WO2023226978A1 - Microbial-derived sucrose synthase and use thereof - Google Patents
Microbial-derived sucrose synthase and use thereof Download PDFInfo
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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
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- sus
- acid substitution
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- 239000002244 precipitate Substances 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
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Classifications
-
- 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
The present invention relates to a modified sucrose synthase (SUS). Specifically, the modified SUS of the present invention has an activity of catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP). In addition, the modified SUS of the present invention has an improved activity of catalyzing the above reaction and/or an improved stability compared to SEQ ID NO: 1. The present invention also relates to a polynucleotide encoding the modified SUS of the present invention, a vector and a host cell expressing the modified SUS of the present invention, and a method for producing an NDP-glucose by means of using the microbial-derived SUS comprising a wild-type SUS and the modified SUS of the present invention, and the host cell expressing the SUS.
Description
本发明涉及酶工程领域。具体而言，本发明涉及微生物来源的蔗糖合酶、其经修饰的变体和在UDP-葡萄糖循环中的应用。The present invention relates to the field of enzyme engineering. In particular, the present invention relates to microbially derived sucrose synthase, modified variants thereof and use in the UDP-glucose cycle.
糖基化修饰是普遍存在的一种对分子修饰的方式，天然的糖基化受体分子包括其他糖、蛋白、脂类等天然聚合物，或其他的天然产物小分子，包括抗生素、色素、甜味剂等。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.
天然的糖基化过程是通过催化剂，如糖基转移酶(GT)，将糖基残基从活化的糖基供体转移到受体分子上。核苷二磷酸(NDP)糖是最常见的糖基供体的类型，如尿苷二磷酸(UDP)葡萄糖、腺苷二磷酸(ADP)葡萄糖等。这些物质在生物体内是作为糖基化的糖基转移中间体流通和消耗的。也就是说，持续性的糖基转移过程需要再生这些核苷二磷酸糖。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.
目前核苷二磷酸糖的再生分为合成型再生和分解型再生。对于合成型再生，对应的糖经激酶催化进行磷酸化获得特定位置活化的磷酸化的糖，所述磷酸化的糖在酶的催化作用下与UDP或ADP反应，生成对应的核苷二磷酸糖。合成型再生过程消耗能量，以及ATP或UTP。分解型再生通过酶催化的糖基转移的逆反应，通过降解某种已有的糖苷键来再生核苷二磷酸糖。例如，蔗糖合酶(SUS)催化如下反应：At present, the regeneration of nucleoside diphosphate sugars is divided into synthetic regeneration and decomposition regeneration. For synthetic 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. For example, sucrose synthase (SUS) catalyzes the following reaction:
蔗糖+NDP—>果糖+NDP-葡萄糖。Sucrose+NDP—>Fructose+NDP-glucose.
该反应消耗相对廉价的蔗糖，产生等摩尔的NDP-葡萄糖。This reaction consumes relatively cheap sucrose and produces equimolar amounts of NDP-glucose.
目前被广泛表征和使用的蔗糖合酶主要来自植物，例如来自拟南芥(Arabidopsis thaliana)的AtSUS1和来自甜菜(Beta vulgaris)的BvSUS1(参见K.et al.“Sucrose synthase:A unique glycosyltransferase for biocatalytic glycosylation process development”，Biotechnology Advances 34(2016)88–111)。这些植物来源的基因在常见的细菌宿主如大肠杆菌中表达量并不理想，而且通常稳定性差。经报道和表征的微生物来源的SUS非常少，仅有的几个例子包括来自Nitrosomonas Europaea、Acidithiobacillus caldus、Denitrovibrio acetiphilus和Melioribacter roseus的蔗糖合酶(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 currently widely characterized and used 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). The expression levels of these plant-derived genes in common bacterial hosts such as Escherichia coli are not ideal, and their stability is usually poor. There are very few reported and characterized SUS from microbial sources, and the only few examples include 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).
因此，需要鉴定具有提高的活性和/或稳定性的SUS，包括野生型SUS和其变体。
发明内容Therefore, there is a need to identify SUS with improved activity and/or stability, including wild-type SUS and variants thereof. Contents of the invention
在第一方面，本发明提供一种经修饰的蔗糖合酶(SUS)多肽，与其野生型SUS多肽相比，包含选自位置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和790的一或多个位置的氨基酸取代，其中所述位置参照SEQ ID NO:1进行编号，其中位置4的氨基酸取代为V，位置24的氨基酸取代为L，位置41的氨基酸取代为D、E或W，位置108的氨基酸取代为C或M，位置114的氨基酸取代为E，位置133的氨基酸取代为K或R，位置136的氨基酸取代为M、T或K，位置161的氨基酸取代为D，位置300的氨基酸取代为M、G或A，位置433的氨基酸取代为C或R，位置473的氨基酸取代为G，位置474的氨基酸取代为C、V或H，位置476的氨基酸取代为V或C，位置479的氨基酸取代为A、R或N，位置482的氨基酸取代为S、T或V，位置483的氨基酸取代为N、H或G，位置513的氨基酸取代为K、Q或I，位置515的氨基酸取代为R或S，位置518的氨基酸取代为Y、V、I、L、F或T，位置529的氨基酸取代为V、T或H，位置533的氨基酸取代为F或L，位置534的氨基酸取代为F、W或Y，位置543的氨基酸取代为L，位置544的氨基酸取代为I，位置585的氨基酸取代为K或R，位置629的氨基酸取代为D，位置630的氨基酸取代为N、H或S，位置640的氨基酸取代为P，位置641的氨基酸取代为R或K，位置644的氨基酸取代为A、V或S，位置664的氨基酸取代为Y，位置676的氨基酸取代为K，位置697的氨基酸取代为V、I或K，位置713的氨基酸取代为L或M，位置715的氨基酸取代为L或V，位置726的氨基酸取代为D或E，位置729的氨基酸取代为D，位置741的氨基酸取代为C或G，位置768的氨基酸取代为P、E或A，位置769的氨基酸取代为P或Q，位置773的氨基酸取代为L，位置788的氨基酸取代为R，位置790的氨基酸取代为K或R，其中所述经修饰的SUS具有催化蔗糖分解，在核苷二磷酸(NDP)存在下产生果糖和NDP-葡萄糖的活性。In a first aspect, 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 The amino acid at position 161 is substituted with D, 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 The amino acid substitution of position 476 is C, V or H, the amino acid substitution of position 476 is V or C, the amino acid substitution of position 479 is A, R or N, the amino acid substitution of position 482 is S, T or V, the amino acid substitution of position 483 is N, H or G, the amino acid at position 513 is substituted with K, Q or I, the amino acid at position 515 is substituted with R or S, the amino acid at position 518 is substituted with Y, V, I, L, F or T, the amino acid at position 529 The amino acid at position 533 is substituted with F or L, the amino acid at position 534 is substituted with F, W or Y, the amino acid at position 543 is substituted with L, the amino acid at position 544 is substituted with I, the amino acid at position 585 The amino acid at position 629 is substituted by D, the amino acid at position 630 is substituted by N, H or S, the amino acid at position 640 is substituted by P, the amino acid at position 641 is substituted by R or K, the amino acid at position 644 is substituted by A, V or S, the amino acid at position 664 is substituted with Y, the amino acid at position 676 is substituted with K, the amino acid at position 697 is substituted with V, I or K, the amino acid at position 713 is substituted with L or M, the amino acid at position 715 is substituted with L or V, the amino acid at position 726 is substituted by D or E, the amino acid at position 729 is substituted by D, the amino acid at position 741 is substituted by C or G, the amino acid at position 768 is substituted by P, E or A, the amino acid at position 769 is substituted by P or Q, the amino acid at position 773 is substituted by L, the amino acid at position 788 is substituted by R, and the amino acid at position 790 is substituted by K or R, wherein the modified SUS has the ability to catalyze sucrose decomposition, and in nucleoside diphosphate (NDP) Generates activity in the presence of fructose and NDP-glucose.
本发明还提供经修饰的SUS多肽，包含SEQ ID NO:2-208之一的氨基酸序列，或者所述经修饰的SUS与2-208之一相比，在除位置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和790之外的位置包含1-10个氨基酸取代，其中所述经修饰的SUS具有催化蔗糖分解，在核苷二磷酸(NDP)存在下产生果糖和NDP-葡萄糖的活性。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).
在第二方面，本发明提供编码本发明的经修饰的SUS多肽的多核苷酸、表达载体和宿主细胞。In a second aspect, the invention provides polynucleotides, expression vectors and host cells encoding modified SUS polypeptides of the invention.
在第三方面，本发明提供一种产生NDP葡萄糖的方法，包括在NDP存在的条件下，使衍生自微生物的SUS多肽或包含所述SUS多肽的宿主细胞与蔗糖接触，其中所述SUS多肽是野生型SUS多肽，如SEQ ID NO:1的SUS多肽或本发明的经修饰的SUS多肽。
发明详述In a third aspect, 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. Detailed description of the invention
本发明主要涉及经修饰的SUS，用于催化蔗糖分解，在NDP存在下产生果糖和NDP-葡萄糖。除非另有说明，本文中使用的术语具有本领域技术人员一般理解的含义。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. Unless otherwise stated, the terms used herein have the meanings generally understood by those skilled in the art.
一、微生物来源的蔗糖合酶及其经修饰的变体1. Sucrose synthase derived from microorganisms and its modified variants
如本文所用，术语“蔗糖合酶”是指催化葡萄糖和果糖反应生产蔗糖的酶，这个反应是可逆的。即，蔗糖合酶也可以催化蔗糖分解，在核苷二磷酸(NDP，例如UDP和ADP)的存在下，产生果糖和NDP-葡萄糖。As used herein, the term "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).
如本文所用，术语“肽”表示通过肽键连接的至少两个氨基酸的链。术语“多肽”在本文中可以与属于“蛋白质”互换使用，是指含有十个或更多个氨基酸残基的链。本文中的所有肽和多肽化学式或序列均是从左至右书写的，表示从氨基末端至羧基末端的方向。As used herein, the term "peptide" refers to a chain of at least two amino acids linked by peptide bonds. The term "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.
术语“氨基酸”包括蛋白质中天然存在的氨基酸和非天然氨基酸。蛋白质中天然存在的氨基酸的单字母和三字母命名采用本领域惯用名，可见于Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd,ed.Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)。
The term "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).
The term "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).
如本文所用，术语“修饰”是包括对多肽的任何化学修饰，也包括对氨基酸序列的修饰，例如氨基酸的取代、缺失、***和/或添加。
As used herein, the term "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.
发明人出人预料地发现，来自Ectothiorhodospira sp.BSL-9的蔗糖合酶EsSUS(SEQ ID NO:1，NCBI登录号WP_063464253)及其突变体具有高活性和高稳定性，适合用于催化蔗糖分解，在NDP(如UDP或ADP)的存在下产生NDP-葡萄糖。The inventor unexpectedly discovered that the sucrose synthase EsSUS (SEQ ID NO: 1, NCBI accession number WP_063464253) and 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).
在一些实施方案中，本发明的经修饰的SUS多肽与其野生型SUS相比，包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸取代，其中所述经修饰的SUS多肽具有催化蔗糖分解，在NDP存在下产生果糖和NDP-葡萄糖的活性。In some embodiments, 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.
在一些实施方案中，本发明的经修饰的SUS多肽与其野生型SUS相比，包含选自位置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和790的一或多个位置的氨基酸取代，其中所述位置参照SEQ ID NO:1进行编号，其中所述经修饰的SUS具有催化蔗糖分解，在核苷二磷酸(NDP)存在下产生果糖和NDP-葡萄糖的活性。优选地，位置4的氨基酸取代为V。优选地，位置24的氨基酸取代为L。优选地，位置41的氨基酸取代为D、E或W。优选地，位置108的氨基酸取代为C或M。优选地，位置114的氨基酸取代为E。优选地，位置133的氨基酸取代为K或R。优选地，位置136的氨基酸取代为M、T或K。优选地，位置161的氨基酸取代为D。优选地，位置300的氨基酸取代为M、G或A。优选地，位置433的氨基酸取代为C或R。优选地，位置473的氨基酸取代为G。优选地，位置474的氨基酸取代为C、V或H。优选地，位置476的氨基酸取代为V或C。优选地，位置479的氨基酸取代为A、R或N。优选地，位置482的氨基酸取代为S、T或V。优选地，位置483的氨基酸取代为N、H或G。优选地，位置513的氨基酸取代为K、Q或I。优选地，位置515的氨基酸取代为R或S。优选地，位置518的氨基酸取代为Y、V、I、L、F或T。优选地，位置529的氨基酸取代为V、T或H。优选地，位置533的氨基酸取代为F或L。优选地，位置534的氨基酸取代为F、W或Y。优选地，位置543的氨基酸取代为L。优选地，位置544的氨基酸取代为I。优选地，位置585的氨基酸取代为K或R。优选地，位置629的氨基酸取代为D。优选地，位置630的氨基酸取代为N、H或S。优选地，位置640的氨基酸取代为P。优选地，位置641的氨基酸取代为R或K。优选地，位置644的氨基酸取代为A、V或S。优选地，位置664的氨基酸取代为Y。优选地，位置676的氨基酸取代为K。优选地，位置697的氨基酸取代为V、I或K。优选地，位置713的氨基酸取代为L或M。优选地，位置715的氨基酸取代为L或V。优选地，位置726的氨基酸取代为D或E。优选地，位置729的氨基酸取代为D。优选地，位置741的氨基酸取代为C或G。优选地，位置768的氨基酸取代为P、E或A。优选地，位置769的氨基酸取代为P或Q。优选地，位置773的氨基酸取代为L。优选地，位置788的氨基酸取代为R。优选地，位置790的氨基酸取代为K或R。In some embodiments, 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). Preferably, the amino acid at position 4 is substituted with V. Preferably, the amino acid substitution at position 24 is L. Preferably, the amino acid substitution at position 41 is D, E or W. Preferably, the amino acid substitution at position 108 is C or M. Preferably, the amino acid at position 114 is substituted with E. Preferably, the amino acid substitution at position 133 is K or R. Preferably, the amino acid substitution at position 136 is M, T or K. Preferably, the amino acid at position 161 is substituted with D. Preferably, the amino acid substitution at position 300 is M, G or A. Preferably, the amino acid substitution at position 433 is C or R. Preferably, the amino acid at position 473 is substituted with G. Preferably, the amino acid substitution at position 474 is C, V or H. Preferably, the amino acid substitution at position 476 is V or C. Preferably, the amino acid substitution at position 479 is A, R or N. Preferably, the amino acid substitution at position 482 is S, T or V. Preferably, the amino acid substitution at position 483 is N, H or G. Preferably, the amino acid substitution at position 513 is K, Q or I. Preferably, the amino acid substitution at position 515 is R or S. Preferably, the amino acid substitution at position 518 is Y, V, I, L, F or T. Preferably, the amino acid substitution at position 529 is V, T or H. Preferably, the amino acid substitution at position 533 is F or L. Preferably, the amino acid substitution at position 534 is F, W or Y. Preferably, the amino acid substitution at position 543 is L. Preferably, the amino acid substitution at position 544 is I. Preferably, the amino acid substitution at position 585 is K or R. Preferably, the amino acid at position 629 is substituted with D. Preferably, the amino acid substitution at position 630 is N, H or S. Preferably, the amino acid at position 640 is substituted with P. Preferably, the amino acid substitution at position 641 is R or K. Preferably, the amino acid substitution at position 644 is A, V or S. Preferably, the amino acid substitution at position 664 is Y. Preferably, the amino acid substitution at position 676 is K. Preferably, the amino acid substitution at position 697 is V, I or K. Preferably, the amino acid substitution at position 713 is L or M. Preferably, the amino acid substitution at position 715 is L or V. Preferably, the amino acid substitution at position 726 is D or E. Preferably, the amino acid at position 729 is substituted with D. Preferably, the amino acid substitution at position 741 is C or G. Preferably, the amino acid substitution at position 768 is P, E or A. Preferably, the amino acid substitution at position 769 is P or Q. Preferably, the amino acid substitution at position 773 is L. Preferably, the amino acid at position 788 is substituted with R. Preferably, the amino acid substitution at position 790 is K or R.
在一些实施方案中，本发明的经修饰的SUS多肽包含位置133和135的氨基酸取
代，位置529和768的氨基酸取代，位置136和768的氨基酸取代，或位置133和529的氨基酸取代。In some embodiments, 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.
在一些实施方案中，本发明的经修饰的SUS多肽包含位置133、136、529、768和790的氨基酸取代，和任选存在的选自位置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和741的一或多个位置的氨基酸取代。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133、136、513、515、518、529、640、641、644、768和790的氨基酸取代。In some embodiments, 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. In some embodiments, modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.
在一些实施方案中，所述野生型SUS多肽包含SEQ ID NO:1的氨基酸序列或其天然变体。In some embodiments, the wild-type SUS polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or a natural variant thereof.
在一些实施方案中，本发明的经修饰的SUS多肽包含SEQ ID NO:2-208之一的氨基酸序列，或由SEQ ID NO:2-208之一的氨基酸序列组成，或者所述经修饰的SUS与2-208之一相比，在除位置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和790之外的位置包含1-10个氨基酸取代，其中所述经修饰的SUS多肽具有催化蔗糖分解，在NDP存在下产生果糖和NDP-葡萄糖的活性。In some embodiments, 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.
在一些实施方案中，与其野生型相比，本发明的经修饰的SUS多肽中的突变是选自如下的氨基酸取代或氨基酸取代的组合(位置参照SEQ ID NO:1进行编号)：In some embodiments, 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:
-A4V；-A4V;
-Q24L；-Q24L;
-S108C；-S108C;
-S108M；-S108M;
-A114E；-A114E;
-N133K；-N133K;
-N133R；-N133R;
-N136M；-N136M;
-N136T；-N136T;
-N136K；-N136K;
-G161D；-G161D;
-T479N；-T479N;
-A482S；-A482S;
-A482T；-A482T;
-A482V；-A482V;
-G529V；-G529V;
-G529T；
-G529T;
-G529H；-G529H;
-G726D；-G726D;
-A741C；-A741C;
-A741G；-A741G;
-L768P；-L768P;
-L768E；-L768E;
-L768A；-L768A;
-T773L；-T773L;
-A790K；-A790K;
-A790R；-A790R;
-E769P；-E769P;
-E769Q；-E769Q;
-L788R；-L788R;
-133R+136M；-133R+136M;
-133K+136M；-133K+136M;
-133R+136K；-133R+136K;
-133K+136K；-133K+136K;
-529T+768E；-529T+768E;
-529T+768E+790R；-529T+768E+790R;
-529H+768A+790K；-529H+768A+790K;
-529V+768A+790K；-529V+768A+790K;
-529V+768E+790K；-529V+768E+790K;
-529H+768A+790R；-529H+768A+790R;
-529T+768E+790K；-529T+768E+790K;
-529H+768E+790R；-529H+768E+790R;
-136M+768A；-136M+768A;
-136M+529H+768E+790R；-136M+529H+768E+790R;
-136M+529T+768A；-136M+529T+768A;
-136M+529V+768A+790K；-136M+529V+768A+790K;
-136M+529V+768E+790R；-136M+529V+768E+790R;
-136K+529V+768E；-136K+529V+768E;
-136K+529T+768E；-136K+529T+768E;
-136K+529V+768A+790K；-136K+529V+768A+790K;
-136K+529T+768E+790K；-136K+529T+768E+790K;
-133R+529V；-133R+529V;
-133R+529V+768E+790R；
-133R+529V+768E+790R;
-133K+529V+768E+790K；-133K+529V+768E+790K;
-133K+529T+768E+790R；-133K+529T+768E+790R;
-133K+529T+768A+790K；-133K+529T+768A+790K;
-133K529H+768E+790K；-133K529H+768E+790K;
-133R+136M+529T+768E+790K；-133R+136M+529T+768E+790K;
-133R+136M+529T+768E+790R；-133R+136M+529T+768E+790R;
-133R+136M+529H+768A+790K；-133R+136M+529H+768A+790K;
-133K+136M+529T+790K；-133K+136M+529T+790K;
-133K+136M+529T+768E+790K；-133K+136M+529T+768E+790K;
-133K+136M+529H+768E+790K；-133K+136M+529H+768E+790K;
-133R+136K+529V+768E+790K；-133R+136K+529V+768E+790K;
-133R+136K+529T+768E+790R；-133R+136K+529T+768E+790R;
-133R+136K+529H+768E+790R；-133R+136K+529H+768E+790R;
-133R+136K+529H+768A+790K；-133R+136K+529H+768A+790K;
-133K+136K+529T+768E+790K；-133K+136K+529T+768E+790K;
-133K+136K+529T+768A+790K；-133K+136K+529T+768A+790K;
-133K+136M+529H+768E+790K+300M；-133K+136M+529H+768E+790K+300M;
-133K+136M+529H+768E+790K+300G；-133K+136M+529H+768E+790K+300G;
-133K+136M+529H+768E+790K+300A；-133K+136M+529H+768E+790K+300A;
-133K+136M+529H+768E+790K+433C；-133K+136M+529H+768E+790K+433C;
-133K+136M+529H+768E+790K+433R；-133K+136M+529H+768E+790K+433R;
-133K+136M+529H+768E+790K+473G；-133K+136M+529H+768E+790K+473G;
-133K+136M+529H+768E+790K+474C；-133K+136M+529H+768E+790K+474C;
-133K+136M+529H+768E+790K+474V；-133K+136M+529H+768E+790K+474V;
-133K+136M+529H+768E+790K+474H；-133K+136M+529H+768E+790K+474H;
-133K+136M+529H+768E+790K+476V；-133K+136M+529H+768E+790K+476V;
-133K+136M+529H+768E+790K+476C；-133K+136M+529H+768E+790K+476C;
-133K+136M+529H+768E+790K+479A；-133K+136M+529H+768E+790K+479A;
-133K+136M+529H+768E+790K+479R；-133K+136M+529H+768E+790K+479R;
-133K+136M+529H+768E+790K+479N；-133K+136M+529H+768E+790K+479N;
-133K+136M+529H+768E+790K+483N；-133K+136M+529H+768E+790K+483N;
-133K+136M+529H+768E+790K+483H；-133K+136M+529H+768E+790K+483H;
-133K+136M+529H+768E+790K+483G；-133K+136M+529H+768E+790K+483G;
-133K+136M+529H+768E+790K+513K；-133K+136M+529H+768E+790K+513K;
-133K+136M+529H+768E+790K+513Q；-133K+136M+529H+768E+790K+513Q;
-133K+136M+529H+768E+790K+515R；
-133K+136M+529H+768E+790K+515R;
-133K+136M+529H+768E+790K+543L；-133K+136M+529H+768E+790K+543L;
-133K+136M+529H+768E+790K+518Y；-133K+136M+529H+768E+790K+518Y;
-133K+136M+529H+768E+790K+518V；-133K+136M+529H+768E+790K+518V;
-133K+136M+529H+768E+790K+518I；-133K+136M+529H+768E+790K+518I;
-133K+136M+529H+768E+790K+518F；-133K+136M+529H+768E+790K+518F;
-133K+136M+529H+768E+790K+518L；-133K+136M+529H+768E+790K+518L;
-133K+136M+529H+768E+790K+518V；-133K+136M+529H+768E+790K+518V;
-133K+136M+529H+768E+790K+518T；-133K+136M+529H+768E+790K+518T;
-133K+136M+529H+768E+790K+640P；-133K+136M+529H+768E+790K+640P;
-133K+136M+529H+768E+790K+641R；-133K+136M+529H+768E+790K+641R;
-133K+136M+529H+768E+790K+644A；-133K+136M+529H+768E+790K+644A;
-133K+136M+529H+768E+790K+644V；-133K+136M+529H+768E+790K+644V;
-133K+136M+529H+768E+790K+664Y；-133K+136M+529H+768E+790K+664Y;
-133K+136M+529H+768E+790K+676k；-133K+136M+529H+768E+790K+676k;
-133K+136M+529H+768E+790K+741G；-133K+136M+529H+768E+790K+741G;
-133K+136M+529H+768E+790K+513I+515S+518I+641R；-133K+136M+529H+768E+790K+513I+515S+518I+641R;
-133K+136M+529H+768E+790K+513I+515S+518I+641K+644V；-133K+136M+529H+768E+790K+513I+515S+518I+641K+644V;
-133K+136M+529H+768E+790K+513I+515S+518I+641R+644V；-133K+136M+529H+768E+790K+513I+515S+518I+641R+644V;
-133K+136M+529H+768E+790K+513I+515S+518I+641R+644A；-133K+136M+529H+768E+790K+513I+515S+518I+641R+644A;
-133K+136M+529H+768E+790K+513K+515S+518Y+641K+644V；-133K+136M+529H+768E+790K+513K+515S+518Y+641K+644V;
-133K+136M+529H+768E+790K+513K+515S+518V；-133K+136M+529H+768E+790K+513K+515S+518V;
-133K+136M+529H+768E+790K+513I+515S+518V+640P+641R+644V；-133K+136M+529H+768E+790K+513I+515S+518V+640P+641R+644V;
-133K+136M+529H+768E+790K+518V+641R+644V；-133K+136M+529H+768E+790K+518V+641R+644V;
-133K+136M+529H+768E+790K+513K+515S+518V+640P+641K；-133K+136M+529H+768E+790K+513K+515S+518V+640P+641K;
-133K+136M+529H+768E+790K+513I+515S+518V+640P+641R；-133K+136M+529H+768E+790K+513I+515S+518V+640P+641R;
-133K+136M+529H+768E+790K+513I+515S+518V+640P+641K+644A；-133K+136M+529H+768E+790K+513I+515S+518V+640P+641K+644A;
-133K+136M+529H+768E+790K+513I+515S+518V+641K+644V；-133K+136M+529H+768E+790K+513I+515S+518V+641K+644V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641K；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641K;
-133K+136M+529H+768E+790K+513I+515S+518V+641K+644S+543L；-133K+136M+529H+768E+790K+513I+515S+518V+641K+644S+543L;
-133K+136M+529H+768E+790K+513I+515S+518F+641R+644V；-133K+136M+529H+768E+790K+513I+515S+518F+641R+644V;
-133K+136M+529H+768E+790K+518F+641R+644V；-133K+136M+529H+768E+790K+518F+641R+644V;
-133K+136M+529H+768E+790K+513I+515S+518F+640P+641K+644A；-133K+136M+529H+768E+790K+513I+515S+518F+640P+641K+644A;
-133K+136M+529H+768E+790K+513K+515S+518Y+641K；-133K+136M+529H+768E+790K+513K+515S+518Y+641K;
-133K+136M+529H+768E+790K+513I+515S+518T+640P+641R+644V；-133K+136M+529H+768E+790K+513I+515S+518T+640P+641R+644V;
-133K+136M+529H+768E+790K+513K+515S+518T+641K+644A；
-133K+136M+529H+768E+790K+513K+515S+518T+641K+644A;
-133K+136M+529H+768E+790K+513I+515R+640P+641R+644V；-133K+136M+529H+768E+790K+513I+515R+640P+641R+644V;
-133K+136M+529H+768E+790K+513I+515S+518T+640P+641K+644V；-133K+136M+529H+768E+790K+513I+515S+518T+640P+641K+644V;
-133K+136M+529H+768E+790K+513I+515R+640P+641K+644A；-133K+136M+529H+768E+790K+513I+515R+640P+641K+644A;
-133K+136M+529H+768E+790K+513I+515S+518Y+640P+641K+644V；-133K+136M+529H+768E+790K+513I+515S+518Y+640P+641K+644V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V(同义突变？)+664Y；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V (synonymous mutation?)+664Y;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+I483H；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+I483H;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41D；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41D;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41W；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41W;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41E；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41E;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534F；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534F;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+544I；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+544I;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+585K；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+585K;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+585R；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+585R;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+629D；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+629D;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630N；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630N;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630H；
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630H;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630S；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630S;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697I；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697I;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697K；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697K;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+713L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+713L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+713M；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+713M;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+726D；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+726D;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+726E；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+726E;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+729D；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+729D;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+585R+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+585R+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697K+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697K+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697V+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697V+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697I；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697I;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697K+715V；
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697K+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585K+629D+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585K+629D+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585R+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585R+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+585R+629D+697K+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+585R+629D+697K+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697I+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697I+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+544I+585R+629D+697V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+544I+585R+629D+697V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+585R+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+585R+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697I；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697I;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585R+697V+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585R+697V+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+585K+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+585K+629D+697V+715V;
-533F+534Y+585K+629D+697I+715V；-533F+534Y+585K+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+585R+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+585R+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585R+629D+697K+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585R+629D+697K+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+585K+629D+697K+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+585K+629D+697K+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+544I+585K+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+544I+585K+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+585K+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+585K+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533
L+534Y+544I+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533 L+534Y+544I+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+585K+629D+697K+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+585K+629D+697K+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585K+629D+697K+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585K+629D+697K+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+629D+697V+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+629D+697V+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+629D+697I+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+629D+697I+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585K+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585K+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+544I+585R+629D+697I+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+544I+585R+629D+697I+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697I+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697I+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+585K+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+585K+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+544I+585K+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+544I+585K+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585R+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585R+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585K+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585K+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697V+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697V+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+697V+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+697V+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533
F+534W+585R+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533 F+534W+585R+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+629D+697K+715L；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+629D+697K+715L;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585R+629D+697V+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585R+629D+697V+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585K+629D+697I+715V；-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585K+629D+697I+715V;
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+697V+715V；和-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+697V+715V; and
-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585R+697V+715V。-133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585R+697V+715V.
如本文所用，术语“野生型SUS”是指天然存在的SUS。在一些实施方案中，所述野生型SUS是来自外硫红螺菌属的SUS，如来自Ectothiorhodospira sp.BSL-9的SUS(EsSUS，SEQ ID NO:1)。As used herein, the term "wild-type SUS" refers to naturally occurring SUS. In some embodiments, the wild-type SUS is a SUS from Ectothiorhodospira sp. BSL-9 (EsSUS, SEQ ID NO: 1).
对于本发明，为确定两个氨基酸序列或两个核酸序列的相同性百分比，以最佳比较为目的比对序列(例如在第一个氨基酸或核酸序列中可导入缺口，以与第二个氨基酸或核酸序列进行最佳比对)。然后比较在相应氨基酸位置或核苷酸位置的氨基酸残基或核苷酸。当第一个序列中的位置在第二个序列中相应位置由相同氨基酸残基或核苷酸占据时，则这些分子在这个位置是相同的。两个序列之间的相同性百分比是所述序列共有的相同位置的数量的函数(即相同性百分比＝相同位置的数量/位置(即重叠位置)的总数量×100)。优选地，这两个序列是相同长度的。For the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, 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. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions/total number of positions (i.e., overlapping positions) x 100). Preferably, the two sequences are of the same length.
本领域技术人员知晓，可以使用不同的计算机程序确定两个序列之间的相同性。Those skilled in the art are aware that different computer programs can be used to determine the identity between two sequences.
“氨基酸相同性百分比”或者“氨基酸序列相同性百分比”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽具有大约指定的相同氨基酸百分比。例如，“95％的氨基酸相同性”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽有95％的氨基酸相同。"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.
在一些实施方案中，所述野生型SUS与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性。In some embodiments, 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.
在一些实施方案中，本发明的经修饰的SUS多肽与其野生型SUS相比，包含选自位置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和790的一或多个位置的氨基酸取代，其中所述位置参照SEQ ID NO:1进行编号，其中所述经修饰的SUS
具有催化蔗糖分解，在核苷二磷酸(NDP)存在下产生果糖和NDP-葡萄糖的活性。优选地，位置4的氨基酸取代为V。优选地，位置24的氨基酸取代为L。优选地，位置41的氨基酸取代为D、E或W。优选地，位置108的氨基酸取代为C或M。优选地，位置114的氨基酸取代为E。优选地，位置133的氨基酸取代为K或R。优选地，位置136的氨基酸取代为M、T或K。优选地，位置161的氨基酸取代为D。优选地，位置300的氨基酸取代为M、G或A。优选地，位置433的氨基酸取代为C或R。优选地，位置473的氨基酸取代为G。优选地，位置474的氨基酸取代为C、V或H。优选地，位置476的氨基酸取代为V或C。优选地，位置479的氨基酸取代为A、R或N。优选地，位置482的氨基酸取代为S、T或V。优选地，位置483的氨基酸取代为N、H或G。优选地，位置513的氨基酸取代为K、Q或I。优选地，位置515的氨基酸取代为R或S。优选地，位置518的氨基酸取代为Y、V、I、L、F或T。优选地，位置529的氨基酸取代为V、T或H。优选地，位置533的氨基酸取代为F或L。优选地，位置534的氨基酸取代为F、W或Y。优选地，位置543的氨基酸取代为L。优选地，位置544的氨基酸取代为I。优选地，位置585的氨基酸取代为K或R。优选地，位置629的氨基酸取代为D。优选地，位置630的氨基酸取代为N、H或S。优选地，位置640的氨基酸取代为P。优选地，位置641的氨基酸取代为R或K。优选地，位置644的氨基酸取代为A、V或S。优选地，位置664的氨基酸取代为Y。优选地，位置676的氨基酸取代为K。优选地，位置697的氨基酸取代为V、I或K。优选地，位置713的氨基酸取代为L或M。优选地，位置715的氨基酸取代为L或V。优选地，位置726的氨基酸取代为D或E。优选地，位置729的氨基酸取代为D。优选地，位置741的氨基酸取代为C或G。优选地，位置768的氨基酸取代为P、E或A。优选地，位置769的氨基酸取代为P或Q。优选地，位置773的氨基酸取代为L。优选地，位置788的氨基酸取代为R。优选地，位置790的氨基酸取代为K或R。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133和135的氨基酸取代，位置529和768的氨基酸取代，位置136和768的氨基酸取代，或位置133和529的氨基酸取代。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133、136、529、768和790的氨基酸取代，和任选存在的选自位置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和741的一或多个位置的氨基酸取代。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133、136、513、515、518、529、640、641、644、768和790的氨基酸取代。优选地，所述野生型SUS与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性。In some embodiments, 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). Preferably, the amino acid at position 4 is substituted with V. Preferably, the amino acid substitution at position 24 is L. Preferably, the amino acid substitution at position 41 is D, E or W. Preferably, the amino acid substitution at position 108 is C or M. Preferably, the amino acid at position 114 is substituted with E. Preferably, the amino acid substitution at position 133 is K or R. Preferably, the amino acid substitution at position 136 is M, T or K. Preferably, the amino acid at position 161 is substituted with D. Preferably, the amino acid substitution at position 300 is M, G or A. Preferably, the amino acid substitution at position 433 is C or R. Preferably, the amino acid at position 473 is substituted with G. Preferably, the amino acid substitution at position 474 is C, V or H. Preferably, the amino acid substitution at position 476 is V or C. Preferably, the amino acid substitution at position 479 is A, R or N. Preferably, the amino acid substitution at position 482 is S, T or V. Preferably, the amino acid substitution at position 483 is N, H or G. Preferably, the amino acid substitution at position 513 is K, Q or I. Preferably, the amino acid substitution at position 515 is R or S. Preferably, the amino acid substitution at position 518 is Y, V, I, L, F or T. Preferably, the amino acid substitution at position 529 is V, T or H. Preferably, the amino acid substitution at position 533 is F or L. Preferably, the amino acid substitution at position 534 is F, W or Y. Preferably, the amino acid substitution at position 543 is L. Preferably, the amino acid substitution at position 544 is I. Preferably, the amino acid substitution at position 585 is K or R. Preferably, the amino acid at position 629 is substituted with D. Preferably, the amino acid substitution at position 630 is N, H or S. Preferably, the amino acid at position 640 is substituted with P. Preferably, the amino acid substitution at position 641 is R or K. Preferably, the amino acid substitution at position 644 is A, V or S. Preferably, the amino acid substitution at position 664 is Y. Preferably, the amino acid substitution at position 676 is K. Preferably, the amino acid substitution at position 697 is V, I or K. Preferably, the amino acid substitution at position 713 is L or M. Preferably, the amino acid substitution at position 715 is L or V. Preferably, the amino acid substitution at position 726 is D or E. Preferably, the amino acid at position 729 is substituted with D. Preferably, the amino acid substitution at position 741 is C or G. Preferably, the amino acid substitution at position 768 is P, E or A. Preferably, the amino acid substitution at position 769 is P or Q. Preferably, the amino acid substitution at position 773 is L. Preferably, the amino acid at position 788 is substituted with R. Preferably, the amino acid substitution at position 790 is K or R. In some embodiments, 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. In some embodiments, 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. In some embodiments, modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790. Preferably, 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.
在一些实施方案中，本发明的经修饰的SUS多肽与其野生型SUS相比，包含选自位置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和790的一或多个位
置的氨基酸取代，其中所述位置参照SEQ ID NO:1进行编号，所述经修饰的SUS与SEQ ID NO:1具有至少90％、91％、92％、93％、94％、95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％或99.5％的序列相同性，其中所述经修饰的SUS具有催化蔗糖分解，在核苷二磷酸(NDP)存在下产生果糖和NDP-葡萄糖的活性。优选地，位置4的氨基酸取代为V。优选地，位置24的氨基酸取代为L。优选地，位置41的氨基酸取代为D、E或W。优选地，位置108的氨基酸取代为C或M。优选地，位置114的氨基酸取代为E。优选地，位置133的氨基酸取代为K或R。优选地，位置136的氨基酸取代为M、T或K。优选地，位置161的氨基酸取代为D。优选地，位置300的氨基酸取代为M、G或A。优选地，位置433的氨基酸取代为C或R。优选地，位置473的氨基酸取代为G。优选地，位置474的氨基酸取代为C、V或H。优选地，位置476的氨基酸取代为V或C。优选地，位置479的氨基酸取代为A、R或N。优选地，位置482的氨基酸取代为S、T或V。优选地，位置483的氨基酸取代为N、H或G。优选地，位置513的氨基酸取代为K、Q或I。优选地，位置515的氨基酸取代为R或S。优选地，位置518的氨基酸取代为Y、V、I、L、F或T。优选地，位置529的氨基酸取代为V、T或H。优选地，位置533的氨基酸取代为F或L。优选地，位置534的氨基酸取代为F、W或Y。优选地，位置543的氨基酸取代为L。优选地，位置544的氨基酸取代为I。优选地，位置585的氨基酸取代为K或R。优选地，位置629的氨基酸取代为D。优选地，位置630的氨基酸取代为N、H或S。优选地，位置640的氨基酸取代为P。优选地，位置641的氨基酸取代为R或K。优选地，位置644的氨基酸取代为A、V或S。优选地，位置664的氨基酸取代为Y。优选地，位置676的氨基酸取代为K。优选地，位置697的氨基酸取代为V、I或K。优选地，位置713的氨基酸取代为L或M。优选地，位置715的氨基酸取代为L或V。优选地，位置726的氨基酸取代为D或E。优选地，位置729的氨基酸取代为D。优选地，位置741的氨基酸取代为C或G。优选地，位置768的氨基酸取代为P、E或A。优选地，位置769的氨基酸取代为P或Q。优选地，位置773的氨基酸取代为L。优选地，位置788的氨基酸取代为R。优选地，位置790的氨基酸取代为K或R。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133和135的氨基酸取代，位置529和768的氨基酸取代，位置136和768的氨基酸取代，或位置133和529的氨基酸取代。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133、136、529、768和790的氨基酸取代，和任选存在的选自位置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和741的一或多个位置的氨基酸取代。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133、136、513、515、518、529、640、641、644、768和790的氨基酸取代。In some embodiments, 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 diphosphate (NDP ) in the presence of fructose and NDP-glucose. Preferably, the amino acid at position 4 is substituted with V. Preferably, the amino acid substitution at position 24 is L. Preferably, the amino acid substitution at position 41 is D, E or W. Preferably, the amino acid substitution at position 108 is C or M. Preferably, the amino acid at position 114 is substituted with E. Preferably, the amino acid substitution at position 133 is K or R. Preferably, the amino acid substitution at position 136 is M, T or K. Preferably, the amino acid at position 161 is substituted with D. Preferably, the amino acid substitution at position 300 is M, G or A. Preferably, the amino acid substitution at position 433 is C or R. Preferably, the amino acid at position 473 is substituted with G. Preferably, the amino acid substitution at position 474 is C, V or H. Preferably, the amino acid substitution at position 476 is V or C. Preferably, the amino acid substitution at position 479 is A, R or N. Preferably, the amino acid substitution at position 482 is S, T or V. Preferably, the amino acid substitution at position 483 is N, H or G. Preferably, the amino acid substitution at position 513 is K, Q or I. Preferably, the amino acid substitution at position 515 is R or S. Preferably, the amino acid substitution at position 518 is Y, V, I, L, F or T. Preferably, the amino acid substitution at position 529 is V, T or H. Preferably, the amino acid substitution at position 533 is F or L. Preferably, the amino acid substitution at position 534 is F, W or Y. Preferably, the amino acid substitution at position 543 is L. Preferably, the amino acid substitution at position 544 is I. Preferably, the amino acid substitution at position 585 is K or R. Preferably, the amino acid at position 629 is substituted with D. Preferably, the amino acid substitution at position 630 is N, H or S. Preferably, the amino acid at position 640 is substituted with P. Preferably, the amino acid substitution at position 641 is R or K. Preferably, the amino acid substitution at position 644 is A, V or S. Preferably, the amino acid substitution at position 664 is Y. Preferably, the amino acid substitution at position 676 is K. Preferably, the amino acid substitution at position 697 is V, I or K. Preferably, the amino acid substitution at position 713 is L or M. Preferably, the amino acid substitution at position 715 is L or V. Preferably, the amino acid substitution at position 726 is D or E. Preferably, the amino acid at position 729 is substituted with D. Preferably, the amino acid substitution at position 741 is C or G. Preferably, the amino acid substitution at position 768 is P, E or A. Preferably, the amino acid substitution at position 769 is P or Q. Preferably, the amino acid substitution at position 773 is L. Preferably, the amino acid at position 788 is substituted with R. Preferably, the amino acid substitution at position 790 is K or R. In some embodiments, 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. In some embodiments, 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. In some embodiments, modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.
在一些实施方案中，与SEQ ID NO:1相比，本发明的经修饰的SUS还包含一或多个氨基酸的保守取代。In some embodiments, the modified SUS of the invention further comprises one or more conservative substitutions of amino acids compared to SEQ ID NO: 1.
术语“保守取代”也称为由“同源”氨基酸残基取代，是指其中氨基酸残基由具有相似
侧链的氨基酸残基置换的取代，例如，碱性侧链的氨基酸(例如赖氨酸、精氨酸和组氨酸)、酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、非荷电极性侧链氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支的侧链氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及芳香侧链氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。The term "conservative substitution", 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), β-branched side chain amino acids (such as threonine, valine, isoleucine ) and aromatic side chain amino acids (such as tyrosine, phenylalanine, tryptophan, histidine).
保守氨基酸取代通常对所得蛋白质的活性的影响最小。这种取代在下文描述。保守取代是用大小、疏水性、电荷、极性、空间特征、芳香性等相似的氨基酸置换一个氨基酸。当希望精细调节蛋白质的特性时，这种取代通常是保守的。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.
如本文所用，“同源”氨基酸残基是指具有相似化学性质的氨基酸残基，所述化学性质涉及疏水性、电荷、极性、空间特征、芳香性特征等。彼此同源的氨基酸的例子包括正电荷的赖氨酸、精氨酸、组氨酸，负电荷的谷氨酸、天冬氨酸，疏水性的甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸，极性的丝氨酸、苏氨酸、半胱氨酸、甲硫氨酸、色氨酸、酪氨酸、天冬酰胺、谷氨酰胺，芳香性的苯丙氨酸、酪氨酸、色氨酸，化学相似侧链基团的丝氨酸与苏氨酸，或者谷氨酰胺和天冬酰胺，或者亮氨酸和异亮氨酸。As used herein, "homologous" amino acid residues refer to amino acid residues with similar chemical properties involving hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, and the like. Examples of 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.
蛋白质中氨基酸保守取代的例子包括：Ser取代Ala，Lys取代Arg，Gln或His取代Asn，Glu取代Asp，Ser取代Cys，Asn取代Gln，Asp取代Glu，Pro取代Gly，Asn或Gln取代His，Leu或Val取代Ile，Ile或Val取代Leu，Arg或Gln取代Lys，Leu或Ile取代Met，Met、Leu或Tyr取代Phe，Thr取代Ser，Ser取代Thr，Tyr取代Trp，Trp或Phe取代Tyr，及Ile或Leu取代Val。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.
在一些实施方案中，与SEQ ID NO:1相比，本发明的经修饰的SUS多肽中的修饰由位置133、136、529、768和790的氨基酸取代，和任选存在的选自位置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和741的一或多个位置的氨基酸取代组成。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133、136、513、515、518、529、640、641、644、768和790的氨基酸取代。优选地，位置41的氨基酸取代为D、E或W。优选地，位置133的氨基酸取代为K或R。优选地，位置136的氨基酸取代为M、T或K。优选地，位置300的氨基酸取代为M、G或A。优选地，位置433的氨基酸取代为C或R。优选地，位置473的氨基酸取代为G。优选地，位置474的氨基酸取代为C、V或H。优选地，位置476的氨基酸取代为V或C。优选地，位置479的氨基酸取代为A、R或N。优选地，位置483的氨基酸取代为N、H或G。优选地，位置513的氨基酸取代为K、Q或I。优选地，位置515的氨基酸取代为R或S。优选地，位置518的氨基酸取代为Y、V、I、L、F或T。优选地，位置529的氨基酸取代为V、T或H。优选地，位置533的氨基酸取代为F或L。优选地，位置534的氨基酸取代为F、W或Y。优选地，位置543的氨基酸取代为L。优选地，位置544的氨
基酸取代为I。优选地，位置585的氨基酸取代为K或R。优选地，位置629的氨基酸取代为D。优选地，位置630的氨基酸取代为N、H或S。优选地，位置640的氨基酸取代为P。优选地，位置641的氨基酸取代为R或K。优选地，位置644的氨基酸取代为A、V或S。优选地，位置664的氨基酸取代为Y。优选地，位置676的氨基酸取代为K。优选地，位置697的氨基酸取代为V、I或K。优选地，位置713的氨基酸取代为L或M。优选地，位置715的氨基酸取代为L或V。优选地，位置726的氨基酸取代为D或E。优选地，位置729的氨基酸取代为D。优选地，位置741的氨基酸取代为C或G。优选地，位置768的氨基酸取代为P、E或A。优选地，位置790的氨基酸取代为K或R。In some embodiments, 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 Composed of amino acid substitutions at one or more positions of , 726, 729 and 741. In some embodiments, modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790. Preferably, the amino acid substitution at position 41 is D, E or W. Preferably, the amino acid substitution at position 133 is K or R. Preferably, the amino acid substitution at position 136 is M, T or K. Preferably, the amino acid substitution at position 300 is M, G or A. Preferably, the amino acid substitution at position 433 is C or R. Preferably, the amino acid at position 473 is substituted with G. Preferably, the amino acid substitution at position 474 is C, V or H. Preferably, the amino acid substitution at position 476 is V or C. Preferably, the amino acid substitution at position 479 is A, R or N. Preferably, the amino acid substitution at position 483 is N, H or G. Preferably, the amino acid substitution at position 513 is K, Q or I. Preferably, the amino acid substitution at position 515 is R or S. Preferably, the amino acid substitution at position 518 is Y, V, I, L, F or T. Preferably, the amino acid substitution at position 529 is V, T or H. Preferably, the amino acid substitution at position 533 is F or L. Preferably, the amino acid substitution at position 534 is F, W or Y. Preferably, the amino acid substitution at position 543 is L. Preferably, the ammonia at position 544 The base acid is substituted with I. Preferably, the amino acid substitution at position 585 is K or R. Preferably, the amino acid at position 629 is substituted with D. Preferably, the amino acid substitution at position 630 is N, H or S. Preferably, the amino acid at position 640 is substituted with P. Preferably, the amino acid substitution at position 641 is R or K. Preferably, the amino acid substitution at position 644 is A, V or S. Preferably, the amino acid substitution at position 664 is Y. Preferably, the amino acid substitution at position 676 is K. Preferably, the amino acid substitution at position 697 is V, I or K. Preferably, the amino acid substitution at position 713 is L or M. Preferably, the amino acid substitution at position 715 is L or V. Preferably, the amino acid substitution at position 726 is D or E. Preferably, the amino acid at position 729 is substituted with D. Preferably, the amino acid substitution at position 741 is C or G. Preferably, the amino acid substitution at position 768 is P, E or A. Preferably, the amino acid substitution at position 790 is K or R.
在一些实施方案中，与SEQ ID NO:1相比，本发明的经修饰的SUS多肽中的修饰由位置133、136、529、768和790的氨基酸取代，和任选存在的选自位置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和741的一或多个位置的氨基酸取代组成，其中位置41的氨基酸取代为D、E或W，位置133的氨基酸取代为K或R，位置136的氨基酸取代为M、T或K，位置300的氨基酸取代为M、G或A，位置433的氨基酸取代为C或R，位置473的氨基酸取代为G，位置474的氨基酸取代为C、V或H，位置476的氨基酸取代为V或C，位置479的氨基酸取代为A、R或N，位置483的氨基酸取代为N、H或G，位置513的氨基酸取代为K、Q或I，位置515的氨基酸取代为R或S，位置518的氨基酸取代为Y、V、I、L、F或T，位置529的氨基酸取代为V、T或H，位置533的氨基酸取代为F或L，位置534的氨基酸取代为F、W或Y，位置543的氨基酸取代为L，位置544的氨基酸取代为I，位置585的氨基酸取代为K或R，位置629的氨基酸取代为D，位置630的氨基酸取代为N、H或S，位置640的氨基酸取代为P，位置641的氨基酸取代为R或K，位置644的氨基酸取代为A、V或S，位置664的氨基酸取代为Y，位置676的氨基酸取代为K，位置697的氨基酸取代为V、I或K，位置713的氨基酸取代为L或M，位置715的氨基酸取代为L或V，位置726的氨基酸取代为D或E，位置729的氨基酸取代为D，位置741的氨基酸取代为C或G，位置768的氨基酸取代为P、E或A，位置790的氨基酸取代为K或R。在一些实施方案中，本发明的经修饰的SUS多肽包含位置133、136、513、515、518、529、640、641、644、768和790的氨基酸取代。In some embodiments, 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, R or N, the amino acid at position 483 is substituted by N, H or G, the amino acid at position 513 is substituted by K, Q or I, the amino acid at position 515 is substituted by 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 at position 543 is substituted with L, the amino acid at position 544 is substituted with I, the amino acid at position 585 is substituted with K or R, the amino acid at position 629 is substituted with D, the amino acid at position 630 is substituted with N, H or S, the amino acid at position 640 The amino acid at position 641 is substituted with P, the amino acid at position 641 is substituted with R or K, the amino acid at position 644 is substituted with A, V or S, the amino acid at position 664 is substituted with Y, the amino acid at position 676 is substituted with K, and the amino acid at position 697 is substituted with V, I or K, the amino acid at position 713 is substituted with L or M, the amino acid at position 715 is substituted with L or V, the amino acid at position 726 is substituted with D or E, the amino acid at position 729 is substituted with D, the amino acid at position 741 is substituted with C or G, the amino acid at position 768 is substituted by P, E or A, and the amino acid at position 790 is substituted by K or R. In some embodiments, modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.
如本文所用，酶的活性指在一定条件下，在单位质量的酶催化的化学反应中，单位时间内底物的减少量或产物的增加量。例如，本发明的经修饰的SUS的活性，用一定条件下，在单位质量的经修饰的SUS多肽和NDP如UDP存在下，单位时间内蔗糖减少的量或NDP-葡萄糖如UDP葡萄糖增加的量来表示。As used herein, 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. For example, 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.
在本文中，酶的活性也可以指酶的相对活性，以感兴趣的酶的活性与催化相同反应的给定的酶的活性的比值表示，如百分比相对活性。As used herein, 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.
在一些实施方案中，本发明的经修饰的SUS的活性以与SEQ ID NO:1相比的百分
比相对活性表示。In some embodiments, the activity of the modified SUS of the invention is expressed as a percentage compared to SEQ ID NO: 1 Specific relative activity expressed.
提高SUS的稳定性对于应用于工业生产也是有利的。在一些实施方案中，所述稳定性是热稳定性，指酶在一定温度(如40-70℃或更高)温育一定时间(如10分钟至1小时)后的保持活性的能力。在一些实施方案中，所述经修饰的SUS具有优于SEQ ID NO:1的多肽的热稳定性。例如，在50-70℃温育，例如，1小时后，本发明的经修饰的SUS的活性是SEQ ID NO:1的多肽的活性的至少100％、105％、110％、120％、130％、150％、170％、200％、250％、300％或更高。或者，本发明的经修饰的SUS具有更高的T50，其中T50是指在该温度温育，例如，一小时后，酶的活性下降50％的温度。在一些实施方案中，本发明的经修饰的SUS的T50比SEQ ID NO:1的多肽高约1、2、3、4、5、6、7、8、9、10℃或更高。Improving the stability of SUS is also beneficial for application in industrial production. In some embodiments, 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). In some embodiments, the modified SUS has better thermal stability than the polypeptide of SEQ ID NO: 1. For example, after incubation at 50-70°C, for example, for 1 hour, 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. Alternatively, 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. In some embodiments, 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.
在一些实施方案中，不经预先温育，所述经修饰的SUS催化蔗糖分解，在NDP存在下产生果糖和NDP-葡萄糖的活性是SEQ ID NO:1的催化上述反应的活性的至少100％、105％、110％、120％、130％、150％、170％、200％、250％、300％、400％、500％、600％、700％、800％、900％、1000％或更高。In some embodiments, without prior incubation, 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.
在一些实施方案中，所述经修饰的SUS具有优于SEQ ID NO:1的多肽的热稳定性，例如，在50-70℃温育1小时后，所述经修饰的SUS的活性是SEQ ID NO:1的多肽的活性的至少100％、105％、110％、120％、130％、150％、170％、200％、250％、300％或更高，或者所述经修饰的SUS的T50比SEQ ID NO:1的多肽高约1、2、3、4、5、6、7、8、9、10℃或更高；且不经预先温育，其催化蔗糖分解，在NDP存在下产生果糖和NDP-葡萄糖的活性是SEQ ID NO:1的催化上述反应的活性的至少100％、105％、110％、120％、130％、150％、170％、200％、250％、300％、400％、500％、600％、700％、800％、900％、1000％或更高。In some embodiments, 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.
二、编码经修饰的SUS的多核苷酸。2. Polynucleotide encoding modified SUS.
如本文所用，术语“多核苷酸”或者“核酸分子”包括DNA分子(例如cDNA或基因组DNA)和RNA分子(例如mRNA)及使用核苷酸类似物产生的DNA或RNA的类似物。所述核酸分子可以是单链或双链的，优选双链DNA。所述核酸的合成可以使用核苷酸类似物或衍生物(例如肌苷或硫代磷酸核苷酸)。这种核苷酸可以用于，例如，制备具有改变的碱基配对能力或者增加的核酸酶抗性的核酸。As used herein, the term "polynucleotide" or "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.
本发明还提供编码本发明的经修饰的SUS的多核苷酸。因此，在本发明中，术语修饰还包括对编码本发明的SUS多肽的多核苷酸的遗传操作。所述修饰可以是核苷酸的取代、缺失、***和/或添加。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.
如本文所用，术语“编码”是指多核苷酸直接指定其蛋白质产物的氨基酸序列。编码序列的边界一般由开放读框确定，所述开放读框通常以ATG起始密码子或另外的起始密码子如GTG和TTG开始，以终止密码子如TAA、TAG和TGA结束。所述编码序列可以是DNA、cDNA或重组核苷酸序列。
As used herein, 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.
此外，涵盖本发明的全部或部分核酸序列的核酸分子可以通过聚合酶链反应(PCR)分离，所述PCR使用基于所述序列中包含的序列信息设计合成的寡核苷酸引物。Furthermore, 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.
本发明的多核苷酸可以使用cDNA、mRNA或者基因组DNA作为模板及合适的寡核苷酸引物根据标准PCR扩增技术进行扩增。如此扩增的核酸可以克隆进合适的载体中，并通过DNA序列分析进行表征。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.
本发明的多核苷酸可以通过标准的合成技术制备，例如使用自动化DNA合成仪制备。Polynucleotides of the invention can be prepared by standard synthesis techniques, for example using an automated DNA synthesizer.
本发明还涉及本文描述的核酸分子的互补链。与其它核苷酸序列互补的核酸分子是与该核苷酸序列充分互补的分子，使得其可以与其他核苷酸序列杂交，从而形成稳定双链体。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.
如本文所用，术语“杂交”是在给定的严格杂交和洗涤条件下，彼此至少大约90％、优选至少大约95％、更优选至少大约96％、更优选至少98％同源的核苷酸序列通常保持彼此杂交。As used herein, the term "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.
本领域技术人员知道各种用于杂交的条件，如严格杂交条件和高度严格杂交条件。参见，例如，Sambrook et al.,1989,Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Press,N.Y.；和Ausubel et al.(eds.),1995,Current Protocols in Molecular Biology,John Wiley&Sons,N.Y.。Those skilled in the art are aware of various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
当然，本发明的多核苷酸不包括仅与poly A序列(如mRNA的3’末端poly(A))或者与互补的一段poly T(或U)残基杂交的多核苷酸。Of course, the 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.
三、表达和生产经修饰的SUS3. Expression and production of modified SUS
为表达本发明的经修饰的SUS，还提供包含本发明的多核苷酸的核酸构建体和载体，如表达载体。To express the modified SUS of the invention, nucleic acid constructs and vectors, such as expression vectors, comprising the polynucleotides of the invention are also provided.
如本文所用，术语“表达”包括多肽生产中包含的任何步骤，包括但不限于转录、转录后修饰、翻译、翻译后修饰和分泌。As used herein, the term "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.
术语“核酸构建体”是指单链或双链的核酸分子，其分离自天然存在的基因或者被修饰为含有天然不存在的核酸区段。当所述核酸构建体含有表达本发明编码序列所需的控制序列时，术语核酸构建体与术语“表达盒”同义。The term "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. The term 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.
术语“表达载体”在本文是指线性或环形DNA分子，其包含编码本发明多肽的多核苷酸，所述多核苷酸与为所述多核苷酸表达而提供的另外的核苷酸，例如，控制序列，可操纵地连接。所述表达载体包括病毒载体或质粒载体。The term "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.
术语“控制序列”在本文是指包括表达编码本发明多肽的多核苷酸所需或有利的所有元件。各控制序列对于编码多肽的核苷酸序列可以是天然的或者是外来的，或者彼此是天然或者外来的。这种控制序列包括但不限于前导序列、聚腺苷酸化序列、前肽序列、启动子、信号肽序列及转录终止子。最低限度，控制序列包括启动子和转录及翻译终止信号。The term "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. Such 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.
例如，所述控制序列可以是合适的启动子序列，一种由宿主细胞识别以表达编码本
发明多肽的多核苷酸的核苷酸序列。所述启动子序列含有介导所述多肽的表达的转录控制序列。所述启动子可以是在所选择的宿主细胞中表现出转录活性的任何核苷酸序列，例如，大肠杆菌(Escherichia coli)lac操纵子。所述启动子还包括突变的、截短的和杂合的启动子，并且可以从与宿主细胞同源或异源的编码胞外或胞内多肽的基因获得。For example, the control sequence may be a suitable promoter sequence, one recognized by the host cell to express the code. The nucleotide sequence of the polynucleotide of the inventive polypeptide. 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.
术语“可操纵地连接”在本文是指这样的构型，其中控制序列置于相对于多核苷酸序列的编码序列的适当位置，由此所述控制序列指导多肽编码序列的表达。The term "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.
编码本发明多肽的多核苷酸可以进行各种操作，以使得多肽表达。在将其***载体之前，根据表达载体对多核苷酸的操作是可取的或必需的。利用重组DNA方法修饰多核苷酸序列的技术为本领域熟知。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.
为了鉴定和选择包含本发明的表达载体的宿主细胞，本发明的载体优选含有一或多个可选择标记，其使得可以对转化、转染、转导等的细胞进行简单的选择。可选择标记是一种基因，其产物提供生物杀灭剂或病毒抗性、重金属抗性、补充营养缺陷型等。例如，细菌的可选择标记是来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或者赋予抗生素抗性如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的标记。In order to identify and select host cells containing the expression vectors of the invention, 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. For example, 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.
本发明的载体可整合进宿主细胞基因组中或者在细胞中不依赖于基因组而自主复制。为了整合进宿主细胞基因组中或者自主复制所需的元件是本领域已知的(参见例如前述Sambrook et al.,1989)。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).
载体DNA可以通过常规转化或转染技术导入原核或真核细胞中。如本文所用，术语“转化”和“转染”是指将外源核酸(例如DNA)导入宿主细胞中的各种本领域公认的技术，可见于例如前述Sambrook et al.,1989；Davis et al.,Basic Methods in Molecular Biology(1986)及其它实验室手册。Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "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.
本发明还涉及重组宿主细胞，其包含本发明的多核苷酸，所述多核苷酸有利地用于SUS多肽的重组产生中。包含本发明多核苷酸的载体被导入宿主细胞中，由此所述载体作为染色体整合体或作为自身复制染色体外载体被保留。本领域技术人员知晓表达蛋白质的常规载体和宿主细胞。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.
在一些实施方案中，本发明的宿主细胞是大肠杆菌细胞，如大肠杆菌BL21(DE3)。在一些实施方案中，所述表达载体是pET-30a(+)。In some embodiments, the host cell of the invention is an E. coli cell, such as E. coli BL21(DE3). In some embodiments, the expression vector is pET-30a(+).
本发明的经修饰的SUS可以与非-SUS多肽(例如异源氨基酸序列)可操纵地连接，形成融合蛋白。例如，在一个实施方案中，所述融合蛋白是GST-SUS融合蛋白，其中SUS序列与GST序列的C-末端融合。这种融合蛋白可帮助重组SUS的纯化。在另一实施方案中，所述融合蛋白是在其N末端含有异源信号序列的SUS蛋白。在某些宿主细胞中(例如哺乳动物和酵母宿主细胞)，可以通过使用异源信号序列增加SUS的表达和/或分泌。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. For example, in one embodiment, 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. In another embodiment, 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.
在一些实施方案中，本发明的宿主细胞也包括表达SEQ ID NO:1的SUS多肽的宿主细胞。In some embodiments, host cells of the invention also include host cells expressing the SUS polypeptide of SEQ ID NO: 1.
四、产生NDP-葡萄糖
4. Produce NDP-glucose
本发明提供一种产生NDP-葡萄糖的方法，包括使SEQ ID NO:1的SUS多肽、本发明的经修饰的SUS或宿主细胞与D-草铵膦接触。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.
在一些实施方案中，本发明的产生NDP-葡萄糖的方法包括如下步骤：In some embodiments, the method of producing NDP-glucose of the present invention includes the steps of:
(a)向反应介质提供SUS活性、蔗糖和NDP如ADP或UDP，和(a) providing SUS activity, sucrose and an NDP such as ADP or UDP to the reaction medium, and
(b)温育所述反应介质以使得蔗糖分解，并产生NDP-葡萄糖，如ADP-葡萄糖或UDP-葡萄糖，(b) incubating the reaction medium to cause sucrose to decompose and produce NDP-glucose, such as ADP-glucose or UDP-glucose,
其中通过SEQ ID NO:1的SUS多肽或本发明的经修饰的SUS或宿主细胞提供所述SUS活性。wherein said SUS activity is provided by the SUS polypeptide of SEQ ID NO: 1 or the modified SUS or host cell of the invention.
在一些实施方案中，使用无细胞催化方法生产L-草铵膦，在步骤(a)中，提供SEQ ID NO:1的SUS多肽或本发明的经修饰的SUS。在一些实施方案中，可以使用游离或固定化的SEQ ID NO:1的SUS多肽或本发明的经修饰的SUS。In some embodiments, 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. In some embodiments, free or immobilized SUS polypeptide of SEQ ID NO: 1 or modified SUS of the invention may be used.
在一些实施方案中，所述温育在50-70℃的温度进行。在一些实施方案中，所述温育在50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70℃或更高的温度进行。In some embodiments, 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℃ or higher temperature.
在一些实施方案中，所述介质是缓冲液，例如PBS、Tris-HCl缓冲液。在一个实施方案中，所述介质是Tris-HCl缓冲液，例如50mM、pH8.0的Tris-HCl缓冲液。In some embodiments, the medium is a buffer, such as PBS, Tris-HCl buffer. In one embodiment, the medium is a Tris-HCl buffer, such as 50 mM Tris-HCl buffer, pH 8.0.
在一些实施方案中，反应介质是部分或全部由细胞培养基组成的介质，所述SUS活性由本发明的宿主细胞提供，所述宿主细胞在所述反应介质中培养。In some embodiments, 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.
在一些实施方案中，将本发明的宿主细胞和/或所述第二宿主细胞在细胞培养基中培养并扩增，然后从细胞培养基分离经扩增的宿主细胞，使用缓冲液或水使生物量重悬浮。在加入所述经扩增的宿主细胞之前、期间或之后在所述缓冲液或水中加入蔗糖和NDP。In some embodiments, 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.
在一些实施方案中，可以使用细菌细胞，例如大肠杆菌细胞。In some embodiments, bacterial cells, such as E. coli cells, may be used.
通过本发明的方法，可以在比现有技术更高的温度下产生NDP-葡萄糖，如ADP-葡萄糖或UDP-葡萄糖。By the method of the present invention, NDP-glucose, such as ADP-glucose or UDP-glucose, can be produced at higher temperatures than in the prior art.
通过以下实施例，本领域技术人员会更清楚地理解本发明。应理解，实施例只是用于说明，而非限制本发明的范围Through the following examples, those skilled in the art will understand the present invention more clearly. It should be understood that the examples are for illustration only and do not limit the scope of the invention.
实施例1、材料和方法Example 1, Materials and Methods
如无特别说明，本发明中使用的实验方法均为常规方法，基因克隆操作具体可参见前述Sambrook et al.,1989。Unless otherwise specified, the experimental methods used in the present invention are all conventional methods. For specific gene cloning operations, please refer to the aforementioned Sambrook et al., 1989.
i)试剂：DNA聚合酶(PrimeSTAR Max DNA Polymerase)和DpnI内切酶购自TaKaRa公司，质粒提取试剂盒购自Axygen公司，蔗糖和MgCl2购自麦克林，UDP(尿苷-5′-二磷酸钠盐)和UDP-G(二磷酸尿苷葡萄糖二钠盐)购自阿拉丁。i) Reagents: DNA polymerase (PrimeSTAR Max DNA Polymerase) and 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) and UDP-G (uridine diphosphate glucose disodium salt) were purchased from Aladdin.
ii)载体和菌株：所使用的表达载体为pET-30a(+)，质粒购自Novagen公司，所使
用的宿主细胞为大肠杆菌BL21(DE3)，购自天根生化科技(北京)有限公司。ii) Vector and strain: The expression vector used was pET-30a(+), and the plasmid was purchased from Novagen. The host cell used was Escherichia coli BL21 (DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
iii)测序与引物合成由苏州泓迅生物科技股份有限公司完成，野生型基因(编码SEQ ID NO:1的核苷酸序列)的合成由通用生物完成。iii) Sequencing and primer synthesis were completed by Suzhou Hongxun Biotechnology Co., Ltd., and the synthesis of the wild-type gene (the nucleotide sequence encoding SEQ ID NO:1) was completed by General Biotechnology.
iv)定点突变：iv) Site-directed mutation:
设计特异性引物对，在所需突变的氨基酸位置对应的碱基引入所需的取代。用提取的突变前质粒(包含野生型GluDH编码序列，pET-30a(+)骨架)为模版，利用Quickchange技术(Nucleic Acids Research,2004,32(14):e115)通过PCR引入突变。PCR扩增结束后，扩增产物用Dpn I消化4h去除模版质粒。将消化产物转化至大肠杆菌BL21(DE3)感受态细胞中，涂布于LB琼脂培养基(含有50mg/L的卡那霉素)、挑单菌落至LB液体培养基(含有50mg/L的卡那霉素)中培养，测序验证突变正确性。经验证的克隆置于-80℃保藏备用。Design specific primer pairs to introduce the desired substitution at the base corresponding to the amino acid position of the desired mutation. Using the extracted pre-mutation plasmid (containing wild-type GluDH coding sequence, pET-30a(+) backbone) as a template, mutations were introduced by PCR using Quickchange technology (Nucleic Acids Research, 2004, 32(14):e115). After PCR amplification, the amplified product was digested with DpnI for 4 hours to remove the template plasmid. Transform the digested products into E. coli BL21 (DE3) competent cells, spread them on LB agar medium (containing 50 mg/L kanamycin), and pick single colonies to LB liquid medium (containing 50 mg/L kanamycin). Namycin) was cultured, and sequencing was performed to verify the correctness of the mutation. The verified clones were stored at -80°C for later use.
v)蛋白质表达及粗酶液的制备：v) Protein expression and preparation of crude enzyme solution:
在LB琼脂培养基上将保藏的克隆活化。然后，将单菌落接种至LB液体培养基(含有50mg/L的卡那霉素)中，37℃震荡温育12h。将1mL培养物转接至50mL新鲜的LB液体培养基(含有50mg/L的卡那霉素)中，37℃震荡温育至OD600达到0.6左右，加入IPTG(终浓度为0.4mM)在25℃温育16h以诱导蛋白质表达。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.
温育后，将培养物以4,000g在4℃离心10min，弃上清，收集大肠杆菌细胞。将收集的大肠杆菌细胞重悬于预冷的15mL pH 6.5的50mM PBS中，在4℃超声破碎大肠杆菌细胞。细胞破碎液以6,000g在4℃离心15min去除沉淀，得到的上清为含重组酶的粗酶液。After incubation, 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.
vi)酶活性测定vi) Enzyme activity measurement
配备下述终浓度的混合液：50mM PBS、50g/L蔗糖、2mM UDP、10mM MgCl2，pH＝6.5。向上述溶液加入如v)中描述的方法制备的蔗糖合酶粗酶液(使用的酶的量根据UDP转化百分比进行调整，确保UDP的转化百分比低于15％)。在指定的温度下，于振荡器上持续震荡(400rpm)1小时，95℃加热10min使蛋白质失活，然后通过高效液相色谱检测UDP-G的浓度，从而测定催化反应初始速度。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%). At 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.
vii)T50测定vii)T50 determination
将酶分别在一系列温度温育一小时，并以在4℃温育一小时的酶作为对照，如vi)所述测量温育后的酶活性，并作图，确定温育后的酶活性是对照活性的50％的温度为T50。对于野生型，温育温度为50-60℃，间隔1℃；对于稳定性提升的突变体，温度根据实际情况提升。Incubate the enzyme at a series of temperatures for one hour, and use the enzyme incubated for one hour at 4°C as a control. Measure the enzyme activity after incubation as described in vi) and plot it to determine the enzyme activity after incubation. The temperature that is 50% of the control activity is T50. For the wild type, 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.
实施例2、表征来自Ectothiorhodospira sp.BSL-9的蔗糖合酶EsSUSExample 2. Characterization of sucrose synthase EsSUS from Ectothiorhodospira sp.BSL-9
将来自Ectothiorhodospira sp.BSL-9的蔗糖合酶EsSUS(SEQ ID NO:1)的编码核酸克隆到pET-30a(+)质粒中，并如实施例1所述进行表达，并测定在50℃和56℃的酶活性，和T50。
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.
结果显示，野生型的EsSUS的酶活性为约1.27U/ml；T50约为54℃；与在50℃相比，在56℃，野生型酶活降低约60％。The results showed that the enzyme activity of wild-type EsSUS was about 1.27U/ml; T50 was about 54°C; compared with 50°C, the wild-type enzyme activity decreased by about 60% at 56°C.
实施例3、在EsSUS中引入单突变以提高热稳定性Example 3. Introduction of single mutations in EsSUS to improve thermal stability
以SEQ ID NO:1的编码核酸为模板，根据实施例1的方法制备包含单突变的突变体。所得突变体如表1所示。根据实施例1描述的方法测定所得到的突变体在56℃的酶活，结果示于表1，其中相对酶活是突变体的活性vs.野生型的活性的比例。Using the coding nucleic acid of SEQ ID NO: 1 as a template, 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.
表1、包含单突变的EsSUS突变体
Table 1. EsSUS mutants containing single mutations
Table 1. EsSUS mutants containing single mutations
可见，在EsSUS中引入突变，获得了有活性的突变体，并且可以提高酶的热稳定性。It can be seen that by introducing mutations into EsSUS, active mutants are obtained and the thermal stability of the enzyme can be improved.
实施例4、在EsSUS中引入多个突变以提高热稳定性Example 4. Introducing multiple mutations into EsSUS to improve thermal stability
以SEQ ID NO:1的编码核酸为模板，根据实施例1的方法制备包含2-5个突变的突变体。所得突变体如表2所示。根据实施例1描述的方法测定所得到酶在56℃的相对酶活，结果示于表2，其中相对酶活是突变体的活性vs.野生型的活性的比例。Using the coding nucleic acid of SEQ ID NO:1 as a template, 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.
表2、包含2-5个突变的EsSUS突变体
Table 2. EsSUS mutants containing 2-5 mutations
Table 2. EsSUS mutants containing 2-5 mutations
如表2所示，与野生型相比，引入多个突变可以将56℃温育1h后的酶的活性提高到超过2倍。As shown in Table 2, compared with 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.
如实施例1所述测定突变体的T50。结果显示，突变体的T50比野生型提高了至少3℃，其中SEQ ID NO:63-70的突变体的T50达到了63-64℃。即，在5个位置(133、136、529、768和790)引入突变显著提高了酶的T50。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.
可见，在EsSUS中引入突变，其效果是可以叠加的，显著提高了酶的热稳定性。It can be seen that the effects of introducing mutations into EsSUS can be superimposed, significantly improving the thermal stability of the enzyme.
实施例5、在EsSUS突变体中引入额外的突变以提高酶活性Example 5. Introducing additional mutations into EsSUS mutants to improve enzyme activity
在SEQ ID NO:64的基础上进一步引入氨基酸取代，并如实施例1所述测定突变体在56℃的酶活性，所得结果示于表3，其中相对酶活是指，突变体的活性vs.SEQ ID NO:64的活性的比例。On the basis of SEQ ID NO:64, amino acid substitutions were further introduced, and the enzyme activity of the mutant at 56°C was measured as described in Example 1. The results are shown in Table 3, where the relative enzyme activity refers to the activity of the mutant vs. .Proportion of activity of SEQ ID NO:64.
表3
table 3
table 3
进一步，在SEQ ID NO:118的基础上进一步引入氨基酸取代，并如实施例1所述测定突变体在56℃的酶活性，所得结果示于表4，其中相对酶活是指，突变体的活性vs.SEQ ID NO:118的活性的比例。Further, 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.
表4
Table 4
Table 4
进一步，如实施例2所述测定所得的突变体的T50，SEQ ID NO:131的突变体的T50为63.5℃，表3和4中的其他突变体的T50也与SEQ ID NO:64相当。Further, the 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.
以上结果表明，引入额外的突变大大提高了活性但没有损害热稳定性，突变的效果至少是叠加的。The above results indicate that introducing additional mutations greatly improves activity without compromising thermal stability, and that the effects of mutations are at least additive.
实施例6、引入进一步的突变以提高酶的热稳定性Example 6. Introducing further mutations to improve the thermostability of the enzyme
进一步，在SEQ ID NO:131的基础上进一步引入氨基酸取代，并如实施例1所述测定突变体在63.5℃的酶活性，所得结果示于表5，其中相对酶活是指，63.5℃温育1h后突变体的活性vs.SEQ ID NO:131的活性的比例。Further, 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.
表5
table 5
table 5
如实施例1所述测定所得的突变体的T50。结果显示，突变体的T50显著高于SEQ ID NO:131，其中SEQ ID NO:159-189和191-203的T50为66-66.5℃。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.
可见，在EsSUS中引入突变，其效果是可以叠加的，显著提高了酶的热稳定性。
It can be seen that the effects of introducing mutations into EsSUS can be superimposed, significantly improving the thermal stability of the enzyme.
Claims (13)
- 一种经修饰的蔗糖合酶(SUS)多肽，A modified sucrose synthase (SUS) polypeptide,与其野生型SUS相比，包含选自位置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和790的一或多个位置的氨基酸取代，其中所述位置参照SEQ ID NO:1进行编号，Compared with its wild-type SUS, including positions selected from 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, one or more amino acid substitutions at positions, wherein said positions are numbered with reference to SEQ ID NO:1,其中位置4的氨基酸取代为V，位置24的氨基酸取代为L，位置41的氨基酸取代为D、E或W，位置108的氨基酸取代为C或M，位置114的氨基酸取代为E，位置133的氨基酸取代为K或R，位置136的氨基酸取代为M、T或K，位置161的氨基酸取代为D，位置300的氨基酸取代为M、G或A，位置433的氨基酸取代为C或R，位置473的氨基酸取代为G，位置474的氨基酸取代为C、V或H，位置476的氨基酸取代为V或C，位置479的氨基酸取代为A、R或N，位置482的氨基酸取代为S、T或V，位置483的氨基酸取代为N、H或G，位置513的氨基酸取代为K、Q或I，位置515的氨基酸取代为R或S，位置518的氨基酸取代为Y、V、I、L、F或T，位置529的氨基酸取代为V、T或H，位置533的氨基酸取代为F或L，位置534的氨基酸取代为F、W或Y，位置543的氨基酸取代为L，位置544的氨基酸取代为I，位置585的氨基酸取代为K或R，位置629的氨基酸取代为D，位置630的氨基酸取代为N、H或S，位置640的氨基酸取代为P，位置641的氨基酸取代为R或K，位置644的氨基酸取代为A、V或S，位置664的氨基酸取代为Y，位置676的氨基酸取代为K，位置697的氨基酸取代为V、I或K，位置713的氨基酸取代为L或M，位置715的氨基酸取代为L或V，位置726的氨基酸取代为D或E，位置729的氨基酸取代为D，位置741的氨基酸取代为C或G，位置768的氨基酸取代为P、E或A，位置769的氨基酸取代为P或Q，位置773的氨基酸取代为L，位置788的氨基酸取代为R，位置790的氨基酸取代为K或R，The amino acid at position 4 is substituted with 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, and the amino acid at position 133 is substituted with The amino acid substitution is K or R, the amino acid substitution at position 136 is M, T or K, the amino acid substitution at position 161 is D, the amino acid substitution at position 300 is M, G or A, the amino acid substitution at position 433 is C or R, position 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 with A, R or N, the amino acid at position 482 is substituted with S, T or V, the amino acid at position 483 is substituted by N, H or G, the amino acid at position 513 is substituted by K, Q or I, the amino acid at position 515 is substituted by R or S, the amino acid at position 518 is substituted by Y, V, I, L , F or T, the amino acid at position 529 is substituted with V, T or H, the amino acid at position 533 is substituted with F or L, the amino acid at position 534 is substituted with F, W or Y, the amino acid at position 543 is substituted with L, the amino acid at position 544 is substituted The amino acid substitution is I, the amino acid substitution at position 585 is K or R, the amino acid substitution at position 629 is D, the amino acid substitution at position 630 is N, H or S, the amino acid substitution at position 640 is P, and the amino acid substitution at position 641 is R or K, the amino acid at position 644 is substituted with A, V or S, the amino acid at position 664 is substituted with Y, the amino acid at position 676 is substituted with K, the amino acid at position 697 is substituted with V, I or K, the amino acid at position 713 is substituted with L or M, the amino acid at position 715 is substituted with L or V, the amino acid at position 726 is substituted with D or E, the amino acid at position 729 is substituted with D, the amino acid at position 741 is substituted with C or G, the amino acid at position 768 is substituted with P or E or A, the amino acid at position 769 is substituted with P or Q, the amino acid at position 773 is substituted with L, the amino acid at position 788 is substituted with R, the amino acid at position 790 is substituted with K or R,其中所述经修饰的SUS具有催化蔗糖分解，在核苷二磷酸(NDP)存在下产生果糖和NDP-葡萄糖的活性。The modified SUS has the activity of catalyzing the decomposition of sucrose and producing fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP).
- 权利要求1的经修饰的SUS多肽，其包含位置133和135的氨基酸取代，位置529和768的氨基酸取代，位置136和768的氨基酸取代，或位置133和529的氨基酸取代。The modified SUS polypeptide of claim 1, comprising 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.
- 权利要求1或2的经修饰的SUS多肽，其包含位置133、136、529、768和790的氨基酸取代。The modified SUS polypeptide of claim 1 or 2, comprising amino acid substitutions at positions 133, 136, 529, 768 and 790.
- 权利要求3的经修饰的SUS多肽，其还包含选自位置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和741的一或多个位置的氨基酸取代。 The modified SUS polypeptide of claim 3, further comprising positions selected from positions 41, 300, 433, 473, 474, 476, 479, 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, Amino acid substitutions at one or more positions 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729 and 741.
- 权利要求3或4的经修饰的SUS多肽，其包含位置133、136、513、515、518、529、640、641、644、768和790的氨基酸取代。The modified SUS polypeptide of claim 3 or 4, comprising amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768 and 790.
- 权利要求1-5任一项的经修饰的SUS多肽，其中所述野生型SUS多肽包含SEQ ID NO:1的氨基酸序列或其天然变体。The modified SUS polypeptide of any one of claims 1-5, wherein the wild-type SUS polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or a natural variant thereof.
- 一种经修饰的SUS多肽，包含SEQ ID NO:2-208之一的氨基酸序列，或者所述经修饰的SUS与2-208之一相比，在除位置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和790之外的位置包含1-10个氨基酸取代，其中所述经修饰的SUS多肽具有催化蔗糖分解，在NDP存在下产生果糖和NDP-葡萄糖的活性。A modified SUS polypeptide, comprising the amino acid sequence of one of SEQ ID NO: 2-208, or the modified SUS compared to one of 2-208, except for positions 4, 24, 41, 108, 114 ,133,136,161,300,433,473,474,476,479,482,483,513,515,518,529,533,534,543,544,585,629,630,640,641,644 , positions other than 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788 and 790 comprise 1-10 amino acid substitutions, wherein the modified SUS polypeptide has the ability to catalyze sucrose decomposition , activity to produce fructose and NDP-glucose in the presence of NDP.
- 一种多核苷酸，编码权利要求1-7任一项的经修饰的SUS多肽。A polynucleotide encoding the modified SUS polypeptide of any one of claims 1-7.
- 一种表达载体，包含权利要求8的多核苷酸。An expression vector comprising the polynucleotide of claim 8.
- 一种宿主细胞，包含权利要求1-7任一项的经修饰的SUS多肽、权利要求8多核苷酸或权利要求14的载体。A host cell comprising the modified SUS polypeptide of any one of claims 1-7, the polynucleotide of claim 8 or the vector of claim 14.
- 一种产生NDP葡萄糖的方法，包括在NDP存在的条件下，使衍生自微生物的SUS多肽或包含所述SUS多肽的宿主细胞与蔗糖接触，其中所述SUS多肽是野生型SUS多肽或权利要求1-7任一项的经修饰的SUS多肽。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 a wild-type SUS polypeptide or claim 1 The modified SUS polypeptide of any one of -7.
- 权利要求11的方法，其中所述野生型SUS多肽包含SEQ ID NO:1的氨基酸序列。The method of claim 11, wherein the wild-type SUS polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
- 权利要求11或12的方法，其在50-70℃的温度进行。 The method of claim 11 or 12, which is carried out at a temperature of 50-70°C.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101495626A (en) * | 2006-07-25 | 2009-07-29 | 拜尔生物科学公司 | Identification of a novel type of sucrose synthase and use thereof in fiber modification |
| WO2017207484A1 (en) * | 2016-05-31 | 2017-12-07 | Universiteit Gent | Mutant sucrose synthases and their uses |
| CN112805295A (en) * | 2018-07-30 | 2021-05-14 | 科德克希思公司 | Engineering glycosyltransferases and methods of glycosylation of steviol glycosides |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101495626A (en) * | 2006-07-25 | 2009-07-29 | 拜尔生物科学公司 | Identification of a novel type of sucrose synthase and use thereof in fiber modification |
| WO2017207484A1 (en) * | 2016-05-31 | 2017-12-07 | Universiteit Gent | Mutant sucrose synthases and their uses |
| CN112805295A (en) * | 2018-07-30 | 2021-05-14 | 科德克希思公司 | Engineering glycosyltransferases and methods of glycosylation of steviol glycosides |
Non-Patent Citations (4)
| 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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