WO2012105711A1 - Procédé pour produire un composé organique - Google Patents

Procédé pour produire un composé organique Download PDF

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WO2012105711A1
WO2012105711A1 PCT/JP2012/052626 JP2012052626W WO2012105711A1 WO 2012105711 A1 WO2012105711 A1 WO 2012105711A1 JP 2012052626 W JP2012052626 W JP 2012052626W WO 2012105711 A1 WO2012105711 A1 WO 2012105711A1
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compound
enzyme
amino acid
activity
group
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PCT/JP2012/052626
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Japanese (ja)
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木野　邦器
友希 中川
大 佐藤
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学校法人早稲田大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
    • 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
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for producing an organic compound. Specifically, the present invention relates to a method for producing an arbitrary organic compound by combining an arbitrary compound and a compound having nucleophilic activity.
  • the enzymatic method when used, it reflects the substrate specificity of the enzyme, so if it matches the target binding direction and binding order, the target is not incurred in cost or time. Can be achieved.
  • the enzyme reaction since the enzyme reaction has high reaction specificity, there is an advantage that generation of unnecessary isomers or intermediate products can be avoided. Therefore, at present, the development of compounds useful as pharmaceuticals and functional foods, in particular, enzymes that can be used for the production of peptides and peptide derivatives is underway.
  • a dipeptide containing proline at the C-terminus (Xaa-Pro) is a compound that is expected to have excellent physiological activity such as exhibiting a blood pressure lowering action.
  • Xaa-Pro a dipeptide containing proline at the C-terminus
  • Patent Document 1 and Non-Patent Documents 1 and 2 a proline-containing dipeptide is used. The generation of has been reported.
  • Patent Document 1 reports the production of a proline-containing dipeptide, but the proline-containing dipeptide has proline bound to the N-terminus rather than the C-terminus expected to be useful as a pharmaceutical or functional food. Limited to the production of
  • Non-Patent Documents 1 and 2 report the production of C-terminal proline-containing dipeptides, but only disclose the production of cyclic proline-containing dipeptides.
  • Diketopiperazine which is a cyclic dipeptide, has attracted attention in recent years for its various functions and has been researched and developed for industrial use.
  • the methods disclosed in Non-Patent Documents 1 and 2 only the cyclic dipeptide can be produced as described above, and the other form is known which has many usefulness as a pharmaceutical or functional food. It is not possible to produce a linear dipeptide.
  • the present invention has been made in view of such circumstances, and has found a synthesis system that can bind target compounds to each other in a desired binding direction and order, and is simpler and more efficient. It aims at providing the manufacturing method of the organic compound by a method. Another object of the present invention is to provide a method for easily supplying industrially useful dipeptides and diketopiperazines.
  • the present inventors have focused on an enzyme or enzyme group having an activity of thioesterifying an arbitrary compound, and as a result, by using such an enzyme or enzyme group, It has been found that the above compound and other compounds can be bonded in a straight chain, and the present invention has been achieved.
  • a method for producing an organic compound comprising preparing an arbitrary compound and a compound having a nucleophilic activity, and the arbitrary compound. In the presence of an enzyme or group of enzymes having the activity to thioesterify the compound, and contacting the compound with the nucleophilic activity with the compound having the nucleophilic activity.
  • a method is provided, characterized in that it binds to the thioester moiety of any compound by nucleophilic attack.
  • the target compound can be produced in an industrially useful yield inexpensively and safely. Further, according to such a configuration, an arbitrary compound and a compound having nucleophilic activity can be bound in the target binding direction depending on the substrate specificity of the enzyme.
  • the arbitrary compound is activated by an enzyme or enzyme group having an activity to thioesterify an arbitrary compound
  • the compound can be subjected to various nucleophilic substitution reactions. It can be used as an electrophile. This is useful because it supports the supply of organic compounds for which there is no practical synthetic method that can be used industrially and can contribute to functional development and application development research of the organic compounds.
  • the enzyme or the enzyme group is a module constituting at least a part of NRPS (non-ribosomal peptide synthetase), and at least an adenylation active domain.
  • NRPS non-ribosomal peptide synthetase
  • the peptidyl carrier protein site, and the arbitrary compound is one that is linked to the peptidyl carrier protein site of the enzyme or group of enzymes through a thioester bond.
  • the arbitrary compound is a compound having a carboxyl group.
  • the compound having a carboxyl group is preferably an amino acid, and the amino acid is particularly preferably selected from the group consisting of tryptophan, tyrosine, methionine, leucine, and valine.
  • the enzyme or group of enzymes is derived from Brevibacterium parabrevis.
  • the enzyme or group of enzymes comprises (A) an enzyme having the amino acid sequence set forth in SEQ ID NO: 1, and (B) a sequence ID number.
  • An enzyme having an activity of binding to (C) an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1, and adenylating a carboxyl group of any compound, and by thioester bond
  • the compound having nucleophilic activity comprises a nitrogen-containing compound containing proline, hydroxyproline, proline derivative, hydroxylamine, and pyrrolidine. It is selected from the group.
  • an organic compound which is a genetic recombination having the ability to express an enzyme according to any one of the following (A) to (F):
  • a step of culturing a body in a medium a step of bringing an arbitrary compound and a compound having nucleophilic activity into contact with the genetic recombinant, and a combination of the arbitrary compound and the compound having nucleophilic activity.
  • the compound having the nucleophilic activity is nucleophilic at the thioester site of the arbitrary compound.
  • a method is provided, characterized in that it binds by attack, wherein the enzyme is (A) an enzyme having the amino acid sequence described in SEQ ID NO: 1, (B) the enzyme described in SEQ ID NO: 1. 1 in the amino acid sequence
  • An enzyme comprising an amino acid sequence containing the above amino acid substitution, deletion, insertion, addition or inversion, adenylating a carboxyl group of any compound, and having an activity of binding to any compound by a thioester bond
  • C It consists of an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and has an activity to adenylate the carboxyl group of any compound and to bind to the any compound through a thioester bond
  • An enzyme, (D) consisting of an amino acid sequence encoded by a polynucleotide comprising a base sequence having 60% or more homology with the base sequence set forth in SEQ ID NO: 3, and adenylating the carboxyl group of any compound; and
  • the genetic recombinant is one that has been recombined with the DNA described in any of (A) to (C) below, (A) DNA having the base sequence represented by SEQ ID NO: 3, (B) a base sequence having 60% or more homology with the base sequence represented by SEQ ID NO: 3, and a carboxyl of any compound A DNA encoding an enzyme having an activity of adenylating a group and binding to the above-mentioned arbitrary compound by a thioester bond; (C) a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 3 and stringent An enzyme that hybridizes under conditions, adenylates the carboxyl group of any compound, and binds to any compound by a thioester bond.
  • Sul DNA DNA that is a.
  • the host of the gene recombinant is E. coli.
  • FIG. 1 is a schematic diagram showing a non-ribosomal peptide synthetase (NRPS) according to the present invention.
  • FIG. 2 is a schematic diagram showing the synthesis of cyclic proline-containing dipeptides by NRPS.
  • FIG. 3 is a schematic diagram showing organic compound synthesis by a nucleophilic substitution reaction in one embodiment of the present invention.
  • FIG. 4 is a chromatogram of dipeptide production by L-tryptophan and L-proline in an embodiment of the present invention.
  • FIG. 5 is a chromatogram of dipeptide production by L-leucine and L-proline and dipeptide production by L-tyrosine and L-proline in an embodiment of the present invention.
  • FIG. 6 is a table showing dipeptide production in one embodiment of the present invention.
  • FIG. 7 is a chromatogram showing organic compound synthesis using pyrrolidine as a substrate in one embodiment of the present invention.
  • FIG. 8 is a graph showing organic synthesis using pyrrolidine as a substrate in one embodiment of the present invention.
  • the method for producing an organic compound according to the present embodiment includes a step of preparing an arbitrary compound and a compound having nucleophilic activity, and an enzyme or enzyme having an activity of thioesterifying the arbitrary compound.
  • the “arbitrary compound” is a compound that undergoes a nucleophilic attack from a compound having a nucleophilic activity, which will be described later, in the method for producing an organic compound by the enzyme according to the present invention. Binds to compounds with nuclear activity.
  • the arbitrary compound has a carboxyl group.
  • an “arbitrary compound” is activated by an enzyme or enzyme group according to the present invention described later, thereby enabling a thioester bond with the enzyme or enzyme group.
  • the activation of the arbitrary compound is not particularly limited as long as the carboxyl group in the arbitrary compound is activated for the reaction, such as phosphorylation or acetylation, but preferably adenyl is used.
  • the arbitrary compound is activated by the enzyme or enzyme group to form a thioester bond with a peptidyl carrier protein site (PCP site) in the enzyme or enzyme group. is there.
  • PCP site is used for consent to a thiolation domain (T domain), unless otherwise specified, and the same site when expressed in any language. Or it refers to a domain.
  • the arbitrary compound according to the present invention is a compound having a carboxyl group, and the carboxyl group is activated, preferably adenylated by the enzyme or enzyme group, and thereby binds to the PCP site. If it is a thing, it will not specifically limit. Accordingly, an organic acid having at least one carboxyl group such as ⁇ -, ⁇ -, and ⁇ -keto acid can be any compound according to the present invention.
  • the “compound having nucleophilic activity” has an unshared electron pair (lone pair), and with respect to the arbitrary compound that forms a thioester with the enzyme or enzyme group according to the present invention.
  • a nucleophilic substitution reaction is performed.
  • the compound having the nucleophilic activity performs a nucleophilic attack on the thioester site of the arbitrary compound, and thereby the binding with the arbitrary compound is achieved.
  • the “compound having nucleophilic activity” according to the present invention is not particularly limited as long as it performs a nucleophilic substitution reaction by having a loan pair as described above.
  • the “compound having nucleophilic activity” is preferably D, L-proline, or a proline derivative containing D, L-hydroxyproline, proline methyl ester, proline amide, and dehydroproline. Or a nitrogen-containing compound containing hydroxylamine or pyrrolidine.
  • the “enzyme or enzyme group having an activity to thioesterify any compound” includes Non-ribosomal peptide synthetase, Acetate-CoA ligase, O-Succinylbenzoate-CoA ligase, Butyrate-CoA ligase, Long-chain-fatty-acid-CoA ligase, Succinate-CoA ligase, Glutarate-CoA ligase, Choleate-CoA ligase, Oxalate-CoA ligase, Malate-CoAligate CoAligate, Biotinga CoAligate CoA ligase, 6-Carboxyhexanoate-CoA ligase, Arachidonate-CoA ligase, Citrate-CoA ligase, Dicarboxylate-CoA ligase, Phytanate-CoA ligase, Benzoate-CoA ligase, O-Succinyl
  • the “enzyme or enzyme group having an activity to thioesterify an arbitrary compound” preferably has a thioester bond with an arbitrary compound as described above to form the arbitrary compound. It catalyzes the bond with the compound having nucleophilic activity.
  • the enzyme or group of enzymes phosphorylates and acetylates the arbitrary compound, and preferably adenylates the carboxyl group of the arbitrary compound.
  • enzyme refers to a molecule having a function of catalyzing a chemical reaction in a living body, unless otherwise specified.
  • an enzyme when an enzyme is composed of a plurality of peptides (subunits), when a plurality of enzymes (complex enzymes) form a cluster to catalyze a series of metabolic processes, a plurality of domains can convert a single enzyme
  • a plurality of domains can convert a single enzyme
  • one or more molecules function as a single enzyme when they are assembled or linked, such as when they are formed, when the one or more molecules alone have enzyme activity, the molecule having the enzyme activity is expressed as “
  • the term “enzyme” refers to a single enzyme associated with or associated with the group or group of enzymes.
  • the “enzyme or enzyme group having an activity to thioesterify any compound” is at least one of non-ribosomal peptide synthetase (NRPS) (FIG. 1). It is a module that constitutes a section.
  • NRPS non-ribosomal peptide synthetase
  • FIG. 1 NRPS is a huge multi-module enzyme complex having a molecular weight of several thousand kDa, and is known for synthesizing short-chain physiologically active peptides without going through ribosomes.
  • a module that is a repeating unit that constitutes NRPS is an adenylation domain (A domain) that recognizes and activates a substrate amino acid, and a thiolation domain (T domain) that forms a thioester with an activated amino acid. ) (Peptidyl carrier protein site (PCP site)) and a condensation domain (C domain) that catalyzes the formation of peptide bonds.
  • Some modules have an isomerization domain (E domain) that converts L-amino acids into D-forms in the process of peptide synthesis, and the peptides produced have diversity.
  • tyrosidin A an antibacterial decapeptide synthesized by Brevibacillus parabrevis.
  • the starter module TycA (the amino acid sequence of TycA is shown in SEQ ID NO: 1 and the base sequence is shown in SEQ ID NO: 3)
  • the first extension module TycB1 (the amino acid sequence of TycB1 is shown in SEQ ID NO: 2
  • a cyclic proline-containing dipeptide cyclo (D-Phe-L-Pro) is synthesized using L-phenylalanine and L-proline as substrates, respectively (the base sequence is shown in SEQ ID NO: 4) (FIG.
  • a peptide excision reaction from NRPS is catalyzed by a thioesterase domain (TE domain) of a terminal module.
  • TE domain thioesterase domain
  • generation of a cyclic peptide by cyclization and hydrolysis of a chain peptide by hydrolysis are performed.
  • cyclo (D-Phe-L-Pro) synthesis reaction it is known that the release of the product from the enzyme main body proceeds non-enzymatically by intramolecular self-cyclization. The possibility of synthesizing a chain dipeptide was also predicted by the structure of the intermediate compound.
  • NRPS The biggest factor that NRPS has not been industrially used to date, although it has the potential to comprehensively synthesize various dipeptides that could not be synthesized enzymatically, is the production of peptides by this method. It is that the nature was extremely low.
  • the yield of reaction products by NRPS is generally as low as ⁇ M, and even in the above-mentioned example of fermentation production of cyclo (D-Phe-L-Pro), the maximum is 38 ⁇ M (9.2 mg / L).
  • NRPS-related research with a view to industrialization is scarce, and mainly research with deep academic and analytical hues. Most of them are related to combinatorial peptide synthesis utilizing the characteristics of NRPS.
  • the enzyme or group of enzymes has at least the above-mentioned adenylation active domain and peptidyl carrier protein site, and has the same or similar effect as the method according to the present invention. It goes without saying that any module, domain, or site constituting NRPS can be included in addition to these domains and sites.
  • the enzyme or group of enzymes is derived from Brevibacillus parabrevis, but as long as the enzyme or group of enzymes or the NRPS has the same function as described above, Any strain may be used as long as it produces an enzyme having an amino acid sequence including amino acid substitution, deletion, insertion, addition, or inversion.
  • “Brevibacillus parabrevis” is “Brevibacillus parabrevis NBRC 3331”, unless otherwise specified, and in some cases “Brevibacillus brevis”, “Bacillus brevis”, “AT” 81, “AT” 85, “AT In some cases, they are the same strain.
  • the arbitrary compound when a module constituting at least a part of NRPS is used as the enzyme or enzyme group, the arbitrary compound is preferably an amino acid.
  • peptide refers to a compound in which two or more amino acids are linked by peptide bonds.
  • an amino acid or peptide includes a methyl group-containing alkyl group, a phosphate group, a sugar chain, and / or an ester bond or other covalent bond modification, it is referred to as an amino acid or peptide derivative, respectively.
  • amino acids may be represented by compound names such as asparagine and glutamine, and may be represented by conventional three letter codes such as Asn and Gln. When represented by a compound name, it is represented using a prefix (or D-) indicating the configuration of the ⁇ -carbon of the amino acid. When expressed in conventional three-letter notation, the three-letter notation represents an L-amino acid unless otherwise specified.
  • an amino acid refers to any compound in which an amino group and a carboxyl group are bonded via at least one carbon atom and can be polymerized by a peptide bond.
  • the amino acids in the present specification are 20 consisting of glycine, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine.
  • a variety of amino acids and their stereoisomers D-amino acids, as well as amino acids that do not constitute proteins, ⁇ -alanine, sarcosine, ornithine, citrulline, creatine, ⁇ -aminobutyric acid, opine, etc. It may be any natural or unnatural amino acid that is not.
  • any of the amino acids described above can be used.
  • the chirality of the amino acid moiety as the arbitrary compound in the organic compound produced by binding with a compound having nucleophilic activity is retained before and after the binding.
  • Xaa-Pro can be synthesized according to the method of the present invention.
  • Xaa on the N-terminal side is an arbitrary amino acid, but preferably, the N-terminal amino acid Xaa is Trp, Tyr, Met, Leu, Val.
  • Xaa may be L-form or D-form
  • Xaa of the generated dipeptide Xaa-Pro depends on the chirality of Xaa.
  • the dipeptide (product) produced by the method according to the present invention can be any of DL, LD, LL, or DD.
  • TycA which is a module constituting at least part of NRPS
  • the amino acid (Xaa) as “any compound” May be L-form or D-form.
  • the enzyme in a normal enzyme reaction, as shown by the substrate specificity of the enzyme that acts on a specific substrate to catalyze a specific reaction, the enzyme has a configuration of atomic groups in the substrate. It distinguishes loci (D and L, cis and trans, etc.) and recognizes the three-dimensional structure.
  • TycA which is a module constituting at least part of NRPS is used as “an enzyme or enzyme group having an activity to thioesterify any compound”
  • Phe which is a substrate of D-Phe-L-Pro
  • D-Phe-L-Pro- DKP D-Phe-L-Pro- DKP
  • L-Trp-L-Pro, L-Tyr-L-Pro, L-Leu-L-Pro, D-Leu-L-Pro , L-Met-L-Pro, D-Met-L-Pro, L-Trp-D-Pro and the like can be synthesized in units of g / L, and cis- When 4-L-HydroxyPro (C4L) or cis-4-D-HydroxyPro (C4D) is used, L-Trp-C4L, L-Trp-C4D, etc.
  • a C-terminal proline dipeptide can be produced easily without purification by-products as compared with chemical synthesis combining complicated reactions, which is a normal production method.
  • a C-terminal proline-containing peptide can be produced as a chain peptide from an unprotected amino acid in a one-step process.
  • by using a gene recombinant as described below production of a C-terminal proline dipeptide does not require ATP or magnesium, and production is possible only by adding a substrate.
  • TycA which is a module constituting at least a part of NRPS
  • TycB1 is a module constituting at least a part of NRPS and having at least an adenylation active domain and a peptidyl carrier protein site.
  • TycA and TycB1 are used in the method according to the present invention, an organic compound is obtained by binding any compound thioester-bonded to the peptidyl carrier protein site of TycB1 to any compound thioester-bonded to the peptidyl carrier protein site of TycA. Is manufactured.
  • TycB1 plays a role of bringing a compound bonded to TycB1 into contact with any compound bonded to TycA in order to enable a catalytic reaction, and any compound having a thioester bond with TycB1. Need not have nucleophilic activity. Therefore, in one embodiment according to the present invention, as long as the “compound having nucleophilic activity” uses a compound that binds by nucleophilic attack to the thioester site of any compound according to the present invention, only TycA is used. Of course, it goes without saying that the synthesis reaction is accelerated even when both TycA and TycB1 are used.
  • the present invention promotes contact (non-enzymatic reaction) with a compound having nucleophilic activity by activating the compound by thioesterifying an arbitrary compound, thereby making it possible to efficiently and economically use various organic compounds.
  • the technology which can be synthesized is provided.
  • the remarkable effects or features of the present invention that can be achieved when only TycA is used are similarly achieved even when TycB1 is used in addition to TycA.
  • TycB1 is used in addition to TycA.
  • an amino acid is used as an “arbitrary compound”, as described above, as an arbitrary compound in an organic compound produced by bonding with a compound having nucleophilic activity, as described above
  • the chirality of the amino acid moiety is retained before and after the binding, but when an organic compound using TycA and TycB1 is synthesized, the chirality of any amino acid moiety in the product is retained.
  • the chirality of the dipeptide produced using TycA and TycB1 was analyzed by analyzing the hydrolyzate with hydrochloric acid and N ⁇ - (5-fluoro-2,4-dinitrophenyl) -L-alaninamide (FDAA). ) To confirm the retention of chirality when judged by separation on the HPLC chromatogram of the dipeptide derivatized with can do.
  • an enzyme or enzyme group having an activity to thioesterify any compound includes (A) an enzyme having the amino acid sequence described in SEQ ID NO: 1, and (B) SEQ ID NO: 1.
  • An amino acid sequence comprising one or more amino acid substitutions, deletions, insertions, additions, or inversions, wherein the carboxyl group of any compound is adenylated, and a thioester bond forms the arbitrary compound.
  • An enzyme having a binding activity consisting of an amino acid sequence having 60% or more homology with the amino acid sequence described in SEQ ID NO: 1, and adenylating a carboxyl group of an arbitrary compound;
  • sequence ID It consists of an amino acid sequence encoded by a base sequence that hybridizes with the base sequence of No.
  • the enzyme possessed by introducing a site-specific mutation into DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1, for example, using a site-directed mutagenesis method well known in the field of molecular biology Can get.
  • the number of substitution, deletion, insertion, addition, or inversion amino acid residues is not particularly limited, but substitution, deletion, insertion, addition, or inversion can be performed by a well-known method such as the above-mentioned site-specific mutation method.
  • the number is about 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
  • the position of the amino acid at which amino acid residues can be deleted or added is such that the enzyme resulting from the deletion or addition is adenylated on the carboxyl group of an arbitrary compound, and the thioester bond is combined with the optional compound. Any position may be used as long as it has an activity of binding, and examples thereof include 10 amino acid residues on the N-terminal side and C-terminal side of the amino acid sequence represented by SEQ ID NO: 1.
  • deletion, substitution or addition may occur simultaneously, and the amino acid to be substituted or added may be a natural type or a non-natural type.
  • the enzyme or enzyme group according to the present invention has a homology with the amino acid sequence represented by SEQ ID NO: 1 of 60% or more, preferably 65% or more, more preferably 70% or more, and still more preferably 75%. Or more, more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more, still more preferably 97% or more, particularly preferably 98% or more, and most preferably 99% or more.
  • An enzyme having a sequence and having an activity of adenylating a carboxyl group of an arbitrary compound and binding to the arbitrary compound through a thioester bond can be exemplified.
  • the homology with the base sequence represented by SEQ ID NO: 3 is 60% or more, preferably 65% or more, more preferably 70% or more, still more preferably 75% or more, More preferably, it is 80% or more, more preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 98% or more, most preferably 99% or more.
  • an enzyme having an activity of adenylating a carboxyl group of an arbitrary compound and binding to the arbitrary compound through a thioester bond is 60% or more, preferably 65% or more, more preferably 70% or more, still more preferably 75% or more, More preferably, it is 80% or more, more preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 98% or more, most preferably 99% or more.
  • the homology between amino acid sequences and base sequences can be determined using BLAST or FASTA.
  • the enzyme or enzyme group according to the present invention comprises an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions with the base sequence shown in SEQ ID NO: 3, and a carboxyl group of any compound And an enzyme having an activity of adenylating and binding to any of the above compounds through a thioester bond.
  • hybridize indicates that DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part of the base sequence of the DNA is useful as a probe for Northern or Southern blot analysis, or has a length that can be used as an oligonucleotide primer for PCR analysis. May be.
  • DNA used as a probe for Northern or Southern blot analysis DNA of at least 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more can be mentioned, and DNA used as an oligonucleotide primer is at least 10 bases. As mentioned above, DNA having 15 bases or more can be mentioned.
  • a DNA hybridization experiment an experiment can be performed according to hybridization conditions well known in the field of molecular biology.
  • the above stringent conditions may be any conditions as long as the effects of the method according to the present invention can be achieved.
  • a filter on which DNA is immobilized and a probe DNA are mixed with 50% formamide, 5 ⁇ SSC (750 mM sodium chloride, 75 mmol / L sodium citrate), 50 mmol / L sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g / L denatured salmon sperm After overnight incubation at 42 ° C. in a solution containing DNA, conditions such as washing the filter in a 0.2 ⁇ SSC solution at about 65 ° C. can be raised, and lower stringent conditions can be used. it can.
  • the enzyme or enzyme group according to the present invention is an enzyme in which the enzyme according to any one of the above (A) to (E) and a specific binding tag peptide are bound, wherein the carboxyl group of any compound is Mention may be made of an enzyme that has the activity of adenylating and binding to any of the above compounds via a thioester bond.
  • tag peptide refers to a part that is molecularly or genetically linked to serve as a marker for a specific protein molecule, and can be of any length. good. Moreover, as long as it does not affect the physiological or physicochemical properties of the protein or peptide to be bound, it can be bound at any position, but it is preferably bound to the end of the target protein and has a low molecular weight. It is. In addition, other types of proteins can be used as tag peptides. Furthermore, by using a tag peptide, it is possible to isolate and immobilize proteins, detect protein-protein interactions, visualize protein molecules, etc. according to their properties.
  • a method for producing an organic compound comprises culturing a gene recombinant having the ability to express the enzyme according to any one of (A) to (F) above in a medium. Culturing an organic compound obtained by combining a step, a step of bringing an arbitrary compound and a compound having a nucleophilic activity into contact with the gene recombinant, and the arbitrary compound and the compound having a nucleophilic activity And the step of collecting the organic compound from the culture, and the compound having the nucleophilic activity binds to the thioester site of the arbitrary compound by nucleophilic attack It is the method characterized by being.
  • the term “gene recombinant” means that an expression vector incorporating a polynucleotide encoding the protein of the present invention is introduced so that a desired trait related to a protein having a desired function can be expressed.
  • a transformant or an organism capable of expressing a desired trait by introducing a predetermined gene into the chromosome from the outside, and a living organism having a nucleic acid obtained by genetic recombination technology or the like and a replica thereof. It is what you point to.
  • the gene recombinant used in the production method of the present invention is not particularly limited as long as it can be used in the production method of the present invention as long as it is a gene recombinant, but any of the above (A) to (F) A genetic recombination obtained by genetic recombination of a host cell by a known method using a genetic recombinant that expresses the enzyme described in 1. and a DNA described in any of (A) to (C) described later. I can raise my body.
  • host cells include prokaryotes such as Escherichia coli, Bacillus subtilis, and filamentous fungi, and eukaryotes such as yeast, fungi, plants, animals, and the like, and preferably microorganisms belonging to the genus Escherichia. be able to.
  • the host of the gene recombinant is Escherichia coli.
  • the DNA to be recombined with the gene recombinant includes (A) DNA having the base sequence represented by SEQ ID NO: 3 and (B) at least 60% homology with the base sequence represented by SEQ ID NO: 3.
  • DNA encoding an enzyme having an activity of adenylating a carboxyl group of an arbitrary compound and binding to the arbitrary compound through a thioester bond (C) a base sequence represented by SEQ ID NO: 3 DNA encoding an enzyme which hybridizes with a DNA having a complementary base sequence under stringent conditions, adenylates the carboxyl group of any compound, and has an activity to bind to the any compound through a thioester bond Is mentioned.
  • these DNAs are 60% or more, preferably 65% or more, more preferably 70% or more, further preferably 75% or more, more preferably 80% or more, more than the base sequence represented by SEQ ID NO: 3. More preferably, 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 98% or more, most preferably 99% or more of the sequences having homology are searched and obtained by the search Based on the base sequence, it can also be obtained from a chromosomal DNA or cDNA library of an organism having the base sequence by various methods well known in the field of genetic engineering or molecular biology.
  • the obtained DNA is cut as it is or with an appropriate restriction enzyme and incorporated into a vector by a conventional method.
  • a commonly used nucleotide sequence analysis method or nucleotide sequence analyzer Can be used to determine the base sequence of the DNA.
  • any method can be used as long as it is a method for introducing DNA into the host cell, and for example, a calcium ion method, a protoplast method, an electroporation method, or the like can be used. .
  • the cultivation of the genetic recombinant contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the genetic recombinant, and the cultivation of the genetic recombinant is efficient.
  • the culture can be performed using a natural medium or a synthetic medium that can be prepared in the following manner.
  • Any carbon source may be used as long as it can be assimilated by a genetically modified substance.
  • Molasses containing glucose, fructose, etc., saccharified liquid of cellulosic biomass, carbohydrates such as glycerol, starch or starch hydrolysate, acetic acid, propion Organic acids such as acids and alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digests thereof, and the like can be used.
  • monopotassium phosphate dipotassium phosphate
  • magnesium phosphate magnesium sulfate
  • sodium chloride ferrous sulfate
  • manganese sulfate copper sulfate
  • calcium carbonate calcium carbonate
  • Cultivation is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the medium as needed during the culture.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside or the like is used for culturing a gene recombinant having an expression vector using the trp promoter.
  • Indoleacrylic acid or the like may be added to the medium.
  • the organic recombinant in the culture supernatant is subjected to a treatment such as centrifugation or pulverization if necessary.
  • a treatment such as centrifugation or pulverization if necessary.
  • derivatives such as sugar adducts thereof can be recovered, but various compounds can be employed as long as the target organic compound can be collected.
  • a predetermined gene can be further incorporated into a recombinant that produces the organic compound according to the target organic compound.
  • the predetermined gene include a gene that promotes activation of the enzyme or enzyme group according to the present invention, a gene of the enzyme group related to the ATP regeneration system, or the decomposition or metabolism of the organic compound in the gene recombinant.
  • the gene which inhibits the function of the gene to do can be mentioned.
  • a knockout strain of this gene can also be employed. For example, when proline is employed as the above-mentioned “compound having nucleophilic activity” and E.
  • pepQ gene-deficient E. coli is used as a gene recombinant, a peptidase called prolidase (PepQ) exists in the E. coli body, and the L-proline-containing peptide is degraded. Therefore, pepQ gene-deficient E. coli can be used for the purpose of increasing the yield.
  • PepQ prolidase
  • an Sfp gene may be further incorporated into the above gene recombinant.
  • the Sfp gene is a gene relating to the synthesis of Surfactin derived from Bacillus subtilis and produces a phosphopantetheine transferase that links Coenzyme A to activate the NRPS module. Therefore, by producing a gene recombinant that co-expresses tycA and sfp, the organic compound according to the present invention can be produced efficiently and inexpensively.
  • conditions for efficiently producing the target organic compound are set by adjusting the sfp gene expression intensity and the copy number of the gene expression plasmid. can do.
  • TycA Forward Primer: ATA CCATGG TAGCAAATCAGGGCCAATC (SEQ ID NO: 5) (underlined portion is a restriction enzyme site by NcoI)
  • the TycA gene amplified by PCR was digested with restriction enzymes NcoI and BamHI and cloned in the same manner into a linearized vector pET21d (+). It was designed so that a vector-derived His ⁇ 6-tag was added at the cloning stage. After confirming the sequence of the prepared plasmid, it was introduced into Escherichia coli BLR (DE3).
  • the NRPS modifying enzyme gene sfp was amplified by PCR using the genomic DNA of Bacillus subtilis OKB105 as a template and the following primer set.
  • Forward Primer ACGGCA CATATG AAGATTTACGG (sequence ID number 7) (underlined part is a restriction enzyme site by NdeI)
  • Reverse Primer ATA GAATTC TTATAAAAGCTCTTCG (SEQ ID NO: 8) (underlined portion is a restriction enzyme site by EcoRI)
  • the amplified fragment was digested with NdeI and EcoRI and cloned into the linearized vector pET21a (+) in the same manner. After confirming the sequence of the prepared plasmid, it was introduced into Escherichia coli BLR (DE3).
  • Each cultured cell was collected by centrifugation (4 ° C., 3,000 ⁇ g, 10 min), and the washing operation with 50 mM Tris-HCl buffer (pH 8.0) was repeated three times. Thereafter, the bacterial cells resuspended in the same buffer were sonicated, and insoluble proteins and residues were removed by centrifugation (4 ° C., 14,000 rpm, 30 min) to prepare a cell-free extract. In the presence of Coenzyme A and MgCl 2 , Sfp was allowed to act on TycA in the cell-free extract to convert it into an active holoenzyme. Each of the enzymes was purified by Ni 2+ affinity chromatography and used for activity measurement. Confirmation of gene expression status and enzyme purification was performed by SDS-PAGE, and protein quantification was performed based on the Bradford method.
  • the FDAA sample was analyzed and quantified using a WH-C18A column (Hitachi Science Systems, 4 ⁇ 150 mm). The following conditions were used for the HPLC analysis conditions.
  • the mobile phase included 50 mmol / l potassium phosphate buffer (pH 2.7, pH adjusted with phosphoric acid), acetonitrile and methanol in a mixing ratio of 90: 5: 5, and 50 mmol / l phosphorus.
  • Mobile phase C was used.
  • the flow rate of the mobile phase is 0.5 ml / min
  • the mixing ratio of the mobile phase A, the mobile phase B, and the mobile phase C is a gradient from 100: 0: 0 to 55: 45: 0 from 0 to 24 minutes, 24 to 30 Minutes are constant at 55: 45: 0, slopes from 55: 45: 0 to 0: 100: 0 for 30-50 minutes, constants at 0: 100: 0 for 50-55 minutes, 0 for 55-60 minutes : 100: 0 to 0: 0: 100 slope, 60-62 minutes constant at 0: 0: 100, 62-62.1 minutes slope from 0: 0: 100 to 100: 0: 0, 62 .1 to 80 minutes were changed to a constant value of 100: 0: 0.
  • the column temperature was 40 ° C., and ultraviolet absorption at 340 nm was measured.
  • FIG. 3 shows that when proline is used as an amino acid having nucleophilic activity, TycA is released by proline nucleophilic attack on any amino acid thioester-bonded to TycA, and any amino acid and proline are separated. It is a schematic diagram showing the scheme to couple
  • FIG. 4a is a chromatogram of dipeptide production using L-tryptophan and L-proline (10 mM) as substrates when only TycA is used as an enzyme. The reaction conditions are as shown in the figure.
  • FIG. 4b shows the results when the proline concentration is 20 mM. As can be seen from these results, by using TycA, L-tryptophan and L-proline as substrates are combined to produce a dipeptide.
  • FIG. 5a is a chromatogram of dipeptide production using L-leucine and L-proline as substrates when only TycA is used as an enzyme.
  • the reaction conditions are the same as in the above L-tryptophan example except that the substrate was changed from L-tryptophan.
  • L-leucine and L-proline were used, 2.13 mM (reaction time: 24 h)
  • a dipeptide which was a product of the two amino acids bound to each other, was obtained.
  • FIG. 5b is a chromatogram of dipeptide formation using L-tyrosine and L-proline as substrates, and the reaction conditions are the same as above.
  • TycA it can be seen that the amino acid as the substrate may be any amino acid, and dipeptide is produced by nucleophilic substitution reaction of proline to any amino acid.
  • FIG. 6 shows the amount of dipeptide produced when the reaction was carried out using only TycA as an enzyme and D-form or hydroxyproline as a substrate.
  • TycA which is an enzyme or an enzyme group according to the present invention
  • any compound used as a substrate and a compound having nucleophilic activity are either optical isomers of L-form or D-form.
  • the organic compound is produced according to the target binding direction.
  • the chirality of tryptophan before the reaction and the Trp in the product (Trp-Pro) are the same, when TycA is used in the method of the present invention, the chirality of any compound It can be seen that is retained.
  • proline derivatives cis-4-L-hydroxyproline (C4L) and cis-4-D-hydroxyproline (C4D) are used as compounds having nucleophilic activity. From this result, when TycA which is the enzyme or enzyme group according to the present invention is used, the target organic compound can be produced even if proline derivatives are used in addition to proline as a compound having nucleophilic activity. I understood.
  • FIG. 7 shows the amount of organic compound produced when the reaction is carried out using only TycA as the enzyme and pyrrolidine as the compound having nucleophilic activity. Note that L-tryptophan is used as a substrate for TycA.
  • FIG. 8 identifies this product.
  • the assumed product is (S) -2-amino-3- (1H-indol-3-yl) -1- (pyrrolidin-1-yl) propan-1-one (L-Trp-Pyrrolidine) And its exact mass is 257.1528. Therefore, the product was roughly purified from the reaction solution by solid phase extraction and subjected to mass spectrometry. Then, as can be seen from the MS spectrum of the roughly purified reaction product shown in this figure, the peak coincides with m / z (258) of the expected protonated ion of the product, and this reaction product is It was suggested that L-tryptophan and pyrrolidine are compounds in which amide bonds are bound.
  • nitrogen-containing compounds containing pyrrolidine can be used as compounds having nucleophilic activity, and binding of such compounds is achieved. Was shown to do.
  • the present invention can be variously modified, and is not limited to the above-described embodiment, and can be variously modified without changing the gist of the invention.

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Abstract

La présente invention concerne un procédé simple et extrêmement efficace pour produire un composé organique par le procédé enzymatique avec lequel il est possible d'effectuer une liaison dans un sens de liaison souhaité. Le procédé se caractérise en ce qu'il se compose d'une étape de préparation d'un composé arbitraire et d'un composé nucléophile et d'une étape destinée à amener le composé arbitraire en contact avec le composé nucléophile en présence d'une enzyme ou d'un groupe d'enzymes ayant une activité en termes de thioestérification du composé arbitraire, le composé nucléophile étant celui qui effectue la liaison par attaque nucléophile de la fraction de thioesters du composé arbitraire.
PCT/JP2012/052626 2011-02-04 2012-02-06 Procédé pour produire un composé organique WO2012105711A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2019208700A1 (fr) * 2018-04-26 2019-10-31 ゼリア新薬工業株式会社 Dipeptide et composition pharmaceutique le contenant

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EVA PFEIFER ET AL.: "Characterization of tyrocidine synthetase 1 (TY1): Requirement of posttranslational modification for peptide biosynthesis", BIOCHEMISTRY, vol. 34, no. 22, 1995, pages 7450 - 7459 *
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RALF DIECKMANN ET AL.: "Expression of an active adenylate-forming domain of peptide synthetases corresponding to acyl-CoA-synthetases", FEBS LETTERS, vol. 357, 1995, pages 212 - 216 *
RALF DIECKMANN ET AL.: "Probing the domain structure and ligand-induced conformational changes by limited proteolysis of tyrocidine synthetase 1", J.MOL.BIOL., vol. 288, 1999, pages 129 - 140 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019208700A1 (fr) * 2018-04-26 2019-10-31 ゼリア新薬工業株式会社 Dipeptide et composition pharmaceutique le contenant
JPWO2019208700A1 (ja) * 2018-04-26 2021-04-30 ゼリア新薬工業株式会社 ジペプチド及びこれを含有する医薬組成物

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