WO2023226978A1 - Saccharose synthase d'origine microbienne et son utilisation - Google Patents

Saccharose synthase d'origine microbienne et son utilisation Download PDF

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WO2023226978A1
WO2023226978A1 PCT/CN2023/095777 CN2023095777W WO2023226978A1 WO 2023226978 A1 WO2023226978 A1 WO 2023226978A1 CN 2023095777 W CN2023095777 W CN 2023095777W WO 2023226978 A1 WO2023226978 A1 WO 2023226978A1
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amino acid
substituted
sus
acid substitution
polypeptide
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PCT/CN2023/095777
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Chinese (zh)
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谢新开
徐伟
范俊英
董爽
曾唯实
孟婷
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苏州引航生物科技有限公司
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Publication of WO2023226978A1 publication Critical patent/WO2023226978A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • 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
    • C12N9/10—Transferases (2.)

Definitions

  • the present invention relates to the field of enzyme engineering.
  • the present invention relates to microbially derived sucrose synthase, modified variants thereof and use in the UDP-glucose cycle.
  • Glycosylation modification is a common way to modify molecules.
  • Natural glycosylation receptor molecules include other sugars, proteins, lipids and other natural polymers, or other natural product small molecules, including antibiotics, pigments, Sweeteners, etc.
  • the natural glycosylation process involves the transfer of glycosyl residues from an activated glycosyl donor to an acceptor molecule through a catalyst, such as glycosyltransferase (GT).
  • Nucleoside diphosphate (NDP) sugar is the most common type of sugar donor, such as uridine diphosphate (UDP) glucose, adenosine diphosphate (ADP) glucose, etc. These substances are circulated and consumed in the organism as glycosylation intermediates of glycosylation. That is, the process of ongoing glycosyl transfer requires the regeneration of these nucleoside diphosphate sugars.
  • nucleoside diphosphate sugars are divided into synthetic regeneration and decomposition regeneration.
  • the corresponding sugar is phosphorylated by kinase catalysis to obtain a phosphorylated sugar activated at a specific position.
  • the phosphorylated sugar reacts with UDP or ADP under the catalysis of the enzyme to generate the corresponding nucleoside diphosphate sugar. .
  • the synthetic regeneration process consumes energy, as well as ATP or UTP.
  • Decomposition-type regeneration regenerates nucleoside diphosphate sugars by degrading an existing glycosidic bond through the reverse reaction of enzyme-catalyzed glycosyl transfer.
  • sucrose synthase (SUS) catalyzes the following reaction:
  • sucrose synthases are mainly from plants, such as AtSUS1 from Arabidopsis thaliana and BvSUS1 from sugar beet (Beta vulgaris) (see K. et al. "Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development", Biotechnology Advances 34 (2016) 88–111).
  • AtSUS1 Arabidopsis thaliana
  • BvSUS1 sugar beet
  • sugar beet Beta vulgaris
  • sucrose synthase from Nitrosomonas Europaea, Acidithiobacillus caldus, Denitrovibrio acetiphilus, and Melioribacter roseus (M. Diricks et al., “Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes”, Appl Microbiol Biotechnol (2015) 99:8465–8474).
  • the invention provides a modified sucrose synthase (SUS) polypeptide, compared with its wild-type SUS polypeptide, comprising positions 4, 24, 41, 108, 114, 133, 136, 161, 300 ,433,473,474,476,479,482,483,513,515,518,529,533,534,543,544,585,629,630,640,641,644,664,676,697,713 , 715, 726, 729, 741, 768, 769, 773, 788 and 790, wherein the positions are numbered with reference to SEQ ID NO: 1, wherein the amino acid substitution at position 4 is V, The amino acid at position 24 is substituted with L, the amino acid at position 41 is substituted with D, E or W, the amino acid at position 108 is substituted with C or M, the amino acid at position 114 is substituted with E, the amino acid at position 133 is substituted with K or R, the amino acid at position 136
  • SUS sucrose syntha
  • the present invention also provides a modified SUS polypeptide, comprising the amino acid sequence of one of SEQ ID NO: 2-208, or the modified SUS compared with one of 2-208, except for positions 4, 24, 41, 108 ,114,133,136,161,300,433,473,474,476,479,482,483,513,515,518,529,533,534,543,544,585,629,630,640,641 , positions other than 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788 and 790 comprise 1-10 amino acid substitutions, wherein the modified SUS has a catalytic sucrose Breaks down to produce fructose and NDP-glucose activity in the presence of nucleoside diphosphate (NDP).
  • NDP nucleoside diphosphate
  • the invention provides polynucleotides, expression vectors and host cells encoding modified SUS polypeptides of the invention.
  • the invention provides a method of producing NDP glucose, comprising contacting a SUS polypeptide derived from a microorganism or a host cell comprising the SUS polypeptide with sucrose in the presence of NDP, wherein the SUS polypeptide is Wild-type SUS polypeptide, such as the SUS polypeptide of SEQ ID NO: 1 or the modified SUS polypeptide of the present invention.
  • the present invention mainly relates to modified SUS for catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP.
  • modified SUS for catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP.
  • sucrose synthase refers to an enzyme that catalyzes the reversible reaction of glucose and fructose to produce sucrose. That is, sucrose synthase can also catalyze the decomposition of sucrose to produce fructose and NDP-glucose in the presence of nucleoside diphosphates (NDP, such as UDP and ADP).
  • NDP nucleoside diphosphates
  • peptide refers to a chain of at least two amino acids linked by peptide bonds.
  • polypeptide is used interchangeably herein with the term “protein” and refers to a chain containing ten or more amino acid residues. All peptide and polypeptide formulas or sequences herein are written from left to right, indicating the direction from the amino terminus to the carboxyl terminus.
  • amino acid includes naturally occurring amino acids and unnatural amino acids in proteins. Single-letter and three-letter nomenclature for naturally occurring amino acids in proteins uses names commonly used in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • modification is intended to include any chemical modification of a polypeptide, and also includes modifications to the amino acid sequence, such as substitutions, deletions, insertions and/or additions of amino acids.
  • sucrose synthase EsSUS SEQ ID NO: 1, NCBI accession number WP_063464253
  • its mutants from Ectothiorhodospira sp.BSL-9 have high activity and high stability, and are suitable for catalyzing the decomposition of sucrose. , producing NDP-glucose in the presence of NDP (such as UDP or ADP).
  • the modified SUS polypeptide of the invention compared to its wild-type SUS, includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, wherein the modified SUS polypeptide has the activity of catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP.
  • the modified SUS polypeptide of the invention compared to its wild-type SUS, comprises positions selected from the group consisting of 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476 ,479,482,483,513,515,518,529,533,534,543,544,585,629,630,640,641,644,664,676,697,713,715,726,729,741 , 768, 769, 773, 788 and 790, wherein the positions are numbered with reference to SEQ ID NO: 1, wherein the modified SUS has the ability to catalyze sucrose decomposition, at nucleoside diphosphate Activity to produce fructose and NDP-glucose in the presence of (NDP).
  • the amino acid at position 4 is substituted with V.
  • the amino acid substitution at position 24 is L.
  • the amino acid substitution at position 41 is D, E or W.
  • the amino acid substitution at position 108 is C or M.
  • the amino acid at position 114 is substituted with E.
  • the amino acid substitution at position 133 is K or R.
  • the amino acid substitution at position 136 is M, T or K.
  • the amino acid at position 161 is substituted with D.
  • the amino acid substitution at position 300 is M, G or A.
  • the amino acid substitution at position 433 is C or R.
  • the amino acid at position 473 is substituted with G.
  • the amino acid substitution at position 474 is C, V or H.
  • the amino acid substitution at position 476 is V or C.
  • the amino acid substitution at position 479 is A, R or N.
  • the amino acid substitution at position 482 is S, T or V.
  • the amino acid substitution at position 483 is N, H or G.
  • the amino acid substitution at position 513 is K, Q or I.
  • the amino acid substitution at position 515 is R or S.
  • the amino acid substitution at position 518 is Y, V, I, L, F or T.
  • the amino acid substitution at position 529 is V, T or H.
  • the amino acid substitution at position 533 is F or L.
  • the amino acid substitution at position 534 is F, W or Y.
  • the amino acid substitution at position 543 is L.
  • the amino acid substitution at position 544 is I.
  • the amino acid substitution at position 585 is K or R.
  • the amino acid at position 629 is substituted with D.
  • the amino acid substitution at position 630 is N, H or S.
  • the amino acid at position 640 is substituted with P.
  • the amino acid substitution at position 641 is R or K.
  • the amino acid substitution at position 644 is A, V or S.
  • the amino acid substitution at position 664 is Y.
  • the amino acid substitution at position 676 is K.
  • the amino acid substitution at position 697 is V, I or K.
  • the amino acid substitution at position 713 is L or M.
  • the amino acid substitution at position 715 is L or V.
  • the amino acid substitution at position 726 is D or E.
  • the amino acid at position 729 is substituted with D.
  • the amino acid substitution at position 741 is C or G.
  • the amino acid substitution at position 768 is P, E or A.
  • the amino acid substitution at position 769 is P or Q.
  • the amino acid substitution at position 773 is L.
  • the amino acid at position 788 is substituted with R.
  • the amino acid substitution at position 790 is K or R.
  • the modified SUS polypeptides of the invention comprise amino acid residues at positions 133 and 135. generation, amino acid substitutions at positions 529 and 768, amino acid substitutions at positions 136 and 768, or amino acid substitutions at positions 133 and 529.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 529, 768, and 790, and optionally selected from positions 41, 300, 433, 473, 474, 476, 479 , 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729 and 741 at one or more positions amino acid substitutions.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.
  • the wild-type SUS polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or a natural variant thereof.
  • the modified SUS polypeptides of the invention comprise or consist of the amino acid sequence of one of SEQ ID NO: 2-208, or the modified SUS is compared to one of 2-208, except at positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518 , 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788 and 790
  • the position of contains 1-10 amino acid substitutions, wherein the modified SUS polypeptide has the activity of catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP.
  • the mutation in the modified SUS polypeptide of the invention is an amino acid substitution or a combination of amino acid substitutions selected from the following (positions are numbered with reference to SEQ ID NO: 1) compared to its wild type:
  • wild-type SUS refers to naturally occurring SUS.
  • the wild-type SUS is a SUS from Ectothiorhodospira sp. BSL-9 (EsSUS, SEQ ID NO: 1).
  • the sequences are aligned for optimal comparison (for example, a gap can be introduced in the first amino acid or nucleic acid sequence to match the second amino acid sequence). or nucleic acid sequence for optimal alignment).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the molecules are identical at a position in the first sequence when the corresponding position in the second sequence is occupied by the same amino acid residue or nucleotide.
  • percent identity number of identical positions/total number of positions (i.e., overlapping positions) x 100).
  • the two sequences are of the same length.
  • Percent amino acid identity or “percent amino acid sequence identity” refers to a comparison of the amino acids of two polypeptides that, when optimally aligned, have approximately a specified percentage of identical amino acids. For example, “95% amino acid identity” refers to a comparison of the amino acids of two polypeptides that, when optimally aligned, are 95% identical.
  • the wild-type SUS is at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96% , 97%, 98% or 99% sequence identity.
  • the modified SUS polypeptide of the invention compared to its wild-type SUS, comprises positions selected from the group consisting of 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476 ,479,482,483,513,515,518,529,533,534,543,544,585,629,630,640,641,644,664,676,697,713,715,726,729,741 , 768, 769, 773, 788 and 790, wherein the positions are numbered with reference to SEQ ID NO: 1, wherein the modified SUS It has the activity of catalyzing the decomposition of sucrose and producing fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP).
  • NDP nucleoside diphosphate
  • the amino acid at position 4 is substituted with V.
  • the amino acid substitution at position 24 is L.
  • the amino acid substitution at position 41 is D, E or W.
  • the amino acid substitution at position 108 is C or M.
  • the amino acid at position 114 is substituted with E.
  • the amino acid substitution at position 133 is K or R.
  • the amino acid substitution at position 136 is M, T or K.
  • the amino acid at position 161 is substituted with D.
  • the amino acid substitution at position 300 is M, G or A.
  • the amino acid substitution at position 433 is C or R.
  • the amino acid at position 473 is substituted with G.
  • the amino acid substitution at position 474 is C, V or H.
  • the amino acid substitution at position 476 is V or C.
  • the amino acid substitution at position 479 is A, R or N.
  • the amino acid substitution at position 482 is S, T or V.
  • the amino acid substitution at position 483 is N, H or G.
  • the amino acid substitution at position 513 is K, Q or I.
  • the amino acid substitution at position 515 is R or S.
  • the amino acid substitution at position 518 is Y, V, I, L, F or T.
  • the amino acid substitution at position 529 is V, T or H.
  • the amino acid substitution at position 533 is F or L.
  • the amino acid substitution at position 534 is F, W or Y.
  • the amino acid substitution at position 543 is L.
  • the amino acid substitution at position 544 is I.
  • the amino acid substitution at position 585 is K or R.
  • the amino acid at position 629 is substituted with D.
  • the amino acid substitution at position 630 is N, H or S.
  • the amino acid at position 640 is substituted with P.
  • the amino acid substitution at position 641 is R or K.
  • the amino acid substitution at position 644 is A, V or S.
  • the amino acid substitution at position 664 is Y.
  • the amino acid substitution at position 676 is K.
  • the amino acid substitution at position 697 is V, I or K.
  • the amino acid substitution at position 713 is L or M.
  • the amino acid substitution at position 715 is L or V.
  • the amino acid substitution at position 726 is D or E.
  • the amino acid at position 729 is substituted with D.
  • the amino acid substitution at position 741 is C or G.
  • the amino acid substitution at position 768 is P, E or A.
  • the amino acid substitution at position 769 is P or Q.
  • the amino acid substitution at position 773 is L.
  • the amino acid at position 788 is substituted with R.
  • the amino acid substitution at position 790 is K or R.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133 and 135, amino acid substitutions at positions 529 and 768, amino acid substitutions at positions 136 and 768, or amino acid substitutions at positions 133 and 529.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 529, 768, and 790, and optionally selected from positions 41, 300, 433, 473, 474, 476, 479 , 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729 and 741 at one or more positions amino acid substitutions.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.
  • the wild-type SUS has a similarity to SEQ ID NO: 1 of at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97% , 98% or 99% sequence identity.
  • the modified SUS polypeptide of the invention compared to its wild-type SUS, comprises positions selected from the group consisting of 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476 ,479,482,483,513,515,518,529,533,534,543,544,585,629,630,640,641,644,664,676,697,713,715,726,729,741 , 768, 769, 773, 788 and one or more bits of 790 Amino acid substitutions at positions, wherein the positions are numbered with reference to SEQ ID NO: 1, and the modified SUS has at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% sequence identity, wherein the modified SUS has the ability to catalyze sucrose decomposition at nucleoside di
  • the amino acid at position 4 is substituted with V.
  • the amino acid substitution at position 24 is L.
  • the amino acid substitution at position 41 is D, E or W.
  • the amino acid substitution at position 108 is C or M.
  • the amino acid at position 114 is substituted with E.
  • the amino acid substitution at position 133 is K or R.
  • the amino acid substitution at position 136 is M, T or K.
  • the amino acid at position 161 is substituted with D.
  • the amino acid substitution at position 300 is M, G or A.
  • the amino acid substitution at position 433 is C or R.
  • the amino acid at position 473 is substituted with G.
  • the amino acid substitution at position 474 is C, V or H.
  • the amino acid substitution at position 476 is V or C.
  • the amino acid substitution at position 479 is A, R or N.
  • the amino acid substitution at position 482 is S, T or V.
  • the amino acid substitution at position 483 is N, H or G.
  • the amino acid substitution at position 513 is K, Q or I.
  • the amino acid substitution at position 515 is R or S.
  • the amino acid substitution at position 518 is Y, V, I, L, F or T.
  • the amino acid substitution at position 529 is V, T or H.
  • the amino acid substitution at position 533 is F or L.
  • the amino acid substitution at position 534 is F, W or Y.
  • the amino acid substitution at position 543 is L.
  • the amino acid substitution at position 544 is I.
  • the amino acid substitution at position 585 is K or R.
  • the amino acid at position 629 is substituted with D.
  • the amino acid substitution at position 630 is N, H or S.
  • the amino acid at position 640 is substituted with P.
  • the amino acid substitution at position 641 is R or K.
  • the amino acid substitution at position 644 is A, V or S.
  • the amino acid substitution at position 664 is Y.
  • the amino acid substitution at position 676 is K.
  • the amino acid substitution at position 697 is V, I or K.
  • the amino acid substitution at position 713 is L or M.
  • the amino acid substitution at position 715 is L or V.
  • the amino acid substitution at position 726 is D or E.
  • the amino acid at position 729 is substituted with D.
  • the amino acid substitution at position 741 is C or G.
  • the amino acid substitution at position 768 is P, E or A.
  • the amino acid substitution at position 769 is P or Q.
  • the amino acid substitution at position 773 is L.
  • the amino acid at position 788 is substituted with R.
  • the amino acid substitution at position 790 is K or R.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133 and 135, amino acid substitutions at positions 529 and 768, amino acid substitutions at positions 136 and 768, or amino acid substitutions at positions 133 and 529.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 529, 768, and 790, and optionally selected from positions 41, 300, 433, 473, 474, 476, 479 , 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729 and 741 at one or more locations amino acid substitutions.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.
  • the modified SUS of the invention further comprises one or more conservative substitutions of amino acids compared to SEQ ID NO: 1.
  • substitutions also known as substitution by a "homologous" amino acid residue, refers to one in which the amino acid residue is substituted by a similar Substitutions of side chain amino acid residue substitutions, for example, basic side chain amino acids (such as lysine, arginine and histidine), acidic side chain amino acids (such as aspartic acid, glutamic acid), Non-charged polar side chain amino acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain amino acids (such as alanine, valine , leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chain amino acids (such as threonine, valine, isoleucine ) and aromatic side chain amino acids (such as tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chain amino acids such as
  • Conservative amino acid substitutions generally have minimal impact on the activity of the resulting protein. This substitution is described below. Conservative substitution is the replacement of an amino acid with an amino acid similar in size, hydrophobicity, charge, polarity, steric characteristics, aromaticity, etc. Such substitutions are often conservative when one wishes to fine-tune the properties of the protein.
  • homologous amino acid residues refer to amino acid residues with similar chemical properties involving hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, and the like.
  • amino acids that are homologous to each other include the positively charged lysine, arginine, and histidine; the negatively charged glutamic acid and aspartic acid; and the hydrophobic glycine, alanine, valine, and leucine.
  • Acid isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine , aromatic phenylalanine, tyrosine, tryptophan, chemically similar side chain groups of serine and threonine, or glutamine and asparagine, or leucine and isoleucine.
  • Examples of conservative substitutions of amino acids in proteins include: Ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for Met, Met, Leu or Tyr for Phe, Thr for Ser, Ser for Thr, Tyr for Trp, Trp or Phe for Tyr, and Ile or Leu replace Val.
  • modified SUS polypeptides of the invention are substituted by amino acids at positions 133, 136, 529, 768 and 790 as compared to SEQ ID NO: 1, and optionally present at position 41 ,300,433,473,474,476,479,483,513,515,518,533,534,543,544,585,629,630,640,641,644,664,676,697,713,715 Composed of amino acid substitutions at one or more positions of , 726, 729 and 741.
  • modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.
  • the amino acid substitution at position 41 is D, E or W.
  • the amino acid substitution at position 133 is K or R.
  • the amino acid substitution at position 136 is M, T or K.
  • the amino acid substitution at position 300 is M, G or A.
  • the amino acid substitution at position 433 is C or R.
  • the amino acid at position 473 is substituted with G.
  • the amino acid substitution at position 474 is C, V or H.
  • the amino acid substitution at position 476 is V or C.
  • the amino acid substitution at position 479 is A, R or N.
  • the amino acid substitution at position 483 is N, H or G.
  • the amino acid substitution at position 513 is K, Q or I.
  • the amino acid substitution at position 515 is R or S.
  • the amino acid substitution at position 518 is Y, V, I, L, F or T.
  • the amino acid substitution at position 529 is V, T or H.
  • the amino acid substitution at position 533 is F or L.
  • the amino acid substitution at position 534 is F, W or Y.
  • the amino acid substitution at position 543 is L.
  • the ammonia at position 544 The base acid is substituted with I.
  • the amino acid substitution at position 585 is K or R.
  • the amino acid at position 629 is substituted with D.
  • the amino acid substitution at position 630 is N, H or S.
  • the amino acid at position 640 is substituted with P.
  • the amino acid substitution at position 641 is R or K.
  • the amino acid substitution at position 644 is A, V or S.
  • the amino acid substitution at position 664 is Y.
  • the amino acid substitution at position 676 is K.
  • the amino acid substitution at position 697 is V, I or K.
  • the amino acid substitution at position 713 is L or M.
  • the amino acid substitution at position 715 is L or V.
  • the amino acid substitution at position 726 is D or E.
  • the amino acid at position 729 is substituted with D.
  • the amino acid substitution at position 741 is C or G.
  • the amino acid substitution at position 768 is P, E or A.
  • the amino acid substitution at position 790 is K or R.
  • the modifications in the modified SUS polypeptides of the invention are substituted by amino acids at positions 133, 136, 529, 768 and 790 as compared to SEQ ID NO: 1, and optionally present at position 41 ,300,433,473,474,476,479,483,513,515,518,533,534,543,544,585,629,630,640,641,644,664,676,697,713,715 , 726, 729 and 741, the amino acid substitution at position 41 is D, E or W, the amino acid substitution at position 133 is K or R, and the amino acid substitution at position 136 is M, T or K, the amino acid at position 300 is substituted with M, G or A, the amino acid at position 433 is substituted with C or R, the amino acid at position 473 is substituted with G, the amino acid at position 474 is substituted with C, V or H, the amino acid at position 476 is substituted with V or C, the amino acid at position 479 is substituted by A
  • enzyme activity refers to the decrease in substrate or increase in product per unit time in a chemical reaction catalyzed by unit mass of enzyme under certain conditions.
  • the activity of the modified SUS of the present invention can be measured by the amount of sucrose decreased per unit time or the amount of NDP-glucose such as UDP glucose increased under certain conditions in the presence of unit mass of modified SUS polypeptide and NDP such as UDP. To represent.
  • enzyme activity may also refer to the relative activity of an enzyme, expressed as the ratio of the activity of the enzyme of interest to the activity of a given enzyme that catalyzes the same reaction, such as percent relative activity.
  • the activity of the modified SUS of the invention is expressed as a percentage compared to SEQ ID NO: 1 Specific relative activity expressed.
  • the stability is thermostability, which refers to the ability of an enzyme to maintain activity after incubation at a certain temperature (such as 40-70°C or higher) for a certain period of time (such as 10 minutes to 1 hour).
  • the modified SUS has better thermal stability than the polypeptide of SEQ ID NO: 1.
  • the activity of the modified SUS of the invention is at least 100%, 105%, 110%, 120%, 130% of the activity of the polypeptide of SEQ ID NO: 1 %, 150%, 170%, 200%, 250%, 300% or higher.
  • the modified SUS of the present invention has a higher T50, where T50 refers to the temperature at which the enzyme activity decreases by 50% after incubation for, for example, one hour.
  • T50 refers to the temperature at which the enzyme activity decreases by 50% after incubation for, for example, one hour.
  • the modified SUS of the invention has a T50 that is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C or higher than the polypeptide of SEQ ID NO: 1.
  • the activity of the modified SUS in catalyzing sucrose decomposition to produce fructose and NDP-glucose in the presence of NDP is at least 100% of the activity of SEQ ID NO: 1 in catalyzing the above reaction. , 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more high.
  • the modified SUS has better thermal stability than the polypeptide of SEQ ID NO: 1, for example, after incubation at 50-70°C for 1 hour, the activity of the modified SUS is SEQ At least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300% or higher of the activity of the polypeptide of ID NO: 1, or the modified SUS
  • the T50 is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C or higher than the polypeptide of SEQ ID NO: 1; and without prior incubation, it catalyzes the decomposition of sucrose in NDP
  • the activity to produce fructose and NDP-glucose in the presence of SEQ ID NO: 1 is at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250% of the activity of catalyzing the above reaction , 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or higher.
  • nucleic acid molecule includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA or RNA produced using nucleotide analogs.
  • the nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA.
  • the nucleic acid may be synthesized using nucleotide analogs or derivatives (eg, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids with altered base pairing abilities or increased nuclease resistance.
  • the invention also provides polynucleotides encoding the modified SUS of the invention. Therefore, in the present invention, the term modification also includes the genetic manipulation of the polynucleotide encoding the SUS polypeptide of the invention. The modifications may be substitutions, deletions, insertions and/or additions of nucleotides.
  • the term "encoding" refers to a polynucleotide that directly specifies the amino acid sequence of its protein product.
  • the boundaries of the coding sequence are generally defined by an open reading frame, which usually begins with the ATG start codon or additional start codons such as GTG and TTG, and ends with a stop codon such as TAA, TAG and TGA.
  • the coding sequence may be a DNA, cDNA or recombinant nucleotide sequence.
  • nucleic acid molecules encompassing all or part of the nucleic acid sequences of the present invention can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the sequence information contained in the sequences.
  • PCR polymerase chain reaction
  • Polynucleotides of the invention can be amplified according to standard PCR amplification techniques using cDNA, mRNA or genomic DNA as templates and appropriate oligonucleotide primers.
  • the nucleic acid so amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • Polynucleotides of the invention can be prepared by standard synthesis techniques, for example using an automated DNA synthesizer.
  • the invention also relates to complementary strands of the nucleic acid molecules described herein.
  • a nucleic acid molecule that is complementary to another nucleotide sequence is a molecule that is sufficiently complementary to that nucleotide sequence that it can hybridize to other nucleotide sequences, thereby forming a stable duplex.
  • hybridizes is nucleotides that are at least about 90%, preferably at least about 95%, more preferably at least about 96%, more preferably at least 98% homologous to each other under given stringent hybridization and wash conditions. Sequences generally remain hybridized to each other.
  • polynucleotides of the present invention do not include polynucleotides that hybridize only to poly A sequences (such as the 3' end poly(A) of mRNA) or to a complementary stretch of poly T (or U) residues.
  • nucleic acid constructs and vectors such as expression vectors, comprising the polynucleotides of the invention are also provided.
  • expression includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.
  • nucleic acid construct refers to a single- or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified to contain nucleic acid segments that do not occur naturally.
  • nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains control sequences required for expression of the coding sequence of the invention.
  • expression vector refers herein to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the invention together with additional nucleotides provided for expression of the polynucleotide, e.g. Control sequences, manipulatively connected.
  • the expression vector includes a viral vector or a plasmid vector.
  • control sequences is intended herein to include all elements necessary or advantageous for expression of a polynucleotide encoding a polypeptide of the invention.
  • Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other.
  • control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include the promoter and transcription and translation termination signals.
  • control sequence may be a suitable promoter sequence, one recognized by the host cell to express the code.
  • the promoter sequence contains transcriptional control sequences that mediate expression of the polypeptide.
  • the promoter can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, for example, the Escherichia coli lac operon.
  • Such promoters also include mutant, truncated and hybrid promoters and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.
  • operably linked refers herein to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence, whereby the control sequence directs expression of the polypeptide coding sequence.
  • Polynucleotides encoding polypeptides of the invention can be subjected to various manipulations to allow expression of the polypeptides. Depending on the expression vector, manipulation of the polynucleotide may be desirable or necessary before inserting it into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.
  • the vectors of the invention preferably contain one or more selectable markers which allow for simple selection of transformed, transfected, transduced, etc. cells.
  • a selectable marker is a gene whose product provides biocide or viral resistance, heavy metal resistance, supplementation of auxotrophs, etc.
  • selectable markers for bacteria are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline.
  • the vectors of the present invention can be integrated into the host cell genome or replicate autonomously in the cell independently of the genome.
  • the elements required for integration into the host cell genome or for autonomous replication are known in the art (see, e.g., Sambrook et al., 1989, supra).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques.
  • transformation and “transfection” refer to various art-recognized techniques for introducing exogenous nucleic acid (e.g., DNA) into a host cell, as can be found, for example, in the aforementioned Sambrook et al., 1989; Davis et al. ., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • the invention also relates to recombinant host cells comprising polynucleotides of the invention which are advantageously used in the recombinant production of SUS polypeptides.
  • a vector comprising a polynucleotide of the invention is introduced into a host cell whereby the vector is retained as a chromosomal integrant or as a self-replicating extrachromosomal vector.
  • Those skilled in the art are aware of conventional vectors and host cells for expressing proteins.
  • the host cell of the invention is an E. coli cell, such as E. coli BL21(DE3).
  • the expression vector is pET-30a(+).
  • the modified SUS of the invention can be operably linked to a non-SUS polypeptide (eg, a heterologous amino acid sequence) to form a fusion protein.
  • a non-SUS polypeptide eg, a heterologous amino acid sequence
  • the fusion protein is a GST-SUS fusion protein, wherein the SUS sequence is fused to the C-terminus of the GST sequence. This fusion protein can aid in the purification of recombinant SUS.
  • the fusion protein is a SUS protein containing a heterologous signal sequence at its N-terminus. In certain host cells (eg, mammalian and yeast host cells), expression and/or secretion of SUS can be increased through the use of heterologous signal sequences.
  • host cells of the invention also include host cells expressing the SUS polypeptide of SEQ ID NO: 1.
  • the invention provides a method for producing NDP-glucose, comprising contacting the SUS polypeptide of SEQ ID NO: 1, the modified SUS of the invention or a host cell with D-glufosinate.
  • the method of producing NDP-glucose of the present invention includes the steps of:
  • NDP-glucose such as ADP-glucose or UDP-glucose
  • SUS activity is provided by the SUS polypeptide of SEQ ID NO: 1 or the modified SUS or host cell of the invention.
  • a cell-free catalytic process is used to produce L-glufosinate-ammonium, and in step (a), the SUS polypeptide of SEQ ID NO: 1 or the modified SUS of the invention is provided.
  • free or immobilized SUS polypeptide of SEQ ID NO: 1 or modified SUS of the invention may be used.
  • the incubation is performed at a temperature of 50-70°C. In some embodiments, the incubation is at 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 , 70°C or higher temperature.
  • the medium is a buffer, such as PBS, Tris-HCl buffer.
  • the medium is a Tris-HCl buffer, such as 50 mM Tris-HCl buffer, pH 8.0.
  • the reaction medium is a medium consisting in part or entirely of cell culture medium, and the SUS activity is provided by the host cells of the invention that are cultured in the reaction medium.
  • the host cells of the invention and/or the second host cells are cultured and expanded in a cell culture medium, and then the expanded host cells are isolated from the cell culture medium using a buffer or water. Biomass resuspension. Sucrose and NDP are added to the buffer or water before, during or after the addition of the expanded host cells.
  • bacterial cells such as E. coli cells, may be used.
  • NDP-glucose such as ADP-glucose or UDP-glucose
  • ADP-glucose or UDP-glucose can be produced at higher temperatures than in the prior art.
  • DNA polymerase PrimeSTAR Max DNA Polymerase
  • DpnI endonuclease were purchased from TaKaRa Company
  • plasmid extraction kit was purchased from Axygen Company
  • sucrose and MgCl 2 were purchased from McLean
  • UDP uridine-5'-di Phosphate sodium salt
  • UDP-G uridine diphosphate glucose disodium salt
  • ii) Vector and strain The expression vector used was pET-30a(+), and the plasmid was purchased from Novagen.
  • the deposited clones were activated on LB agar medium. Then, a single colony was inoculated into LB liquid medium (containing 50 mg/L kanamycin) and incubated at 37°C for 12 h with shaking. Transfer 1 mL of culture to 50 mL of fresh LB liquid medium (containing 50 mg/L kanamycin), incubate with shaking at 37°C until OD600 reaches about 0.6, add IPTG (final concentration: 0.4mM) and incubate at 25°C Incubate for 16h to induce protein expression.
  • LB liquid medium containing 50 mg/L kanamycin
  • IPTG final concentration: 0.4mM
  • the culture was centrifuged at 4,000 g for 10 min at 4°C, the supernatant was discarded, and E. coli cells were collected. Resuspend the collected E. coli cells in 15 mL of pre-chilled 50 mM PBS, pH 6.5, and disrupt the E. coli cells by sonication at 4°C. The cell disruption solution was centrifuged at 6,000g for 15 minutes at 4°C to remove the precipitate, and the supernatant obtained was a crude enzyme solution containing recombinant enzyme.
  • Equipped with a mixture of the following final concentrations: 50mM PBS, 50g/L sucrose, 2mM UDP, 10mM MgCl2, pH 6.5.
  • To the above solution add the crude enzyme solution of sucrose synthase prepared as described in v) (the amount of enzyme used is adjusted according to the UDP conversion percentage to ensure that the UDP conversion percentage is less than 15%).
  • the specified temperature continue shaking on a oscillator (400 rpm) for 1 hour, heat at 95°C for 10 minutes to inactivate the protein, and then detect the concentration of UDP-G by high-performance liquid chromatography to determine the initial speed of the catalytic reaction.
  • the temperature that is 50% of the control activity is T50.
  • the incubation temperature is 50-60°C with an interval of 1°C; for mutants with improved stability, the temperature is increased according to actual conditions.
  • sucrose synthase EsSUS SEQ ID NO: 1
  • BSL-9 The coding nucleic acid of sucrose synthase EsSUS (SEQ ID NO: 1) from Ectothiorhodospira sp. BSL-9 was cloned into pET-30a(+) plasmid and expressed as described in Example 1, and measured at 50°C and Enzyme activity at 56°C, and T50.
  • a mutant containing a single mutation was prepared according to the method of Example 1.
  • the resulting mutants are shown in Table 1.
  • the enzyme activity of the obtained mutant at 56°C was measured according to the method described in Example 1, and the results are shown in Table 1, where the relative enzyme activity is the ratio of the activity of the mutant vs. the activity of the wild type.
  • a mutant containing 2-5 mutations was prepared according to the method of Example 1.
  • the resulting mutants are shown in Table 2.
  • the relative enzyme activity of the obtained enzyme at 56°C was measured according to the method described in Example 1, and the results are shown in Table 2, where the relative enzyme activity is the ratio of the activity of the mutant vs. the activity of the wild type.
  • the introduction of multiple mutations can increase the activity of the enzyme after incubation at 56°C for 1 h to more than 2 times.
  • the T50 of the mutants was determined as described in Example 1. The results showed that the T50 of the mutants was increased by at least 3°C compared with the wild type, and the T50 of the mutant of SEQ ID NO: 63-70 reached 63-64°C. That is, introducing mutations at five positions (133, 136, 529, 768, and 790) significantly increased the T50 of the enzyme.
  • amino acid substitutions were further introduced on the basis of SEQ ID NO: 118, and the enzyme activity of the mutant at 56°C was measured as described in Example 1. The results are shown in Table 4, where the relative enzyme activity refers to the mutant's enzyme activity. Activity vs. activity ratio of SEQ ID NO:118.
  • T50 of the mutant obtained was measured as described in Example 2.
  • the T50 of the mutant of SEQ ID NO:131 was 63.5°C.
  • the T50 of other mutants in Tables 3 and 4 were also equivalent to SEQ ID NO:64.
  • amino acid substitutions were further introduced on the basis of SEQ ID NO: 131, and the enzyme activity of the mutant at 63.5°C was measured as described in Example 1. The results are shown in Table 5, where the relative enzyme activity refers to the temperature at 63.5°C. The ratio of the activity of the mutant vs. the activity of SEQ ID NO:131 after 1 hour of incubation.
  • the T50 of the resulting mutants was determined as described in Example 1. The results showed that the T50 of the mutant was significantly higher than that of SEQ ID NO:131, among which the T50 of SEQ ID NO:159-189 and 191-203 was 66-66.5°C.

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Abstract

La présente invention concerne une saccharose synthase (SUS) modifiée. Plus particulièrement, la SUS modifiée de la présente invention présente une activité de catalyse de la décomposition du saccharose pour produire du fructose et du NDP-glucose en présence de nucléoside diphosphate (NDP). En outre, la SUS modifiée de la présente invention présente une activité améliorée de catalyse de la réaction susmentionnée et/ou une stabilité améliorée par comparaison avec la SEQ ID NO : 1. La présente invention concerne également un polynucléotide codant pour la SUS modifiée de la présente invention, un vecteur et une cellule hôte exprimant la SUS modifiée de la présente invention, et un procédé de production d'un NDP-glucose grâce à l'utilisation de la SUS microbienne comprenant une SUS de type sauvage et la SUS modifiée de la présente invention, et de la cellule hôte exprimant la SUS.
PCT/CN2023/095777 2022-05-23 2023-05-23 Saccharose synthase d'origine microbienne et son utilisation WO2023226978A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101495626A (zh) * 2006-07-25 2009-07-29 拜尔生物科学公司 新型蔗糖合酶的鉴定及其在纤维改性中的用途
WO2017207484A1 (fr) * 2016-05-31 2017-12-07 Universiteit Gent Saccharoses synthases mutantes et leurs utilisations
CN112805295A (zh) * 2018-07-30 2021-05-14 科德克希思公司 工程化糖基转移酶和甜菊醇糖苷葡糖基化方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101495626A (zh) * 2006-07-25 2009-07-29 拜尔生物科学公司 新型蔗糖合酶的鉴定及其在纤维改性中的用途
WO2017207484A1 (fr) * 2016-05-31 2017-12-07 Universiteit Gent Saccharoses synthases mutantes et leurs utilisations
CN112805295A (zh) * 2018-07-30 2021-05-14 科德克希思公司 工程化糖基转移酶和甜菊醇糖苷葡糖基化方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 13 October 2019 (2019-10-13), ANONYMOUS : "sucrose synthase [Ectothiorhodospira sp. BSL-9]", XP093112080, retrieved from NCBI Database accession no. WP_063464253.1 *
DATABASE Protein 13 October 2019 (2019-10-13), ANONYMOUS: "sucrose synthase [Ectothiorhodospira mobilis]", XP093112074, retrieved from NCBI Database accession no. WP_090484796.1 *
DATABASE Protein 7 December 0931 (0931-12-07), ANONYMOUS : "MULTISPECIES: sucrose synthase [unclassified Ectothiorhodospira]", XP093112077, retrieved from NCBI Database accession no. WP_238620542.1 *
DATABASE Protein 7 March 2022 (2022-03-07), ANONYMOUS : "sucrose synthase [Ectothiorhodospira variabilis]", XP093112071, retrieved from NCBI Database accession no. WP_239821389.1 *

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