WO2021219124A1 - Thréonine transaldolase modifiée et son application - Google Patents

Thréonine transaldolase modifiée et son application Download PDF

Info

Publication number
WO2021219124A1
WO2021219124A1 PCT/CN2021/091392 CN2021091392W WO2021219124A1 WO 2021219124 A1 WO2021219124 A1 WO 2021219124A1 CN 2021091392 W CN2021091392 W CN 2021091392W WO 2021219124 A1 WO2021219124 A1 WO 2021219124A1
Authority
WO
WIPO (PCT)
Prior art keywords
ltta
substituted
modified
amino acid
present
Prior art date
Application number
PCT/CN2021/091392
Other languages
English (en)
Chinese (zh)
Inventor
谢新开
梁晓亮
黄晓飞
Original Assignee
苏州引航生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州引航生物科技有限公司 filed Critical 苏州引航生物科技有限公司
Publication of WO2021219124A1 publication Critical patent/WO2021219124A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to the field of enzyme engineering. Specifically, the present invention relates to a modified L-threonine transaldolase (LTTA) and its application in the production of 3-phenyl-L-serine and its derivatives.
  • LTTA modified L-threonine transaldolase
  • 3-Phenyl-L-serine and its derivatives are a very important class of organic synthesis intermediates, which are widely used in drug synthesis.
  • (2S,3R)-p-methylsulfonylphenylserine is a key intermediate for the synthesis of veterinary drugs florfenicol and thiamphenicol
  • (2S,3R)-p-nitrophenylserine can be used as an antibacterial drug chloramphenicol Key intermediates.
  • 3-phenyl-L-serine and its derivatives can also prepare chiral aziridines through simple transformation (David Tanner, Chiral Aziridines-Their Synthesis and Use in Stereoselective Transformations, Angew. Chem., Int.
  • 3-phenyl-L-serine derivatives are mainly prepared by organic synthesis. Such methods often have disadvantages such as many steps, low yield, and poor stereoselectivity. Biologists have also made some attempts to prepare such compounds. For example, Steinreiber et al. (Johannes Steinreiber et al., Threonine aldolases—an emerging tool for organic synthesis, Tetrahedron, 63, 918-926 (2007)) used benzaldehyde derivatives. Synthesize 3-phenyl-L-serine derivatives with glycine under the action of aldolase, the reaction formula is as follows:
  • LTTA is used to catalyze the reaction between p-methylsulfonyl benzaldehyde and L-threonine, such as (2S,3R)-p-methylsulfonyl phenylserine, see CN 109836362A.
  • LTTA can catalyze the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives and acetaldehyde.
  • the general reaction formula is as shown in formula (I) ( See, for example, PCT/CN2019/123974):
  • the present invention provides a modified L-threonine transaldolase (LTTA), wherein the modified LTTA comprises amino acid substitutions at one or more positions compared to its starting LTTA, and It has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to generate 3-phenyl-L-serine or its derivatives.
  • LTTA modified L-threonine transaldolase
  • the modified LTTA includes an amino acid substitution at position 70, preferably substitution to H, N or S, more preferably substitution to H, and the position is numbered with reference to SEQ ID NO: 2.
  • the modified LTTA further comprises amino acids at one or more positions selected from positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229, and 407 replace.
  • position 35 is substituted with A, G or S, more preferably S.
  • position 38 is substituted with F.
  • position 48 is substituted with A or C.
  • position 57 is substituted with S, M or T, more preferably M.
  • position 59 is substituted with A or Y.
  • position 94 is substituted with E.
  • position 116 is substituted with R, S or N.
  • position 141 is substituted with C.
  • position 181 is substituted with Q.
  • position 185 is substituted with G.
  • position 205 is selected from S, Q or A.
  • position 229 is substituted with C.
  • position 407 is substituted with R.
  • the modified LTTA comprises the amino acid sequence of one of SEQ ID NO: 13-72 or consists of the amino acid sequence of one of SEQ ID NO: 13-72; or is the same as SEQ ID NO: 13-65 Compared with the amino acid sequence of one of 69-72, the modified LTTA has positions 35, 38, 48, 57, 59, 70, 94, 116, 141, 181, 185, 205, 229 and 407. The position further contains 1-10 amino acid substitutions; or compared with the amino acid sequence of one of SEQ ID NO: 66-68, the modified LTTA also contains 1- 10 amino acid substitutions.
  • the present invention provides a polynucleotide encoding the modified LTTA of the present invention, and a vector comprising the polynucleotide of the present invention.
  • the present invention provides a host cell containing the modified LTTA of the present invention, its encoding polynucleotide or a vector containing the polynucleotide.
  • the present invention also provides a method for producing 3-phenyl-L-serine and its derivatives, which comprises combining the modified LTTA of the present invention or the host cell of the present invention with benzaldehyde or its derivatives, and L-threonine contact.
  • L-threonine transaldolase and "LTTA” have the ability to catalyze the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or derivatives and acetaldehyde (ie The reaction of formula (I)) activity.
  • the present invention provides a modified LTTA polypeptide. Compared with the original LTTA polypeptide, the modified LTTA has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine.
  • peptide means a chain of at least two amino acids connected by peptide bonds.
  • polypeptide is used interchangeably with the term “protein” herein and refers to a chain containing ten or more amino acid residues. All peptide and polypeptide chemical formulas or sequences herein are written from left to right, indicating the direction from the amino terminal to the carboxy terminal.
  • amino acid includes naturally occurring amino acids and unnatural amino acids in proteins.
  • the one-letter and three-letter names of amino acids naturally occurring in proteins are commonly used in the field, 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 refers to any chemical modification of a polypeptide, such as amino acid substitutions, deletions, insertions, and/or additions.
  • the modified LTTA of the present invention contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 compared to its starting LTTA. , 16, 17, 18, 19, 20 or more amino acid substitutions, wherein the modified LTTA of the present invention has an improved catalyzed reaction between benzaldehyde or its derivatives and L-threonine compared with the original LTTA Activity of 3-phenyl-L-serine or derivatives.
  • the modified LTTA comprises 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1- 9. 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acid substitutions, wherein the modified LTTA of the present invention has an improvement compared with the original LTTA
  • the modified LTTA of the present invention contains an amino acid substitution at position 70 compared to its starting LTTA, and the position is numbered with reference to SEQ ID NO: 2, preferably the substitution is H, N or S, more preferably Replaced by H.
  • the modified LTTA of the present invention further comprises one or more positions selected from positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229 and 407.
  • Amino acid substitution Preferably, position 35 is substituted with A, G or S, more preferably S.
  • position 38 is substituted with F.
  • position 48 is substituted with A or C.
  • position 57 is substituted with S, M or T, more preferably M.
  • position 59 is substituted with A or Y.
  • position 94 is substituted with E.
  • position 116 is substituted with R, S or N.
  • position 141 is substituted with C.
  • position 181 is substituted with Q.
  • position 185 is substituted with G.
  • position 205 is selected from S, Q or A.
  • position 229 is substituted with C.
  • position 407 is substituted with R.
  • the modified LTTA of the present invention contains amino acid substitutions at positions 35 and 70, and the positions are numbered with reference to SEQ ID NO: 2, wherein position 35 is substituted with S and position 70 is substituted with H. More preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57 and 70, wherein position 35 is substituted with S, position 57 is substituted with M, and position 70 is substituted with H.
  • the modified LTTA comprises amino acid substitutions at positions 35, 57, 59, 70, 94 and 141, wherein position 35 is substituted with S, position 57 is substituted with M, position 59 is substituted with A, and position 70 is substituted with H, position 94 is substituted with E, and position 141 is substituted with C.
  • the modified LTTA of the present invention has an amino acid substitution or a combination of amino acid substitutions selected from the group consisting of the following amino acid substitutions or combinations of amino acid substitutions (positions are numbered with reference to SEQ ID NO: 2):
  • the LTTA polypeptide on which amino acid modifications are made is referred to as the starting LTTA.
  • the starting LTTA can be wild-type LTTA or a variant of wild-type LTTA.
  • the polypeptide of SEQ ID NO: 2 is the "starting LTTA"; and if the polypeptide of SEQ ID NO: 2 is modified (for example, SEQ ID NOs: 13-65 and 69-72) start to be modified, compared to the modified LTTA, the variant polypeptide is the "starting LTTA".
  • wild-type LTTA refers to naturally occurring LTTA.
  • the wild-type LTTA is LTTA from Pseudomonas.
  • the wild-type LTTA is shown in SEQ ID NO: 2, 4, or 6.
  • SEQ ID NO: 2 is the amino acid sequence of LTTA from Pseudomonas fluorescens (Genbank accession number AQZ26585.1)
  • SEQ ID NO: 4 is the amino acid sequence of LTTA from Pseudomonas sp.34E 7 (Genbank accession No. CRN02517.1)
  • SEQ ID NO: 6 is the amino acid sequence of LTTA from Pseudomonas sp.
  • the wild-type LTTA is an LTTA from Chitiniphilus shinanonensis, and its sequence is shown in SEQ ID NO: 8 (Genbank accession number WP_018749561).
  • the sequences are aligned for the purpose of optimal comparison (for example, gaps 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. When a position in the first sequence is occupied by the same amino acid residue or nucleotide in the corresponding position in the second sequence, then these molecules are the same at this position.
  • percent identity number of identical positions/total number of positions (i.e. overlapping positions) x 100).
  • the two sequences are the same length.
  • Percent amino acid identity or “percent amino acid sequence identity” refers to comparing the amino acids of two polypeptides, and when optimally aligned, the two polypeptides have approximately the specified percentage of identical amino acids. For example, “95% amino acid identity” refers to comparing the amino acids of two polypeptides. When the two polypeptides are optimally aligned, 95% of the amino acids of the two polypeptides are identical.
  • the modified LTTA polypeptide of the present invention has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85% compared to SEQ ID NO: 2, 4, 6 or 8. , 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity.
  • the modified LTTA of the present invention includes 1, 2, 3, 4, 5, 6, 7, 8 compared to its starting LTTA (for example, SEQ ID NO: 2, 4, 6, or 8). , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, wherein the modified LTTA of the present invention has improved catalysis compared with the original LTTA The activity of reacting benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives.
  • the modified LTTA contains 1-20, 1-15, 1-14, 1-13, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acid substitutions.
  • the modified LTTA of the present invention contains an amino acid substitution at position 70 compared to its starting LTTA, and the position is numbered with reference to SEQ ID NO: 2, preferably the substitution is H, N or S, more preferably Replaced by H.
  • the modified LTTA has 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 LTTA of the present invention further comprises one or more positions selected from positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229 and 407.
  • Amino acid substitution Preferably, position 35 is substituted with A, G or S, more preferably S.
  • position 38 is substituted with F.
  • position 48 is substituted with A or C.
  • position 57 is substituted with S, M or T, more preferably M.
  • position 59 is substituted with A or Y.
  • position 94 is substituted with E.
  • position 116 is substituted with R, S or N.
  • position 141 is substituted with C.
  • position 181 is substituted with Q.
  • position 185 is substituted with G.
  • position 205 is selected from S, Q or A.
  • position 229 is substituted with C.
  • position 407 is substituted with R.
  • the modified LTTA has 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 LTTA of the present invention contains amino acid substitutions at positions 35 and 70, and the positions are numbered with reference to SEQ ID NO: 2, wherein position 35 is substituted with S and position 70 is substituted with H. More preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57 and 70, wherein position 35 is substituted with S, position 57 is substituted with M, and position 70 is substituted with H.
  • the modified LTTA comprises amino acid substitutions at positions 35, 57, 59, 70, 94 and 141, wherein position 35 is substituted with S, position 57 is substituted with M, position 59 is substituted with A, and position 70 is substituted with H, position 94 is substituted with E, and position 141 is substituted with C.
  • the modified LTTA has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, compared with its starting LTTA. 97%, 98% or 99% sequence identity.
  • the modified LTTA of the present invention contains an amino acid substitution at position 70 compared to its starting LTTA, and the position is numbered with reference to SEQ ID NO: 2, preferably the substitution is H, N or S, more preferably Substituted to H, wherein the modified LTTA of the present invention has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives compared to its starting LTTA .
  • the initial LTTA and SEQ ID NO: 2, 4, 6, or 8 have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% % Or 99% sequence identity.
  • the modified LTTA of the present invention further comprises one or more positions selected from positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229 and 407.
  • Amino acid substitution Preferably, position 35 is substituted with A, G or S, more preferably S.
  • position 38 is substituted with F.
  • position 48 is substituted with A or C.
  • position 57 is substituted with S, M or T, more preferably M.
  • position 59 is substituted with A or Y.
  • position 94 is substituted with E.
  • position 116 is substituted with R, S or N.
  • position 141 is substituted with C.
  • position 181 is substituted with Q.
  • position 185 is substituted with G.
  • position 205 is selected from S, Q or A.
  • position 229 is substituted with C.
  • position 407 is substituted with R.
  • the modified LTTA of the present invention contains amino acid substitutions at positions 35 and 70, and the positions are numbered with reference to SEQ ID NO: 2, wherein position 35 is substituted with S and position 70 is substituted with H. More preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57 and 70, wherein position 35 is substituted with S, position 57 is substituted with M, and position 70 is substituted with H.
  • the modified LTTA comprises amino acid substitutions at positions 35, 57, 59, 70, 94 and 141, wherein position 35 is substituted with S, position 57 is substituted with M, position 59 is substituted with A, and position 70 is substituted with H, position 94 is substituted with E, and position 141 is substituted with C.
  • amino acid residue substitution refers to a substitution in which the amino acid residue is replaced by an amino acid residue having a similar side chain, for example, an amino acid with a basic side chain (e.g., lysine) , Arginine and histidine), acidic side chain amino acids (e.g. aspartic acid, glutamic acid), non-charged electroactive side chain amino acids (e.g. glycine, asparagine, glutamine, serine, threonine) Acid, tyrosine, cysteine), non-polar side chain amino acids (e.g.
  • a basic side chain e.g., lysine
  • acidic side chain amino acids e.g. aspartic acid, glutamic acid
  • non-charged electroactive side chain amino acids e.g. glycine, asparagine, glutamine, serine, threonine
  • Acid tyrosine, cysteine
  • non-polar side chain amino acids
  • ⁇ -branched side chain amino acids e.g. threonine, valine, isoleucine
  • aromatic side chain amino acids e.g. tyrosine, phenylalanine, tryptophan, histidine
  • Conservative amino acid substitutions generally have the least impact on the activity of the resulting protein. This substitution is described below. Conservative substitution is to replace an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, spatial characteristics, and aromaticity. When it is desired to fine-tune the properties of the protein, such substitutions are usually conservative.
  • homologous amino acid residues refer to amino acid residues with similar chemical properties related to hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, and the like.
  • amino acids that are homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, and leucine Acid, isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine , Aromatic phenylalanine, tyrosine, tryptophan, serine and threonine with chemically similar side chain groups, or glutamine and asparagine, or leucine and isoleucine.
  • Examples of conservative amino acid substitutions in proteins include: Ser replaces Ala, Lys replaces Arg, Gln or His replaces Asn, Glu replaces Asp, Ser replaces Cys, Asn replaces Gln, Asp replaces Glu, Pro replaces Gly, Asn or Gln replaces His, Leu Or Val replaces Ile, Ile or Val replaces Leu, Arg or Gln replaces Lys, Leu or Ile replaces Met, Met, Leu or Tyr replaces Phe, Thr replaces Ser, Ser replaces Thr, Tyr replaces Tr, Trp or Phe replaces Tyr, and Ile or Leu replaces Val.
  • the modified LTTA comprises the amino acid sequence of one of SEQ ID NO: 13-72 or consists of the amino acid sequence of one of SEQ ID NO: 13-72; or is the same as SEQ ID NO: 13-65 Compared with the amino acid sequence of one of 69-72, the modified LTTA has positions 35, 38, 48, 57, 59, 70, 94, 116, 141, 181, 185, 205, 229 and 407.
  • the position contains 1-10 amino acid substitutions; or compared with the amino acid sequence of one of SEQ ID NO: 66-68, the modified LTTA contains 1-10 positions other than positions 35, 57 and 70 Amino acid substitutions, wherein the modified LTTA of the present invention has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives compared with the original LTTA.
  • the modified LTTA is at positions 35, 38, 48, 57, 59, 70, 94, 116.
  • the positions other than, 141, 181, 185, 205, 229 and 407 also contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; or with SEQ ID NO: Compared with the amino acid sequence of one of 66-68, the modified LTTA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 at positions other than positions 35, 57, and 70. Or more amino acid substitutions.
  • the activity of an enzyme refers to a decrease in substrate or an increase in product per unit time in a chemical reaction catalyzed by an enzyme per unit mass under certain conditions.
  • the activity of the modified LTTA of the present invention under certain conditions (such as the reaction conditions listed in the following examples), under the catalysis of a unit mass of the modified LTTA, 3-phenyl-L generated in a unit time -Serine or its derivatives are expressed in terms of amount.
  • the activity of an enzyme can also refer to the relative activity of the 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 percentage relative activity.
  • the activity of the modified LTTA of the present invention is expressed as a ratio to the activity of the LTTA of SEQ ID NO: 2.
  • the modified LTTA has an activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives is at least that of the LTTA of SEQ ID NO: 2 1, 1.05, 1.1, 1.2, 1.3, 1.5, 1.7, 2, 2.5, 3, 3.5, 4 times or more.
  • product selectivity means that when the reaction product contains two or more possible stereoisomers, one of the products is produced more. If the reaction is an enzymatic reaction, the product selectivity is also referred to as the product selectivity of the enzyme.
  • the carbons at positions 2 and 3 of the derivatives of 3-phenyl-L-serine are all chiral carbons, which will produce four stereoisomers, namely P(2S,3R ), P(2R,3S), (P(2S,3S) and P(2R,3R).
  • the product selectivity of LTTA can be used under certain reaction conditions (such as the reaction conditions listed in the following examples) diastereomeric ratio (DR) and enantiomers
  • DR diastereomeric ratio
  • ee% are calculated using the following formulas (I) and (II).
  • the DR of the modified LTTA of the present invention is increased, for example, to at least 95.5:4.5, at least 96:4, at least 96.5:3.5, at least 97:3, at least 97.5: 2.5, at least 98:2, at least 98.5:1.5, at least 99:1 or higher.
  • the ee% of the modified LTTA of the present invention is >99.9%.
  • the benzaldehyde derivative is p-methylsulfonyl benzaldehyde.
  • the derivative of 3-phenyl-L-serine is (2S,3R)-p-methylsulfonylphenylserine.
  • nucleic acid molecule includes DNA molecules (such as cDNA or genomic DNA) and RNA molecules (such as 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 synthesis of the nucleic acid may use nucleotide analogs or derivatives (for example, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids with altered base pairing ability or increased nuclease resistance.
  • the present invention also provides polynucleotides encoding the modified LTTA of the present invention. Therefore, in the present invention, the term modification also includes genetic manipulation of the polynucleotide encoding the LTTA polypeptide of the present invention. The modification may be a substitution, deletion, insertion and/or addition of nucleotides.
  • the term "encoding" refers to the amino acid sequence of a polynucleotide directly specifying its protein product.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which usually starts with the ATG start codon or other start codons such as GTG and TTG, and ends with stop codons such as TAA, TAG, and TGA.
  • the coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.
  • nucleic acid molecules covering all or part of the nucleic acid sequence of the present invention can be separated by polymerase chain reaction (PCR), which uses the design of synthetic oligonucleotide primers based on the sequence information contained in the sequence.
  • PCR polymerase chain reaction
  • the polynucleotide of the present invention can be amplified using cDNA, mRNA or genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • the polynucleotide of the present invention can be prepared by standard synthesis techniques, for example, by using an automated DNA synthesizer.
  • the invention also relates to the complementary strands of the nucleic acid molecules described herein.
  • a nucleic acid molecule that is complementary to other nucleotide sequences is a molecule that is sufficiently complementary to the nucleotide sequence so that it can hybridize with other nucleotide sequences to form a stable duplex.
  • hybridization refers to nucleotides that are at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other under given stringent hybridization and washing conditions. The sequences generally remain hybridized to each other.
  • the polynucleotide of the present invention does not include a polynucleotide that only hybridizes to a poly A sequence (such as the 3'end poly (A) of mRNA) or a complementary stretch of poly T (or U) residues.
  • nucleic acid constructs and vectors containing the polynucleotide of the present 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-stranded or double-stranded nucleic acid molecule, which is isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not occur in nature.
  • nucleic acid construct contains the control sequences required to express the coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette”.
  • expression vector refers herein to a linear or circular DNA molecule, which comprises a polynucleotide encoding a polypeptide of the present invention, the polynucleotide and additional nucleotides provided for the expression of the polynucleotide, for example, Control sequence, operably connected.
  • the expression vector includes a viral vector or a plasmid vector.
  • control sequence refers herein to include all elements necessary or advantageous for the expression of the polynucleotide encoding the polypeptide of the present invention.
  • Each control sequence may be natural or foreign to the nucleotide sequence encoding the polypeptide, or natural 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 terminator. At a minimum, control sequences include promoters and transcription and translation termination signals.
  • control sequence may be a suitable promoter sequence, a nucleotide sequence recognized by the host cell to express the polynucleotide encoding the polypeptide of the present invention.
  • the promoter sequence contains transcription control sequences that mediate the expression of the polypeptide.
  • the promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, the Escherichia coli lac operon.
  • the promoters also include mutated, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.
  • operably linked refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence, whereby the control sequence directs the expression of the polypeptide coding sequence.
  • the polynucleotide encoding the polypeptide of the present invention can be subjected to various operations to allow expression of the polypeptide. Before inserting it into the vector, it is desirable or necessary to manipulate the polynucleotide according to the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.
  • the vector of the present invention preferably contains one or more selectable markers, which allow simple selection of transformed, transfected, transduced, etc. cells.
  • a selectable marker is a gene whose product provides biocide or virus resistance, heavy metal resistance, supplementation of auxotrophs, etc.
  • the bacterial selectable marker is the dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.
  • the vector of the present invention can be integrated into the genome of the host cell or can replicate autonomously in the cell without relying on the genome. Elements required for integration into the host cell genome or autonomous replication are known in the art (see, for example, the aforementioned Sambrook et al., 1989).
  • 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 foreign nucleic acids (such as DNA) into host cells, which can be found in, for example, the aforementioned Sambrook et al., 1989; Davis et al. .,Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • the present invention also relates to a recombinant host cell, which contains the polynucleotide of the present invention, which is advantageously used for the recombinant production of LTTA polypeptides.
  • the vector containing the polynucleotide of the present 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 know conventional vectors and host cells for expressing proteins.
  • the host cell of the present invention is an E. coli cell, such as E. coli BL21 (DE3).
  • the expression vector is pET-30a(+).
  • the present invention provides a method for preparing 3-phenyl-L-serine or a derivative thereof, which comprises contacting the modified LTTA or host cell of the present invention with benzaldehyde or a derivative thereof and L-threonine.
  • the method for preparing 3-phenyl-L-serine or a derivative thereof of the present invention includes the following steps:
  • acetaldehyde reductase and reduced nicotinamide adenine dinucleotide (NADH) are added to the reaction medium to reduce acetaldehyde to ethanol.
  • the acetaldehyde reductase is an acetaldehyde reductase from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO: 10.
  • glucose dehydrogenase and glucose are added to the reaction medium to achieve coenzyme (NADH) regeneration.
  • NADH coenzyme
  • the glucose dehydrogenase is derived from Bacillus subtilis (Bacillus subtilis), and its amino acid sequence is shown in SEQ ID NO: 12.
  • formate or formate and formate dehydrogenase are added to the reaction medium to achieve coenzyme (NADH) regeneration.
  • the formate dehydrogenase is derived from Candida boidinii, and its amino acid sequence is shown in SEQ ID NO: 73.
  • the reaction medium is a buffer, such as PBS or Tris-HCl buffer.
  • the reaction medium is a PBS buffer, such as 0.1M, pH 7.0 PBS buffer.
  • the incubation is performed at 25-40°C, preferably 28-35°C, such as 30°C.
  • DNA polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonuclease were purchased from TaKaRa;
  • the plasmid extraction kit was purchased from Axygen;
  • P-Methylsulfonyl benzaldehyde was purchased from Aladdin, item number M185093, purity 98%;
  • L-threonine was purchased from Macleans, catalog number C10393311, analytically pure;
  • Pyridoxal phosphate was purchased from Aladdin, item number P136795, purity ⁇ 98%;
  • Magnesium chloride was purchased from Aladdin, catalog number A2006034, analytically pure;
  • Oxidized nicotinamide adenine dinucleotide (NAD) was purchased from Aladdin, catalog number N196974, with a purity of 95%.
  • the expression vector used was pET-30a(+), the plasmid was purchased from Novagen, and the host cell used was E. coli BL21 (DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • the E. coli cells transformed with the plasmid containing the target gene were inoculated into LB liquid medium containing 50mg/L of kanamycin (50mL medium in a 250mL bottle, peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH7.0), incubate overnight at 37°C with shaking.
  • kanamycin 50mL medium in a 250mL bottle, peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH7.0
  • E. coli cells were collected.
  • the collected E. coli cells were resuspended in 20 mL of pre-cooled phosphate buffered saline (PBS) at pH 7.0, and the E. coli cells were sonicated at 4°C.
  • PBS phosphate buffered saline
  • the cell disruption solution was centrifuged at 6,000 g at 4°C for 15 min to remove the precipitate, and the supernatant obtained was a solution containing the recombinant enzyme, which was used to catalyze the reaction.
  • the enzyme solution can also be freeze-dried into enzyme powder and stored at 4°C for later use.
  • the E. coli cells transformed with the plasmid containing the target gene were inoculated into the wells of a 96-well shallow-well plate, and each well was filled with 100 ⁇ L of LB medium containing 50 ⁇ g/mL kanamycin.
  • the culture was incubated in a shaker for 18 hours (250 rpm, 30°C and 85% relative humidity). Transfer 20 ⁇ L of the incubated culture to the wells of a 96-well deep well plate filled with 180 ⁇ L of TB medium containing 50 ⁇ g/mL kanamycin.
  • the plate was incubated at 30°C and 250 rpm for 2 hours, and then IPTG (final concentration of 0.4 mM) was added for induction, and incubated in a shaker at 30°C and 250 rpm for 20 hours.
  • the E. coli cells were collected by centrifugation, and 300 ⁇ L of lysate (20mM phosphate buffer pH 7.0, 1mM MgSO 4 , 1mg/mL lysozyme and 0.5mg/mL polymyxin B sulfate) was added. The mixture was shaken at room temperature for 2h, centrifuged and the supernatant was taken for enzyme activity determination.
  • the reactants were added to the wells of a 96-well plate, mixed briefly, and the SpectraMax190 (Molecular Devices) light absorption microplate reader was used to detect the activity of LTTA and its mutants by changing the absorbance at 340 nm.
  • HPLC parameters are as follows:
  • HPLC parameters are as follows:
  • LTTA01 SEQ ID NO:1
  • a mutant was prepared according to the method of Example 1.
  • the resulting mutants are shown in Table 1.
  • the expression LTTA01 and its mutants were cultured by shaking flasks, and enzyme powder was prepared.
  • the activities of LTTA01 and its mutants were measured according to Method 2 in section vi) of Example 1, and the results are shown in Table 1, where the relative activity refers to the activity of the mutant/the activity of LTTA01.
  • N35S-Y38F-G48A-Q94E-K116S-F70H 59 2.65 N35S-C57T-F59A-F70H-Q94E-M141C 60 4.02 N35S-C57S-F59A-F70H-Q94E-M141C 61 3.95 N35S-C57M-F59A-F70H-Q94E-M141C 62 4.32 N35S-F59A-F70H-Q94E-M141C-W185G 63 2.19 N35S-F59A-F70H-Q94E-M141C-K407R 64 2.69 N35S-F59A-F70H-Q94E-M141C-M205A 65 4.02
  • Example 3 Preparation and detection of mutants of L-threonine transaldolase from different sources
  • a mutant containing the substitution combination N35S-C57M-F70H was prepared based on the following LTTA encoding nucleic acid:
  • LTTA02 encoding nucleic acid SEQ ID NO: 3;
  • LTTA04 Chitiniphilus shinanonensis: LTTA04, encoding nucleic acid SEQ ID NO: 7.
  • Example 2 According to the method of Example 1, the expression LTTA01 and its mutants were cultured by shaking flasks, and enzyme powder was prepared. The activity of each wild-type LTTA and its mutants was measured according to method 2 in section vi) of Example 1, and the results are shown in Table 2, where the relative activity refers to the activity of the mutant/the activity of LTTA01.
  • Example 4 Introduce different substitutions at positions 35, 57 and 70 to prepare single mutants and compare their enzyme activities
  • LTTA01 SEQ ID NO:1
  • SEQ ID NO:1 nucleic acid encoding LTTA01
  • Table 1 the expression of LTTA01 and its mutants were cultured in a 96-well plate.
  • the activity of LTTA01 and the mutant was measured according to Method 1 in Section vi) of Example 1, and the results are shown in Table 3, where the relative activity refers to the activity of the mutant/the activity of LTTA01.
  • Example 5 The effect of introducing different mutations in LTTA01 on enzyme activity and product selectivity
  • LTTA01 SEQ ID NO:1
  • a mutant was prepared according to the method of Example 1.
  • the expression LTTA01 and its mutants were cultured by shaking flasks, and enzyme powder was prepared.
  • the activity and product selectivity of LTTA01 and the mutant were determined according to Method 3 in Article vi) of Example 1, and the results are shown in Table 4.
  • the relative activity refers to the activity of the mutant/the activity of LTTA01, and the product selectivity is represented by DR , And for each enzyme, ee%>99.9%.
  • mutants containing F70H or N35S are higher than that of wild type, and the product selectivity of mutants containing N35S and F70H mutations is further improved.
  • the mutant containing the single mutation of N35S was not as active as WT under the above reaction conditions, but it did not significantly affect the enzyme activity after being combined with F70H.
  • Example 6 The effect of introducing different mutations at position 70 of LTTA01 on enzyme activity and product selectivity
  • LTTA01 SEQ ID NO:1
  • a mutant was prepared according to the method of Example 1, and a substitution was introduced at position 70 of LTTA01.
  • the expression LTTA01 and its mutants were cultured by shaking flasks, and enzyme powder was prepared.
  • the activity and product selectivity of LTTA01 and the mutant were determined according to Method 3 in Article vi) of Example 1, and the results are shown in Table 5.
  • the relative activity refers to the activity of the mutant/the activity of LTTA01, and the product selectivity is represented by DR , And for each enzyme, ee%>99.9%.

Landscapes

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

Abstract

L'invention concerne une L-thréonine transaldolase (LTTA) Par rapport au début, la LTTA modifiée présente une activité catalytique améliorée pour une réaction entre le benzaldéhyde ou un dérivé de celui-ci et la L-thréonine par mise en contact. L'invention concerne également un polynucléotide pour Le codage de la LTTA modifiée de la présente invention, un vecteur et une cellule hôte exprimant la LTTA modifiée de la présente invention, et un procédé de production de 3-Phényl-L-sérine et d'un dérivé de celle-ci à l'aide de la LTTA modifiée de la présente invention et de la cellule hôte.
PCT/CN2021/091392 2020-04-30 2021-04-30 Thréonine transaldolase modifiée et son application WO2021219124A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010361933.9 2020-04-30
CN202010361933 2020-04-30

Publications (1)

Publication Number Publication Date
WO2021219124A1 true WO2021219124A1 (fr) 2021-11-04

Family

ID=78243031

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/091392 WO2021219124A1 (fr) 2020-04-30 2021-04-30 Thréonine transaldolase modifiée et son application

Country Status (2)

Country Link
CN (2) CN113583989B (fr)
WO (1) WO2021219124A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118480524A (zh) * 2024-05-07 2024-08-13 江南大学 一株l-苏氨酸转醛酶突变体及其高效合成氯霉素中间体

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024077428A1 (fr) * 2022-10-10 2024-04-18 武汉远大弘元股份有限公司 Enzyme ayant une activité de synthèse d'acide d-aminé et son utilisation
CN118240795B (zh) * 2024-05-28 2024-08-27 中国科学院天津工业生物技术研究所 L-苏氨酸转醛醇酶突变体及其在合成手性β-羟基-α-氨基酸中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109836362A (zh) * 2017-11-29 2019-06-04 苏州引航生物科技有限公司 一种制备手性(2s,3r)-对甲砜基苯丝氨酸乙酯的方法
CN110540977A (zh) * 2019-09-05 2019-12-06 福建昌生生物科技发展有限公司 一种l-苏氨酸转醛酶在氟苯尼考手性中间体合成中的应用
CN110904188A (zh) * 2019-12-23 2020-03-24 福州大学 一种l-苏氨酸转醛酶突变体的高通量快速筛选方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10655153B2 (en) * 2017-03-14 2020-05-19 Washington University Chemoenzymatic synthesis of peptide beta-lactones and beta-hydroxy acids
CN110951799B (zh) * 2019-12-23 2021-07-30 福州大学 “一菌多酶”全细胞不对称合成(2s,3r)-对甲砜基苯丝氨酸的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109836362A (zh) * 2017-11-29 2019-06-04 苏州引航生物科技有限公司 一种制备手性(2s,3r)-对甲砜基苯丝氨酸乙酯的方法
CN110540977A (zh) * 2019-09-05 2019-12-06 福建昌生生物科技发展有限公司 一种l-苏氨酸转醛酶在氟苯尼考手性中间体合成中的应用
CN110904188A (zh) * 2019-12-23 2020-03-24 福州大学 一种l-苏氨酸转醛酶突变体的高通量快速筛选方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 19 December 2017 (2017-12-19), ANONYMOUS : "MULTISPECIES: hypothetical protein [Pseudomonas]", XP055867098, retrieved from NCBI Database accession no. WP_065949345 *
SCOTT THOMAS A., HEINE DANIEL, QIN ZHIWEI, WILKINSON BARRIE: "An L-threonine transaldolase is required for L-threo-β-hydroxy-α-amino acid assembly during obafluorin biosynthesis", NATURE COMMUNICATIONS, vol. 8, no. 1, 1 August 2017 (2017-08-01), XP055867095, DOI: 10.1038/ncomms15935 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118480524A (zh) * 2024-05-07 2024-08-13 江南大学 一株l-苏氨酸转醛酶突变体及其高效合成氯霉素中间体

Also Published As

Publication number Publication date
CN113583989A (zh) 2021-11-02
CN118530962A (zh) 2024-08-23
CN113583989B (zh) 2024-06-25

Similar Documents

Publication Publication Date Title
WO2021219124A1 (fr) Thréonine transaldolase modifiée et son application
US11667896B2 (en) Modified DAAO enzyme and application thereof
CN111944775B (zh) 经修饰的7β-羟基甾体脱氢酶及其应用
JP6279478B2 (ja) ヒダントイナーゼの突然変異体
JP2022547523A (ja) １０－アセチル－３，７－ジヒドロキシフェノキサジンに対するペルオキシダーゼ活性
CN111100851B (zh) 醇脱氢酶突变体及其在手性双芳基醇化合物合成中的应用
WO2022007881A1 (fr) Glutamate déshydrogénase modifiée et son utilisation
TWI852256B (zh) 經修飾的麩胺酸脫氫酶及其應用
WO2024183112A1 (fr) Procédé d'expression de d-aminoacide oxydase et son utilisation
KR101617371B1 (ko) 메타게놈 유래 신규 베이어 빌리거 모노옥시게나아제 BVMOgm1
KR101619864B1 (ko) 메타게놈 유래 신규 베이어 빌리거 모노옥시게나아제 BVMOsm1
CN118667785A (zh) 经修饰的高丝氨酸o-乙酰基转移酶及其应用
WO2024010785A1 (fr) Enzymes cétoréductases pour la synthèse d'indanes substitués par un 1,3-diol
TW202413637A (zh) 具有增加的酮還原酶活性的突變型酮還原酶以及涉及其之方法及用途

Legal Events

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

Ref document number: 21797898

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202217067950

Country of ref document: IN

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21797898

Country of ref document: EP

Kind code of ref document: A1