodiments, X 46 of SEQ ID NO:15 is H. In some embodiments, X 46 of SEQ ID NO:15 is S.
  • the amino acid molecule comprises a sequence having SEQ ID NO:16, X 47 GGRHGX 48 GX 49 X 50 DQIENRX 51 IEA, in which X 47 is I or V; X 48 is L or M; X 49 is M or V; X 50 is A or S; and X 51 is I or V.
  • X 47 of SEQ ID NO:16 is I. In some embodiments, X 47 of SEQ ID NO:16 is V.
  • X 48 of SEQ ID NO:16 is L. In some embodiments, X 48 of SEQ ID NO:16 is M.
  • X 49 of SEQ ID NO:16 is M. In some embodiments, X 49 of SEQ ID NO:16 is V.
  • X 50 of SEQ ID NO:16 is A. In some embodiments, X 50 of SEQ ID NO:16 is S.
  • X 51 of SEQ ID NO:16 is I. In some embodiments, X 51 of SEQ ID NO:16 is V.
  • the amino acid molecule comprises a sequence having SEQ ID NO:17, KSRGIYEAPG.
  • the amino acid molecule comprises a sequence having SEQ ID NO:18, X 52 ALX 53 X 54 X 55 AX 56 ERLX a X 57 X 58 IHNEDT, in which X 52 is L or M; X 53 is F or L; X 54 is F, H or Y; X 55 is A or I; X 56 is F or Y; X a is an amino acid; X 57 is N, S or T; and X 58 is A or G.
  • X 52 of SEQ ID NO:18 is L. In some embodiments, X 52 of SEQ ID NO:18 is M.
  • X 53 of SEQ ID NO:18 is F. In some embodiments, X 53 of SEQ ID NO:18 is L.
  • X 54 of SEQ ID NO:18 is F. In some embodiments, X 54 of SEQ ID NO:18 is H. In some embodiments, X 54 of SEQ ID NO:18 is Y.
  • X 55 of SEQ ID NO:18 is A. In some embodiments, X 55 of SEQ ID NO:18 is I.
  • X 56 of SEQ ID NO:18 is F. In some embodiments, X 56 of SEQ ID NO:18 is Y.
  • X 57 of SEQ ID NO:18 is N. In some embodiments, X 57 of SEQ ID NO:18 is S. In some embodiments, X 57 of SEQ ID NO:18 is T.
  • X 58 of SEQ ID NO:18 is A. In some embodiments, X 58 of SEQ ID NO:18 is G.
  • the amino acid molecule comprises a sequence having SEQ ID NO:19, LGX 59 LX 60 YX 61 GRWX 62 DX 63 QX 64 X 65 MX 66 RX 67 , in which X 59 is K or R; X 60 is L or M; X 61 is A, E or Q; X 62 is F or L; X 63 is P or S; X 64 is A, S or G; X 65 is I, L or M; X 66 is I, L or V; and X 67 is D or E.
  • X 59 of SEQ ID NO: 19 is K. In some embodiments, X 59 of SEQ ID NO: 19 is R.
  • X 60 of SEQ ID NO: 19 is L. In some embodiments, X 60 of SEQ ID NO: 19 is M.
  • X 61 of SEQ ID NO: 19 is A. In some embodiments, X 61 of SEQ ID NO: 19 is E. In some embodiments, X 61 of SEQ ID NO: 19 is Q.
  • X 62 of SEQ ID NO: 19 is F. In some embodiments, X 62 of SEQ ID NO: 19 is L.
  • X 63 of SEQ ID NO: 19 is P. In some embodiments, X 63 of SEQ ID NO: 19 is S.
  • X 64 of SEQ ID NO: 19 is A. In some embodiments, X 64 of SEQ ID NO: 19 is S. In some embodiments, X 64 of SEQ ID NO: 19 is G.
  • X 65 of SEQ ID NO: 19 is I. In some embodiments, X 65 of SEQ ID NO: 19 is L. In some embodiments, X 65 of SEQ ID NO: 19 is M.
  • X 66 of SEQ ID NO: 19 is I. In some embodiments, X 66 of SEQ ID NO: 19 is L. In some embodiments, X 66 of SEQ ID NO: 19 is V.
  • X 67 of SEQ ID NO: 19 is D. In some embodiments, X 67 of SEQ ID NO: 19 is E.
  • the amino acid molecule comprises a sequence having SEQ ID NO:20, LRRGX a DX 68 X 69 X 70 , in which X a is an amino acid; X 68 is F or Y; X 69 is S or T; and X 70 is I or L.
  • X 68 of SEQ ID NO: 20 is F. In some embodiments, X 68 of SEQ ID NO: 20 is Y.
  • X 69 of SEQ ID NO: 20 is S. In some embodiments, X 69 of SEQ ID NO: 20 is T.
  • X 70 of SEQ ID NO: 20 is I. In some embodiments, X 70 of SEQ ID NO: 20 is L.
  • the amino acid molecule comprises a sequence having SEQ ID NO:21, X 71 X 72 X 73 X 74 X a X 75 X 76 X 77 LX 78 MEX 79 , in which X 71 is A, D or N; X 72 is F or L; X 73 is S or T; X 74 is F or Y; X a is an amino acid; X 75 is A, P or S; X 76 is A, E or D; X 77 is K or R; X 78 is S or T; and X 79 is K or R.
  • X 71 of SEQ ID NO: 21 is A. In some embodiments, X 71 of SEQ ID NO: 21 is D. In some embodiments, X 71 of SEQ ID NO: 21 is N.
  • X 72 of SEQ ID NO: 21 is F. In some embodiments, X 72 of SEQ ID NO: 21 is L.
  • X 73 of SEQ ID NO: 21 is S. In some embodiments, X 73 of SEQ ID NO: 21is T.
  • X 74 of SEQ ID NO: 21 is F. In some embodiments, X 74 of SEQ ID NO: 21 is Y.
  • X 75 of SEQ ID NO: 21 is A. In some embodiments, X 75 of SEQ ID NO: 21 is P. In some embodiments, X 75 of SEQ ID NO: 21 is S.
  • X 76 of SEQ ID NO: 21 is A. In some embodiments, X 76 of SEQ ID NO: 21 is E. In some embodiments, X 76 of SEQ ID NO: 21 is D.
  • X 77 of SEQ ID NO: 21 is K. In some embodiments, X 77 of SEQ ID NO: 21 is R.
  • X 78 of SEQ ID NO: 21 is S. In some embodiments, X 78 of SEQ ID NO: 21 is T.
  • X 79 of SEQ ID NO: 21 is K. In some embodiments, X 79 of SEQ ID NO: 21 is R.
  • the amino acid molecule comprises a sequence having SEQ ID NO:22, DRIGQLX 80 MRX 81 X 82 DX 83 X a DX 84 R, in which X 80 is H or T; X 81 is L, N or T; X 82 is L or N; X 83 is I, L or V; X a is an amino acid; and X 84 is S or T.
  • X 80 of SEQ ID NO:22 is H. In some embodiments, X 80 of SEQ ID NO: 22 is T.
  • X 81 of SEQ ID NO:22 is L. In some embodiments, X 80 of SEQ ID NO: 22 is N. In some embodiments, X 80 of SEQ ID NO:22 is T.
  • X 82 of SEQ ID NO:22 is L. In some embodiments, X 82 of SEQ ID NO: 22 is N.
  • X 83 of SEQ ID NO:22 is I. In some embodiments, X 83 of SEQ ID NO: 22 is L. In some embodiments, X 83 of SEQ ID NO:22 is V.
  • X 84 of SEQ ID NO:22 is S. In some embodiments, X 84 of SEQ ID NO: 22 is T.
  • the amino acid molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 sequence(s) selected from the group consisting of SEQ ID NO: 6-22.
  • the amino acid molecule comprises all of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.
  • the amino acid molecule described herein may have a length of at least 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, or 171 amino acids and/or 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188, 187, 186, 185, 184, 183, 182, 181, 180, 179, 178, 177, 176, 175, 174, 173, 172, 171, 170, 169, 168, 167, 166, 165, 164, 163, 162, 161, 160, 159, 158, 157, 156, 155, 154, 153, 152, 151 amino acids or less
  • the amino acid molecule comprises a sequence having at least 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to A. capsulatum sequence, SEQ ID NO: 23.
  • the amino acid molecule comprises a sequence corresponding to at least 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of positions 1-153 and 167-445 of SEQ ID NO: 23.
  • the amino acid molecule comprises a sequence corresponding to positions 1-153 and 167-445 of SEQ ID NO: 23.
  • the amino acid molecule comprises a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to A. faecalis sequence, SEQ ID NO: 24.
  • the amino acid molecule comprises a sequence corresponding to at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of positions 1-153 and 167-444 of SEQ ID NO: 24.
  • the amino acid molecule comprises a sequence corresponding to positions 1-153 and 167-444 of SEQ ID NO: 24.
  • the amino acid molecule comprises a sequence having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to B. pyrrocinia sequence, SEQ ID NO: 26.
  • the amino acid molecule comprises a sequence corresponding to at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of positions 1-153 and 167-445 of SEQ ID NO: 26.
  • the amino acid molecule comprises a sequence corresponding to positions 1-153 and 167-445 of SEQ ID NO: 26.
  • the amino acid molecule comprises a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to C. necator sequence, SEQ ID NO: 29.
  • the amino acid molecule comprises a sequence corresponding to at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of positions 1-153 and 167-441 of SEQ ID NO: 29.
  • the amino acid molecule comprises a sequence corresponding to positions 1-153 and 167-441 of SEQ ID NO: 29.
  • the amino acid molecule comprises a sequence having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to E. coli sequence SEQ ID NO: 30.
  • the amino acid molecule comprises a sequence corresponding to at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % of positions 1-153 and 167-447 of SEQ ID NO: 30.
  • the amino acid molecule comprises a sequence corresponding to positions 1-153 and 167-447 of SEQ ID NO: 30.
  • the amino acid molecule comprises a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to N. weaveri sequence, SEQ ID NO: 32.
  • the amino acid molecule comprises a sequence corresponding to at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of positions 1-157 and 171-448 of SEQ ID NO: 32.
  • the amino acid molecule comprises a sequence corresponding to positions 1-157 and 171-448 of SEQ ID NO: 32.
  • the amino acid molecule comprises a sequence having at least 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 23; having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 24; having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 25; having at least 80, 81, 82, 83, 84, 85, 86, 87, 88,
  • the present disclosure provides a host cell or microorganism expressing the amino acid molecule disclosure herein.
  • the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid molecule disclosed herein.
  • the present disclosure provides a nucleic acid molecule comprising a sequence having at least 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to A.
  • capsulatum sequence SEQ ID NO: 33; a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to A.
  • faecalis sequence SEQ ID NO: 34; a sequence having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to B.
  • pyrrocinia sequence SEQ ID NO: 35; a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to C.
  • necator sequence SEQ ID NO: 36; a sequence having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to E. coli sequence SEQ ID NO: 37; or a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to N. weaveri sequence, SEQ ID NO: 38, wherein the nucleic acid molecule does not occur in nature.
  • a vector comprising a nucleic acid molecule as disclosed herein.
  • a "vector” refers to and may include a DNA construct containing a nucleotide sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host.
  • the expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
  • the vector After transformation into an appropriate host cell, the vector may replicate or function independently of the host genome, and may be integrated into the genome itself.
  • the vector used in the present disclosure is not particularly limited, and any vector known in the art may be used.
  • Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as a phage vector or cosmid vector
  • a pDZ system a pBR system, a pUC system, a pBluescript II system, a pGEM system, a pTZ system, a pCL system, and a pET system may be used as a plasmid vector.
  • pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like may be used. Additional information about the vectors may be found in U.S. Patent Application Publication No. 2023/0134555, which is incorporated by reference in its entirety.
  • a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion.
  • the insertion of a polynucleotide into a chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.
  • a selection marker for confirming chromosome insertion may be further included.
  • the selection marker is used to select cells transformed with a vector, that is, to confirm the insertion of a target nucleic acid molecule, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides may be used. In an environment treated with a selective agent, only the cells expressing the selection marker can survive or exhibit other expression traits, and thus the transformed cells can be selected.
  • the modification of part or all of the polynucleotide in the host cell or microorganism may be induced by (a) homologous recombination using a vector for chromosome insertion into host cells or microorganisms, or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and/or (b) light such as ultraviolet light and radiation and/or chemical treatment, but is not limited thereto.
  • the method for modifying part or all of the gene may include a method by DNA recombination technology.
  • nucleotide sequence or vector including a nucleotide sequence homologous to the target gene into the host cell or microorganism to cause homologous recombination, part or all of the gene may be deleted.
  • the injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.
  • polynucleotide in the polynucleotide according to some embodiments of the present disclosure, various modifications may be made in the coding region within a range in which the amino acid sequence of the protein is not changed in consideration of codon degeneracy or preferred codons in organisms to express the protein described herein.
  • Another aspect of the present disclosure is to provide a polynucleotide comprising a nucleic acid sequence encoding the protein described above, wherein the polynucleotide does not occur in nature.
  • sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and a default gap penalty established by the program being used may be used together.
  • Substantially homologous or identical sequences are generally capable of hybridizing with all or part of the sequence under moderate or high stringent conditions. It is apparent that hybridization also includes hybridization with a polynucleotide containing a common codon in a polynucleotide or a codon taking codon degeneracy into account.
  • Whether or not any two polynucleotide or polypeptide sequences have homology, similarity, or identity may be determined, for example, using known computer algorithms such as the "FASTA” program using default parameters as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444.
  • the homology, similarity or identity may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.
  • the homology, similarity or identity of polynucleotides or polypeptides may be determined by comparing sequence information, for example, using a GAP computer program such as Needleman et al. (1970), J Mol Biol. 48: 443, for example, as known in Smith and Waterman, Adv. Appl. Math. (1981) 2: 482.
  • a GAP program may be defined as the value acquired by dividing the total number of symbols in the shorter of two sequences by the number of similarly aligned symbols (namely, nucleotides or amino acids).
  • Default parameters for the GAP program may include (1) binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and weighted comparison matrix of Gribskov et al., (1986) Nucl. Acids Res. 14: 6745 as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for an end gap.
  • binary comparison matrix containing values of 1 for identity and 0 for non-identity
  • weighted comparison matrix of Gribskov et al., (1986) Nucl. Acids Res. 14: 6745 as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical
  • a host cell in a further aspect, includes both wild-type unmodified host cells or host cells in which genetic modification has occurred naturally or “transformation” has occurred artificially.
  • the host cell described herein may be a microorganism.
  • microorganism (or strain) includes both wild-type unmodified microorganisms or microorganisms in which genetic modification has occurred naturally or "transformation” has occurred artificially.
  • the term "unmodified host cell” or “unmodified microorganism” does not exclude strains containing mutations that may occur naturally in the host cells or microorganisms, and may refer to a wild-type strain or a natural strain itself, or a strain before the trait is changed by genetic mutation due to natural or artificial factors.
  • the unmodified microorganism may refer to a strain in which the activity of the protein described herein is not enhanced or has not yet been enhanced.
  • the "unmodified microorganism” may be used interchangeably with "strain before modification", “microorganism before modification”, “unmutated strain”, “unmodified strain”, “unmutated microorganism”, or "reference microorganism”.
  • the term "transformation” refers to introducing a vector including a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell.
  • the transformed polynucleotide may include both: a transformed polynucleotide that is located by being inserted into the chromosome of the host cell and a transformed polynucleotide that is located outside the chromosome as long as they can be expressed in the host cell.
  • the polynucleotide includes DNA and/or RNA encoding a target polypeptide.
  • the polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements required for self-expression.
  • the expression cassette may usually include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal.
  • the expression cassette may be in the form of an expression vector capable of self-replication.
  • the polynucleotide may be introduced into a host cell in its own form and be operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • the engineered host cell or microorganism described herein may comprise one, two, three or more vectors, each comprising a different nucleotide sequence. In some embodiments, the engineered host cell or microorganism described herein may comprise one, two, three or more vectors, each comprising a nucleotide sequence encoding a different amino acid molecules.
  • Artificially modified host cells or microorganisms refer to host cells or microorganisms in which a specific mechanism is weakened or enhanced by causes such as insertion of an exogenous gene or enhancement or inactivation of the activity of an endogenous gene, containing genetic modification to produce a desired polypeptide, protein, or product.
  • Engineered host cells, microorganisms or strains in accordance with some embodiments herein may be transformed to express recombinant nucleic acid sequences and/or produce recombinant amino acid molecules or argininosuccinate synthase proteins.
  • the present disclosure provides a host cell, microorganism or strain expressing an argininosuccinate synthase disclosed herein.
  • the host cell or microorganism may be an engineered host cell or microorganism, in which the host cell or microorganism has been transformed, modified, or mutated artificially by techniques known to those of skill in the art to yield a host cell or microorganism that does not occur in nature.
  • an amino acid sequence for the amino acid molecule or argininosuccinate synthase described herein is extrinsic to the host cell.
  • an amino acid molecule or an argininosuccinate synthase disclosed herein is extrinsic to the host cell.
  • the "extrinsic" sequence means that the sequence does not occur in the host cell without any modification, and in particular, without any artificial modification.
  • the host cell may be modified to produce an amino acid molecule or an argininosuccinate synthase of another organism or a non-naturally occurring recombinant amino acid molecule or argininosuccinate synthase that does not naturally occur in the host cell (i.e., without any modification).
  • an amino acid sequence for an amino acid molecule or argininosuccinate synthase described herein is an intrinsic amino acid sequence of the microorganism.
  • the "intrinsic" sequence means that the sequence can occur in the host cell without any modification.
  • the host cell may be modified to produce a recombinant amino acid molecule or an argininosuccinate synthase having an intrinsic sequence of the host cell.
  • a nucleotide sequence encoding the argininosuccinate synthase protein is an extrinsic nucleotide sequence of the host cell. In further embodiments of the host cell according to the present disclosure, a nucleotide sequence encoding the argininosuccinate synthase protein is an intrinsic nucleotide sequence of the host cell.
  • the recombinant or modified host cell may be a host cell having increased L-arginine producing ability compared to a natural wild-type host cell, because the activity of the protein described herein or a polynucleotide encoding the same is improved in the recombinant or modified host cell compared to that in the natural wild-type host cell, but is not limited thereto.
  • the L-arginine producing recombinant or modified host cell e.g., of the genus Corynebacterium
  • the L-arginine producing ability of the recombinant or modified host cell may be increased by about 0.3% or more, specifically by about 0.5% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.7% or more, about 11% or more, about 12% or more, about 12.3% or more, about 13% or more, about 14% or more, about 14.4% or more, about 15% or more, about 15.1% or more, about 16% or more, or about 16.9% or more (the upper limit thereof is not particularly limited and the producing ability may be increased by, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, or about 20% or less) compared to the L-arginine producing ability of the parent strain before mutation or unmodified host cell.
  • the L-arginine production by the engineered host cell described herein is increased at least by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 50% and/or about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 or less %.
  • the producing ability is not limited thereto as long as it has an increased amount of + value compared to the producing ability of the parent strain before mutation or an unmodified host cell thereof.
  • the parent strain may be a cell prior to transforming an amino acid molecule described herein.
  • the L-arginine producing ability of the host cell having the increased producing ability may be increased by about 1.005 times or more, about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.04 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.10 times or more, about 1.107 times or more, about 1.11 times or more, about 1.12 times or more, about 1.123 times or more, about 1.13 times or more, about 1.14 times or more, about 1.144 times or more, about 1.15 times or more, about 1.151 times or more, about 1.16 times or more, about 1.169 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more (the upper limit thereof is not particularly limited and the producing ability may be increased by, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to the L-arginine producing ability of
  • the term "about” is relative to the actual value stated, as will be appreciated by those of skill in the art, and allows for approximations, inaccuracies and limits of measurement under the relevant circumstances.
  • the terms “about,” “substantially,” and “approximately” may provide an industry-accepted tolerance for their corresponding terms and/or relativity between items, such as a tolerance of from less than one percent to ten percent of the actual value stated, and other suitable tolerances.
  • the actual value stated may, for example, be that of lengths of nucleotide sequences, degrees of errors, dimensions, quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, and like and/or ranges thereof.
  • the tolerance may occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations.
  • the term “about” also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term “about” the claims appended hereto include equivalents to these quantities.
  • the term “about” further may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within, for example, 50, 25, 10, 9, 8,7, 6, 5,4, 3, 2, 1 percent or less of the stated reference value.
  • the present disclosure provides an engineered host cell expressing a recombinant amino acid molecule, including a recombinant protein that is an argininosuccinate synthase.
  • the amino acid molecule has an amino acid sequence comprising SEQ ID NO: 1 disclosed herein.
  • the engineered host cell described above may be an engineered microorganism.
  • the engineered microorganism is Corynebacterium.
  • the engineered microorganism is Corynebacterium glutamicum.
  • the engineered microorganism is Corynebacterium stationis.
  • the engineered microorganism is Corynebacterium crudilactis.
  • the engineered microorganism is Corynebacterium deserti.
  • the engineered microorganism is Corynebacterium efficiens.
  • the engineered microorganism is Corynebacterium callunae.
  • the engineered microorganism is Corynebacterium singulare. In some embodiments, the engineered microorganism is Corynebacterium halotolerans. In some embodiments, the engineered microorganism is Corynebacterium striatum. In some embodiments, the engineered microorganism is Corynebacterium ammoniagenes. In some embodiments, the engineered microorganism is Corynebacterium pollutisoli. In some embodiments, the engineered microorganism is Corynebacterium imitans. In some embodiments, the engineered microorganism is Corynebacterium testudinoris.
  • the engineered microorganism is Corynebacterium flavescens. In some embodiments, the engineered microorganism is Corynebacterium crenatum. In some embodiments, the engineered microorganism is Corynebacterium suranareeae. In some embodiments, the engineered microorganism is Escherichia. In some embodiments, the engineered microorganism is E. coli.
  • the engineered host cell produces L-arginine. In some embodiments, the engineered microorganism produces L-arginine.
  • the engineered host cell or microorganism is transformed with an argR gene comprising a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 100% sequence identity to SEQ ID NO: 39.
  • the engineered host cell or microorganism is transformed with an argB gene with M54V comprising a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 100% sequence identity to SEQ ID NO: 40.
  • the recombinant protein has an extrinsic amino acid sequence of the hot cell or microorganism. In other embodiments, the recombinant protein has an intrinsic amino acid sequence of the host cell or microorganism.
  • the present disclosure provides use of the engineered host cell or microorganism disclosed herein for producing L-arginine.
  • a method for producing L-arginine comprises culturing an engineered host cell expressing a recombinant protein disclosed herein.
  • the host cell is cultured in a medium.
  • the term "culture” means growing the host cell described herein under properly controlled environmental conditions.
  • the culture process according to some embodiments of the present disclosure may be performed under appropriate medium and culture conditions known in the art. Such a culture process may be easily adjusted and used by those skilled in the art depending on the selected host cell.
  • the culture may be batch, continuous and/or fed-batch culture, but is not limited thereto.
  • the term "medium” refers to a material in which nutrients required for culturing the host cell described herein are mixed as a main component, and supplies nutrients and growth factors, including water, which are essential for survival and growth.
  • any medium may be used without particular limitation as long as it is a medium used for culturing conventional host cells, but the host cell described herein may be cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, vitamins and/or the like under an aerobic condition while the temperature, pH, and the like are adjusted.
  • a culture medium for host cells of the genus Corynebacterium may be found in the literature ["Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington D.C., USA, 1981)].
  • compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, and the like may be added to the medium in an appropriate manner to adjust the pH of the medium.
  • an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation.
  • Oxygen or oxygen-containing gas may be injected into the medium in order to maintain the aerobic state of the medium; or gas may not be injected or nitrogen, hydrogen or carbon dioxide gas may be injected in order to maintain the anaerobic and microaerobic conditions, but the control of atmosphere is not limited thereto.
  • the culturing is performed with at least one source of carbon.
  • suitable sources of carbon include, but are not limited to, carbohydrates, sugar alcohols, organic acids, and amino acids.
  • Carbohydrates may include glucose, saccharose, lactose, fructose, sucrose, and maltose, among others.
  • Sugar alcohols may include mannitol and sorbitol, among others.
  • Organic acids may include pyruvic acid, lactic acid, and citric acid, among others, amino acids may include glutamic acid, methionine, and lysine, among others.
  • the culturing is performed with at least one source of nitrogen.
  • suitable sources of nitrogen include inorganic nitrogen sources, organic nitrogen sources, and combinations thereof.
  • Inorganic nitrogen sources include, but are not limited to, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, and ammonium nitrate.
  • Organic nitrogen sources include, but are not limited to, organic acids including ammonium acetate and ammonium carbonate; amino acids such as glutamic acid, methionine, and glutamine; and other sources of organic nitrogen including peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysates, fish or decomposition products thereof, and defatted soybean cake or decomposition products thereof. These nitrogen sources may be used singly or in combination of two or more, but the manner of use is not limited thereto.
  • the culturing is performed with at least one of monobasic potassium phosphate, dibasic potassium phosphate, monobasic sodium phosphate, dibasic sodium phosphate, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like. Additionally, amino acids, vitamins, suitable precursors and/or the like may be added to the medium.
  • These sources of carbon, sources of nitrogen, additives, or precursors thereof may be added to the medium batchwise or continuously. However, the manner of addition is not limited thereto.
  • Another embodiment of the method of producing L-arginine as disclosed herein provides that the culturing is performed with at least one selected from the group consisting of potassium phosphate, dipotassium phosphate, a sodium-containing salt corresponding thereto, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate.
  • the culturing is performed at a temperature maintained between about 20 °C to about 45 °C. In some embodiments, the culturing is performed at a temperature maintained between about 25 °C to about 40 °C. In some embodiments, the culturing is performed at a temperature maintained from about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 °C to about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 °C. In still further embodiments, the culturing may be conducted for from about 10 to about 160 hours. In still further embodiments, the culturing may be conducted for from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 to about 100, 110, 120, 130, 140, 150 or 160 hours. However, the culture conditions are not limited thereto.
  • Another aspect of the present disclosure provides a method of increasing L-arginine production by a host cell.
  • the method comprises transforming the host cell to produce the engineered host cell expressing a recombinant amino acid molecule disclosed herein.
  • the method further includes preparing the host cell described herein, preparing a medium for culturing the host cell, or a combination thereof (in any order), for example, before the culturing step.
  • L-arginine produced by the culture according to some embodiments of the present disclosure may be secreted into the medium or may remain in the cells.
  • the method for producing L-arginine comprising culturing an engineered host cell expressing a recombinant amino acid molecule such as, for example, a recombinant argininosuccinate synthase as set forth herein, the method further includes recovering L-arginine from the medium after a culturing step (medium subjected to culture) or from the cultured host cell. The recovery step may be further comprised after the culture step.
  • the production method further comprises recovering L-arginine from the cultured engineered host cell and/or from the medium in which the engineered host cell is cultured.
  • Suitable methods for recovering the L-arginine include protocols known in the art according to the method for culturing a host cell described herein, for example, a batch, continuous or fed-batch culture method.
  • centrifugation, filtration, treatment with a crystallized protein precipitating agent salting-out method
  • extraction sonication
  • ultrafiltration dialysis
  • various kinds of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, and affinity chromatography, HPLC, or any combination thereof
  • the desired L-arginine may be recovered from the medium or host cell using a suitable method known in the art.
  • the production method further comprises a purification step.
  • the purification may be performed using a suitable method known in the art.
  • the recovery step and the purification step may be performed continuously or discontinuously in any order, or may be performed simultaneously or by being integrated into one step, but the manner of performance is not limited thereto.
  • the protein, polynucleotide, vector, host cell and the like are as described in the other aspects herein.
  • compositions for L-arginine production comprising a microorganism of the genus Corynebacterium ; a medium in which the microorganism has been cultured; or a combination thereof.
  • the composition according to some embodiments of the present disclosure may further contain arbitrary suitable excipients commonly used in compositions for L-arginine production, and such excipients may include, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, and isotonic agents.
  • Another aspect provides a use of the L-arginine product characterized by one or more elements disclosed in the application.
  • a further aspect provides a use of the method characterized by one or more elements disclosed in the application.
  • the amino acid residues essential for the binding to substrates such L- citrulline, aspartate and ATP in Corynebacterium glutamicum were identified through protein sequence alignment between the argininosuccinate synthase of Corynebacterium glutamicum (SEQ ID NO: 28) and a heterologous argininosuccinate synthase with a known structure. Although the tertiary structure of argininosuccinate synthase of Corynebacterium glutamicum has not been identified, with regard to the argininosuccinate synthase derived from Escherichia coli (SEQ ID NO: 30), the tertiary crystal structure and active site residues therein have been reported.
  • argininosuccinate synthases of Acidobacterium capsulatum, Alcaligenes faecalis, Burkholderia pyrrocinia, Cupriavidus necator, Escherichia coli, and Neisseria weaveri 's argininosuccinate synthases have a 5-amino-acid extension around essential residues in the binding site, which is not present in Corynebacterium glutamicum ( e.g. , see FIG. 2 and SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26 SEQ ID NO: 29 SEQ ID NO: 30 and SEQ ID NO: 32, respectively).
  • the site where the amino acid extension has occurred is located at the entrance of the active site on the tertiary structure of the protein and is particularly adjacent to the substrate binding site. Additionally, this amino acid extension may be involved in the formation of a helical secondary structure. While, in the Corynebacterium glutamicum -derived protein where this amino acid extension is absent, the corresponding site exists in the form of a loop without a specific secondary structure, in the E. coli -derived protein where this extension is present, the site exists in the form of an alpha helix structure. Additionally, he distance between the corresponding region and the substrate may increase in the tertiary structure of the protein due to the difference in sequence between this extended portion and the portion preceding it.
  • the active site entrance may be expanded due to the formation of a secondary structure, and easier access to the active site of the substrate can be facilitated, which may contribute to the improvement of catalytic efficiency of the protein.
  • Argininosuccinate synthase from Escherichia coli may be functional as a tetramer.
  • the amino acid extension may be located near interface between argininosuccinate synthase monomers and thus capable of affecting tetramer formation.
  • a protein percent identity matrix for the ten kinds of argininosuccinate synthase was also generated using the Clustal 2.1 program (Madeira, Fabio et al., Nucleic acids research 50(W1) W276-W279, 2022) and shown in Table 2 below.
  • argininosuccinate synthases having the amino acid extension were included, and the protein sequences of 20 argininosuccinate synthases having a 5 amino acid extension at the corresponding position were additionally collected using the NCBI BLAST program (Altschul, S F et al., Nucleic acids research 25(17) 3389-402, 1997) and included (SEQ ID NO: 1 through SEQ ID NO: 16). These sequences were searched for motifs using the MEME program, a protein motif search program (Bailey, T L, and C Elkan. Proceedings. International Conference on Intelligent Systems for Molecular Biology 2, 28-36, 1994).
  • MOTIF 01 [ILV]Px[AEQ][KR][IV]G[IL]AFSGGLDTS[ATV]A[ILV] (SEQ ID NO: 6) MOTIF 02 [KN]GAx[CP][CY][AST]YTA[DN][IL]GQ[APY]DE (SEQ ID NO: 7) MOTIF 03 [AS][RV][IL][IV][ED][CG][KR]xL[AV] (SEQ ID NO: 8) MOTIF 04 [GN]AF[HN]xx[ST][AG]G(SEQ ID NO: 9) MOTIF 05 YFNTTP[IL]GRAV[AT][AG]T[LM]LV(SEQ ID NO: 10) MOTIF 06 T[FY]KGNDIERF (SEQ ID NO: 11) MOTIF 07 YRYGL[LV][ATV]N (SEQ ID NO: 12) MOTIF 08 YKPWLDxxF[IV]XEL (
  • vectors were prepared, each having an argG gene encoding the argininosuccinate synthase enzyme as disclosed herin.
  • the argG genes were each derived from Acidobacterium capsulatum (SEQ ID NO:33) , Alkaligenes faecalis (SEQ ID NO:34) , Burkholderia pyrrocinia (SEQ ID NO:35) , Bacillus amyloliquefaciens (SEQ ID NO:41) , Corynebacterium ammoniagenes (SEQ ID NO:42) , Corynebacterium glutamicum (SEQ ID NO:43) , Cupriavidus necator (SEQ ID NO:36) , Escherichia coli (SEQ ID NO:37) , Mycobacterium smegmatis (SEQ ID NO:44), and Neisseria weaveri (SEQ ID NO:38).
  • each argG gene can be inserted into the chromosome of Corynebacterium glutamicum by homologous recombination, particularly at the BBD29_RS08210 site, and expressed under the constitutive Po2 promoter (Korean Patent No. 10-1632642).
  • Two DNA fragments containing promoter and homologous region targeting BBD29_RS08210 were prepared.
  • One DNA fragment was for the 5'-homologous region targeting BBD29_RS08210 and a promoter, the other DNA fragment was for the 3'-homologous region targeting BBD29_RS08210.
  • PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and the primer pairs (SEQ ID NOS: 45 and 46, and SEQ ID NOS: 47 and 48) shown in Table 4 to obtain a DNA fragment, which includes sequences of a 5'-homologous region of BBD29_RS08210 with a Po2 promoter and a 3'-homologous region of BBD29_RS08210 (hereinafter, "BBD29_RS08210 5'-DNA fragment” and "BBD29_RS08210 3'-DNA fragment").
  • the PCR was performed as follows: 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 2 minutes. Then, a DNA fragment of the argG gene was prepared.
  • PCR was performed using the primer pairs shown in Table 4 (SEQ ID NOS: 49 and 50 with the genomic DNA of Acidobacterium capsulatum strain KACC 14500, SEQ ID NOS: 51 and 52 with the genomic DNA of Bacillus amyloliquefaciens strain KACC 12067, SEQ ID NOS: 53 and 54 with the genomic DNA of Burkholderia pyrrocinia strain KACC 12018, SEQ ID NOS: 55 and 56 with the genomic DNA of Corynebacterium ammoniagenes strain ATCC 6872, SEQ ID NOS: 57 and 58 with the genomic DNA of Corynebacterium glutamicum strain ATCC 13869, SEQ ID NOS: 59 and 60 with the genomic DNA of Cupriavidus necator strain KCTC 22469, SEQ ID NOS: 61 and 62 with the genomic DNA of Escherichia coli K-12 substrain MG1655, SEQ ID NOS: 63 and 64 with the genomic DNA of Mycobacterium smegmati
  • the genomic DNAs used were distributed by KCTC and KACC.
  • the PCR was performed as follows: 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 2 minutes.
  • DNA fragments of the argG gene hereinafter, argG ( A. capsulatum ), argG ( B. amyloliquefaciens ), argG ( B. pyrrocinia ), argG ( C. ammoniagenes ), argG ( C. glutamicum ), argG ( C. necator ), argG ( E. coli ), argG ( M. smegmatis ), and argG ( N. weaveri ) were obtained.
  • the obtained DNA fragments and the linearized vectors were cloned.
  • the linear vectors were used after treating pDCM2 (Korean Patent Application Publication No. 10-2020-0136813), which cannot replicate in Corynebacterium glutamicum , with SmaI restriction enzyme.
  • the thus-obtained BBD29_RS08210 5'-DNA fragment, BBD29_RS08210 3'-DNA fragment, and each of the DNA fragments of argG gene were cloned by fusion.
  • the fusion cloning was performed using the In-Fusion® HD Cloning Kit (Clontech), and the resulting clones were transformed into E.
  • a Corynebacterium glutamicum strain CJR2 having the ability to produce L-arginine was prepared. This preparation is to introduce two mutations ( ⁇ argR, argB (M54V)) serially into wild-type Corynebacterium glutamicum ATCC13869 (Ikeda, Masato et al., Applied and Environmental Microbiology 75(6)1635-41, 2009).
  • vectors introducing argR deletion (SEQ ID NO:39) and argB (M54V) mutation (SEQ ID NO: 40) were prepared. See FIG. 6.
  • PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and the primer pairs shown in Table 5 (SEQ ID NOS: 67 and 68, and SEQ ID NOS: 69 and 70), and overlapping PCR was performed using the primer pair of SEQ ID NOS: 67 and 70 so as to obtain homologous recombination fragments having a sequence of the argR deletion mutation (SEQ ID NO:39).
  • PCR was performed using the primer pairs shown in Table 5 (SEQ ID NOS: 71 and 72, and SEQ ID NOS: 73 and 74), and overlapping PCR was performed using SEQ ID NOS: 71 and 74. to obtain homologous recombination fragments having a sequence of the argB (M54V) mutation (SEQ ID NO:40).
  • the PCR was performed as follows: 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 2 minutes.
  • an argR deletion mutation was introduced into wild-type Corynebacterium glutamicum ATCC13869 and transformed by the electric pulse method using the pDCM2- ⁇ argR plasmid prepared above (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Subsequently, secondary recombination was performed in a solid plate medium containing 4% sucrose followed by PCR using a primer pair (SEQ ID NOS: 67 and 70) targeting the transformed strains whose secondary recombination was completed, thereby confirming that a deletion mutation was introduced into the argR gene on the chromosome. In particular, the PCR was performed as follows: 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 2 minutes. The transformed strain was named CJR1.
  • the argB (M54V) mutation was introduced into the Corynebacterium glutamicum CJR1 in the same manner as above.
  • the pDCM2- argB (M54V) plasmid prepared above was used, and it was confirmed that the M54V mutation was introduced into the argB gene on the chromosome by performing PCR using a primer pair (SEQ ID NOS: 71 and 74) targeting the transformant whose secondary recombination was completed.
  • the transformed strain was named CJR2.
  • the solid plate medium was as follows: Composite Plate Medium (pH 7.0), glucose 10 g, peptone 10 g, beef extract 5 g, yeast extract 5 g, brain heart infusion 18.5 g, NaCl 2.5 g, urea 2 g, sorbitol 91 g, agar 20 g (per 1 L of distilled water).
  • Example 3-2 Preparation of strains introduced with argininosuccinate synthase (argG) based on CJR2 strain
  • Example 3-2 In order to compare the L-arginine producing ability of the CJR2 strains, in which the argG gene is introduced, prepared in Example 3-2 (i.e., CJR2-argG ( A. capsulatum ), CJR2-argG ( B. amyloliquefaciens ), CJR2-argG ( B. pyrrocinia ), CJR2-argG ( C. ammoniagenes ), CJR2-argG ( C. glutamicum ), CJR2-argG ( C. necator ), CJR2-argG ( E. coli ), CJR2-argG ( M. smegmatis ), and CJR2-argG ( N. weaveri )), the concentration of L-arginine in the culture medium was analyzed by culturing by the method described below.
  • Corynebacterium glutamicum CJR2 i.e., the parent strain
  • the strains prepared in Example 3-2 were each inoculated into a 250 mL corner-baffle flask containing 25 mL of a production medium and then cultured with shaking at 200 rpm at 30°C for 44 hours.
  • Each of the composition of the production medium is as shown below.
  • smegmatis not including the 5-amino-acid extension were introduced, were each measured to be 4.9 g/L and 0.7 g/L; therefore, compared to the former group, the L-arginine concentration was 23% lower on average, and the L-citrulline concentration was 350% higher on average. The average yield improvement of these strains compared to their parent strain was 3.3%. This indicates that the argininosuccinate synthase having the amino acid extension is relatively excellent in terms of catalytic efficiency compared to the enzymes not including the amino acid extension.

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Abstract

La présente invention concerne une molécule d'acide aminé, des cellules hôtes exprimant la molécule d'acide aminé, l'utilisation de cellules hôtes pour produire de la L-arginine, des molécules d'acide nucléique avec des séquences codant pour la molécule d'acide aminé, des vecteurs comprenant des molécules d'acide nucléique avec des séquences codant pour la molécule d'acide aminé, des procédés de production de L-arginine, et des procédés pour augmenter la production de L-arginine par une cellule hôte.
PCT/KR2023/013895 2023-09-15 2023-09-15 Molécule d'acide aminé recombiné, cellules hôtes pour la production de l-arginine, et procédés de production de l-arginine les utilisant Pending WO2025058109A1 (fr)

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Citations (6)

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Publication number Priority date Publication date Assignee Title
US20030233675A1 (en) * 2002-02-21 2003-12-18 Yongwei Cao Expression of microbial proteins in plants for production of plants with improved properties
US20050260581A1 (en) * 2001-02-12 2005-11-24 Chiron Spa Gonococcal proteins and nucleic acids
WO2009037329A2 (fr) * 2007-09-21 2009-03-26 Basf Plant Science Gmbh Plantes à rendement amélioré
US8541208B1 (en) * 2004-07-02 2013-09-24 Metanomics Gmbh Process for the production of fine chemicals
US9085765B2 (en) * 2005-08-20 2015-07-21 Scarab Genomics Llc Reduced genome E. coli
WO2019122936A1 (fr) * 2017-12-22 2019-06-27 Cancer Research Technology Limited Protéines de fusion

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050260581A1 (en) * 2001-02-12 2005-11-24 Chiron Spa Gonococcal proteins and nucleic acids
US20030233675A1 (en) * 2002-02-21 2003-12-18 Yongwei Cao Expression of microbial proteins in plants for production of plants with improved properties
US8541208B1 (en) * 2004-07-02 2013-09-24 Metanomics Gmbh Process for the production of fine chemicals
US9085765B2 (en) * 2005-08-20 2015-07-21 Scarab Genomics Llc Reduced genome E. coli
WO2009037329A2 (fr) * 2007-09-21 2009-03-26 Basf Plant Science Gmbh Plantes à rendement amélioré
WO2019122936A1 (fr) * 2017-12-22 2019-06-27 Cancer Research Technology Limited Protéines de fusion

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