WO2022045481A1 - Composition pour la production de glycolaldéhyde à partir de formaldéhyde, et procédé de production d'acide glycolique, d'éthylène glycol ou d'éthanolamine à partir de formaldéhyde à l'aide de la composition - Google Patents

Composition pour la production de glycolaldéhyde à partir de formaldéhyde, et procédé de production d'acide glycolique, d'éthylène glycol ou d'éthanolamine à partir de formaldéhyde à l'aide de la composition Download PDF

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WO2022045481A1
WO2022045481A1 PCT/KR2020/018314 KR2020018314W WO2022045481A1 WO 2022045481 A1 WO2022045481 A1 WO 2022045481A1 KR 2020018314 W KR2020018314 W KR 2020018314W WO 2022045481 A1 WO2022045481 A1 WO 2022045481A1
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gcl
formaldehyde
glycolaldehyde
novel
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김정선
박진병
서필원
김준홍
김지원
조혜진
김채윤
김예나
백윤진
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전남대학교산학협력단
이화여자대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • the present invention relates to a technology for producing glycolaldehyde, glycolic acid, ethylene glycol and ethanolamine, and more specifically, a composition for producing glycolaldehyde from formaldehyde using an enzyme that is a biocatalyst while using formaldehyde as a starting material, and It relates to a method for preparing ethylene glycol glycol or ethanolamine from formaldehyde using the composition.
  • Glycolaldehyde, glycolic acid, ethylene glycol and ethanolamine are used as monomers for bioplastics in the textile industry, as monomers for dyes and tanning agents, as flavoring and preservatives and emulsifiers in the food industry, as skin care agents in the pharmaceutical industry, as detergents in industry and at home. Although it is widely used as an adhesive, etc., toxic substances including strong acids are used during the manufacturing process, and the selectivity and yield of the reaction are not high, causing various problems such as emission of pollutants and use of high energy (Appl Microbiol Biotechnol (2019) 103: 2525-2535).
  • GCL glyoxylate carboligase
  • glycolaldehyde composition including GCL and its derivatives, capable of producing glycolaldehyde from formaldehyde in an environmentally friendly manner.
  • Another object of the present invention is a novel GCL having a sequence altered through genetic manipulation to more effectively produce glycolaldehyde from formaldehyde, a nucleic acid molecule encoding the same, a vector containing the nucleic acid molecule, and transformation containing the vector to provide a body.
  • Another object of the present invention is to obtain high yield of glycolaldehyde, glycolic acid, ethylene glycol and ethanolamine from formaldehyde in an environmentally friendly manner by using a glycolaldehyde composition comprising GCL and its derivatives or novel GCL, transformant, etc.
  • a glycolaldehyde composition comprising GCL and its derivatives or novel GCL, transformant, etc.
  • the object of the present invention is not limited to the object mentioned above, and even if not explicitly mentioned, the object of the invention that can be recognized by a person of ordinary skill in the art from the description of the detailed description of the invention to be described later may also be included. .
  • the present invention provides a composition for producing glycolaldehyde comprising glyoxylate carboligase (GCL) and a derivative thereof.
  • GCL glyoxylate carboligase
  • the GCL and its derivatives catalyze a reaction in which two molecules of formaldehyde are condensed into a molecule of glycolaldehyde.
  • the GCL is derived from E. coli.
  • the K M , k cat and k cat /K M values of the GCL are 62 mM, 1.00 s -1 and 16.0 M -1 ⁇ s -1 , respectively.
  • the present invention also provides a novel glyoxylate carboligase (GCL) comprising the amino acid sequence shown in SEQ ID NO: 1.
  • the novel GCL catalyzes the condensation of two molecules of formaldehyde into glycolaldehyde molecules to produce glycolaldehyde.
  • the K M , k cat and k cat /K M values of the novel GCL are 24 mM, 0.8 s -1 and 33.1 M -1 ⁇ s -1 , respectively.
  • the present invention also provides a nucleic acid molecule having a nucleotide sequence encoding the novel GCL described above.
  • the present invention also provides a vector comprising a nucleic acid molecule encoding the novel GCL described above.
  • the present invention provides a transformant comprising the vector described above.
  • the transformant is Escherichia coli.
  • the E. coli is caveolin-1 (CAV1)-mediated endocytosing E. coli (CAV1-mediated endocytosing E. coli ).
  • the transformant further comprises a vector containing a nucleic acid molecule encoding an aminotransferase.
  • the nucleic acid molecule encoding the aminotransferase is a nucleic acid molecule encoding an aminotransferase ( 3HMU ) derived from Ruegeria pomeroyi or an aminotransferase (Af) derived from Agrobacterium fabrum .
  • -AT is a nucleic acid molecule encoding
  • the present invention also provides a method for preparing glycolaldehyde from formaldehyde comprising the step of contacting formaldehyde with any one of the above-mentioned compositions, any one of the above-mentioned novel GCLs, and at least one of the above-mentioned transformants. .
  • the step is carried out at 30°C to 65°C.
  • the K M , k cat and k cat /K M values of GCL which catalyze the reaction in which two molecules of formaldehyde are condensed into glycolaldehyde molecules in the step, are 62 mM, 1.00 s -1 and 16.0, respectively. M -1 ⁇ s -1 or less.
  • the present invention provides a first step of forming glycolaldehyde by contacting formaldehyde with at least one of the above-mentioned composition, any one of the above-mentioned novel GCL, and the above-mentioned transformant; and a second step of adding ⁇ -ketoglutaric semialdehyde dehydrogenase (KGSADH) or ALDH to the reactant obtained in the first step.
  • KGSADH ⁇ -ketoglutaric semialdehyde dehydrogenase
  • ALDH ⁇ -ketoglutaric semialdehyde dehydrogenase
  • the KGSADH is derived from Azospirillum brasilense, and K M , k cat and k cat /K M values are 4 mM, 24 s -1 and 6.1 mM -1 ⁇ s -1 , respectively.
  • KGSADH when added in the second step, more than 70% of the formaldehyde is converted to glycolic acid.
  • the present invention provides a first step of contacting formaldehyde with any one of the above-mentioned compositions, any one of the above-mentioned novel GCLs, and at least one of the above-mentioned transformants to form glycolaldehyde, and the first step obtained in the first step. It provides a method for preparing ethylene glycol comprising a second step of adding 1,3-propanediol dehydrogenase (DhaT) or ADH to a reactant.
  • DhaT 1,3-propanediol dehydrogenase
  • the DhaT is derived from Klebsiella pneumoniae , and K M , k cat and k cat /K M values are 12.5 mM, 1.6 s -1 and 0.13 mM -1 ⁇ s -1 , respectively.
  • the present invention provides a first step of forming glycolaldehyde by contacting formaldehyde with at least one of the above-mentioned composition, any one of the above-mentioned novel GCL, and the above-mentioned transformant; and a second step of adding an aminotransferase and an amine donor to the reactant obtained in the first step; provides a method for preparing ethanolamine comprising.
  • the aminotransferase is an aminotransferase ( 3HMU ) derived from Ruegeria pomeroyi or an aminotransferase (Af-AT) derived from Agrobacterium fabrum .
  • 3HMU aminotransferase
  • Ad-AT aminotransferase
  • the present invention provides a method for preparing ethanolamine from formaldehyde, comprising the step of contacting formaldehyde with at least one of the aforementioned transformants and an amine donor.
  • glycolaldehyde composition of the present invention described above can produce glycolaldehyde from formaldehyde in an environmentally friendly manner, including GCL and its derivatives.
  • novel GCL of the present invention has a sequence altered through genetic manipulation to more effectively produce glycolaldehyde from formaldehyde, and a nucleic acid molecule encoding it, a vector containing the nucleic acid molecule, and transformation containing the vector body can be provided.
  • the production method of the present invention is one of glycolaldehyde, glycolic acid, ethylene glycol and ethanolamine from formaldehyde in an environmentally friendly way using a glycolaldehyde composition including GCL and its derivatives, or novel GCL, transformant, etc. More than that can be produced in high yield.
  • Figure 1a is a photograph of the result of purifying EcGCL expressed in E. coli as a single band by SDS-PAGE
  • Figure 1b is a Hydrogenobacter thermophilus -derived acetolactate synthase (HtALS), Vibrio vulnificus -derived acetolactate synthase (VvALS), Escherichia coli -derived decarboxylyl- (EcOCD), Deinococcus metallilatus-derived GCL (DmGCL), and E. coli -derived GCL (EcGCL) are graphs comparing the carbonization activity.
  • HtALS Hydrogenobacter thermophilus -derived acetolactate synthase
  • VvALS Vibrio vulnificus -derived acetolactate synthase
  • EcOCD Escherichia coli -derived decarboxylyl-
  • DmGCL Deinococcus metallilatus-derived GCL
  • 3 is a graph showing the thermal stability of E. coli-derived GCL.
  • Figure 4a is a schematic diagram showing the comparison of the structures of FLS and two GCLs
  • Figure 4b is a schematic diagram showing that the observed overall structural features of E. coli GCL are well preserved in the structure of D. metallilatus GCL.
  • 5 is a graph showing the result of whole-cell bioconversion from formaldehyde to glycolaldehyde by recombinant E. coli BL21 Star (DE3) co-expressed with caveolin-1 (Cav1) with GCL.
  • 6a and 6b are graphs of results showing the cascade reaction over time in which formaldehyde is converted to glycolic acid and ethylene glycol by KGSADH and DhaT, respectively.
  • FIG. 7 is a graph showing the results of the cascade reaction over time in which formaldehyde is converted to ethanolamine by GCL and Af-TA.
  • FIG. 8 shows a reaction scheme for preparing glycolic acid, ethylene glycol, and ethanolamine after producing glycolaldehyde from formaldehyde using GCL and its derivatives according to the present invention.
  • Figure 9a shows the reaction scheme of the natural substrate catalyzed by GCL, glyoxylate
  • Figure 9b shows the reaction scheme of the natural substrate, 3-hydroxypropanol, catalyzed by KGSADH and DhaT.
  • first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component.
  • the technical feature of the present invention is the discovery for the first time that a new use of glyoxylate carboligase (GCL), that is, to catalyze the formation of glycolaldehyde from formaldehyde, is environmentally friendly by using GCL and its derivatives. After producing the aldehyde, glycolic acid, ethylene glycol and ethanolamine are prepared.
  • GCL glyoxylate carboligase
  • composition for producing glycolaldehyde of the present invention includes glyoxylate carboligase (GCL) and its derivatives.
  • glyoxylate carboligase is an enzyme that catalyzes the condensation of two molecules of glyoxylate as shown in the following reaction scheme to give 2-hydroxy-3-oxopropanoate (also called tartronic half aldehyde),
  • GCL and its derivatives catalyze the reaction of condensing two molecules of formaldehyde into glycolaldehyde molecules, and using this, a method for producing glycolaldehyde from formaldehyde was developed. am.
  • GCL and its derivatives included in the composition for producing glycolaldehyde of the present invention are not limited as long as they can catalyze the above-described reaction, and in one embodiment, GCL derived from E. coli and its derivatives may be used.
  • GCL glyoxylate carboligase
  • the K M , k cat and k cat /K M values of GCL and its derivatives included in the composition for producing glycolaldehyde of the present invention are 62 mM, 1.00 s -1 and 16.0 M -1 ⁇ s -1 , respectively.
  • novel glyoxylate carboligase (GCL) of the present invention may be composed of the amino acid sequence described in SEQ ID NO: 1 .
  • the novel GCL is multiplied according to a structure-based strategy of neutralizing the charged surface at the entrance moiety to the active site and narrowing the pathway from the surface to the active site to obtain GCL mutants with superior kinetic parameters than the wild-type enzyme, E. coli-derived GCL.
  • This is the amino acid sequence of the mutant showing the best kinetic parameter characteristics among many GCL mutants obtained by performing the experiment of
  • the novel GCL consisting of the amino acid sequence shown in SEQ ID NO: 1 has a sequence in which Arg484 and Asn283 are substituted with Met484 and Gln283, respectively, compared to the wild-type enzyme by point mutation.
  • the novel GCL also catalyzes the condensation of two molecules of formaldehyde into glycolaldehyde molecules to produce glycolaldehyde.
  • the K M , k cat and k cat /K M values of the novel GCL are 24 mM, 0.8 s -1 and Since 33.1 M ⁇ 1 ⁇ s ⁇ 1 , it can be seen that it has about twice as much superior properties as compared to the above-described wild-type GCL.
  • the novel GCL includes functional equivalents and functional derivatives thereof.
  • “Functional equivalent” of the novel GCL as defined in the present invention refers to a protein that exhibits substantially the same physiological activity as the enzyme consisting of the first sequence of the SEQ ID NO: 1.
  • the term "homogeneous physiological activity” has the ability to catalyze the condensation of two molecules of formaldehyde to perform a reaction to convert it into glycolaldehyde, as shown in FIG. 8, and is at least 50% or more, preferably 70%, More preferably, it refers to having an amino acid sequence homology of 90% or more.
  • “functional equivalent” includes amino acid sequence variants in which some or all of the amino acids of the native protein are substituted, or some of the amino acids are deleted or added. Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions for naturally occurring amino acids are; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) ) and sulfur-containing amino acids (Cys, Met).
  • “Functional derivative” refers to a novel fragment of GCL, a protein comprising the fragment, or a protein to which modifications are applied to increase or decrease the physicochemical properties of the protein.
  • fragment refers to an amino acid sequence corresponding to a part of a protein, which has a common element of origin, structure and mechanism of action within the scope of the present invention.
  • Derivatives to which GCL is modified to change stability, storage properties, solubility, etc. are also included in the scope of the present invention. Methods for making functional derivatives of such proteins are known.
  • the nucleic acid molecule having a nucleotide sequence encoding the novel GCL of the present invention is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules. Also includes analogs in which the base region is modified. Accordingly, it preferably has, but is not limited to, a nucleotide sequence encoding the first sequence in SEQ ID NO: 1, as long as it encodes a protein having substantially the same activity, a part of the nucleotide sequence contains a substituted or deleted nucleotide sequence, It can be directly isolated from Thermophilus by a gene cloning method and then chemically synthesized by point mutation or using a DNA synthesizer.
  • the greatest feature of the novel GCL of the present invention is that it has a performance of 24 mM, 0.8 s -1 and 33.1 M -1 ⁇ s -1 of K M , k cat and k cat /K M values, respectively, when performing the reaction. It is clear to those skilled in the art that the novel GCL of the present invention is not limited to the amino acid sequences described in the accompanying sequence listing.
  • the nucleic acid molecule encoding the novel GCL of the present invention can be expressed using any suitable prokaryotic or eukaryotic expression system well known in the art.
  • the vector system of the present invention may be constructed through various methods known in the art, and may be typically constructed as a vector for cloning or a vector for expression.
  • the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. Since the nucleic acid molecule of the present invention is derived from a prokaryotic cell, it is preferable to use a prokaryotic cell as a host in consideration of the convenience of culture and the like.
  • the expression is E. coli, such as E. coli MV1184, E. coli BL21, E. coli JM109(DE3), E. coli NM522, or yeast such as Saccharomyces cerevisiae , Saccharomyces diastati Curs ( Saccharomyces diastaticus ) and the like may be performed, and preferably performed in E. coli BL21.
  • E. coli MV1184 such as E. coli MV1184, E. coli BL21, E. coli JM109(DE3), E. coli NM522, or yeast such as Saccharomyces cerevisiae , Saccharomyces diastati Curs ( Saccharomyces diastaticus ) and the like may be performed, and preferably performed in E. coli BL21.
  • yeast such as Saccharomyces cerevisiae , Saccharomyces diastati Curs ( Saccharomyces diastaticus ) and the like may
  • the novel GCL gene and caveolin-1 (CAV1) gene are inserted into the hexahistidine-labeled expression vector pET-21a derivative, and the new GCL and keveolin-1 are jointly expressed in CAV1-mediated endo
  • E. coli cytosing CAV1-mediated endocytosing E. coli
  • endocytosing E. coli can be an excellent biocatalyst.
  • a transformant containing a vector containing a nucleic acid molecule encoding a novel GCL and a vector containing a nucleic acid molecule encoding an aminotransferase is prepared, and the novel GCL and aminotransferase are jointly expressed as a biocatalyst. It can be used in the preparation of ethanolamine.
  • the nucleic acid molecule encoding the aminotransferase is a nucleic acid molecule encoding an aminotransferase ( 3HMU ) derived from Ruegeria pomeroyi or an aminotransferase (Af-AT) derived from Agrobacterium fabrum . It may be a nucleic acid molecule that
  • the microbial transformant transformed by the vector that is, the host microorganism containing the desired expression vector
  • the vector is cultured under conditions that maximize the expression of the gene to be expressed and at the same time maintain the optimal growth of the microorganism, followed by culturing.
  • Fresh GCL was recovered and purified from water.
  • the method for producing glycolaldehyde from formaldehyde of the present invention comprises at least one of formaldehyde, any one of the above-mentioned compositions for producing glycolaldehyde, any one of the above-mentioned novel GCL, and any one of the above-mentioned transformants.
  • the K M , k cat and k cat /K M values of GCL which catalyze a reaction in which two molecules of formaldehyde are condensed into glycolaldehyde molecules in the step, are 62 mM, respectively. , 1.00 s ⁇ 1 and 16.0 M ⁇ 1 ⁇ s ⁇ 1 or less, and the above step may be performed at 30° C. to 60° C. This is because GCL and its derivatives have excellent thermal stability, as can be seen from the experimental examples to be described later.
  • one or more of the above-mentioned composition for producing glycolaldehyde, any one of the above-mentioned novel GCL, and any one of the above-mentioned transformants is brought into contact with formaldehyde and glycolaldehyde.
  • KGSADH is derived from Azospirillum brasilense
  • K M , k cat and k cat /K M values are 4 mM, 24 s -1 and 6.1 mM -1 ⁇ s -1 , respectively. and, when KGSADH is added in the second step, more than 70% of the formaldehyde is converted to glycolic acid.
  • one or more of the above-mentioned composition for producing glycolaldehyde, any one of the above-mentioned novel GCL, and any one of the above-mentioned transformants is brought into contact with formaldehyde and glycolaldehyde.
  • DhaT 1,3-propanediol dehydrogenase
  • alcohol dehydrogenase eg, E. coli-derived FucO, YqhD
  • DhaT is derived from Klebsiella pneumoniae , and K M , k cat and k cat /K M values are 12.5 mM, 1.6 s -1 and 0.13 mM -1 s -1 , respectively, and when DhaT is added in the second step, More than 70% of the formaldehyde is converted to ethylene glycol.
  • the ethanolamine production method of the present invention is a method for producing ethanolamine by contacting formaldehyde with one or more of the above-mentioned composition for producing glycolaldehyde, any one of the above-mentioned novel GCL, and any one of the above-mentioned transformants to contact glycolaldehyde.
  • amine donor may be determined depending on the type of aminotransferase used.
  • the method for preparing ethanolamine of the present invention comprises at least one of a transformant containing formaldehyde, a vector containing a nucleic acid molecule encoding a novel GCL and a vector containing a nucleic acid molecule encoding an aminotransferase, and contacting the amine donor.
  • a transformant containing both vectors is used as a biocatalyst
  • glycolaldehyde is formed by the new expressed GCL
  • ethanolamine is formed from the amine donor and glycolaldehyde by the expressed aminotransferase.
  • more than 70% of the formaldehyde can be converted to ethanolamine.
  • GCL mutants with better kinetic parameters than the wild-type enzyme
  • a structure-based strategy was employed that neutralizes the charged surface at the entrance moiety to the active site and narrows the pathway from the surface to the active site. Since the two GCLs are highly conserved not only at the amino acid sequence level but also at the structural level, the generation of GCL derivatives by introduction of point mutations was performed with E. coli GCL.
  • the double mutants replacing Arg484 and Asn283 with Met484 and Gln283 were K M , k cat and k cat /K M with 24 mM, 0.8 s -1 and 33.1 M -1 s -1 , respectively. Enzyme activity with a value was shown.
  • a novel GCL consisting of the amino acid sequence described in SEQ ID NO: 1 was completed.
  • the new GCL which is a variant, has about twice the substrate affinity of that of the wild-type enzyme E. coli GCL, it shows about twice the activity for formaldehyde while maintaining the k cat value. It can be seen that it has better properties when producing glycolaldehyde.
  • acetolactate synthase HtALS
  • VvALS acetolactate synthase
  • EcOCA oxalyl-CoA decarboxylase
  • GCL glyoxylatecarboligase
  • Reaction temperature is one of the main parameters of biotransformation. Therefore, the effect of the reaction temperature on the carboligating activity of E. coli GCL was investigated as follows, and the results are shown in FIG. 2 .
  • the enzyme was added at a concentration of 1 mg/mL in a reaction medium containing 25 mM formaldehyde and sufficient amounts of magnesium (II) ions, thiaminepyrophosphate and flavinadenine dinucleotide. Initial bioconversion rates were determined based on product concentration by HPLC.
  • E. coli GCL has carboligating activity even at a temperature of 50° C. or higher was investigated as follows, and the results are shown in FIG. 3 .
  • the wild-type enzyme, GCL showed K M , k cat and k cat /K M values of 62 mM, 1.00 s -1 and 16.0 M -1 s -1 , respectively. , it can be seen that it shows very good reaction kinetics compared to other known glycolaldehyde producing enzymes (eg, GALS (Nat. Commun. (2019) 26:1378)).
  • the novel GCL composed of the amino acid sequence described in SEQ ID NO: 1 showed that K M , k cat and k cat /K M values were 24 mM, 0.8 s -1 and 33.1 M -1 ⁇ s -1 , respectively.
  • GCL can catalyze the condensation reaction of two glyoxylate molecules to produce tartronate semialdehyde and carbon dioxide through successive decarboxylation and reduction reactions (see FIG. 9a ). So far, only one GCL structure has been reported and shares structural features with the typical pyruvate oxidase family of ThDP enzymes (Nat. Chem. Biol. (2008) 15: 900-906). GCL lacks a conserved Glu residue that interacts with the nitrogen atom of the ThDP moiety, but its absence does not interfere with catalysis. By measuring the crystal structure of GCL from D. metallilatus , which has much less catalytic activity for formaldehyde condensation than E. coli GCL, the structures of FLS and E. coli and D. metallilatus GCL were compared, and schematic diagrams thereof are shown in FIGS. 4A and 4B.
  • E. coli GCL As shown in Fig. 4a, the observed overall structural features of E. coli GCL were D. It is well conserved in the metallilatus GCL structure (mean-squared deviation of 0.805 ⁇ out of 526 aligned residues). The ⁇ -helix (Ala474-Leu494 in E. coli GCL ) provides a residue for interaction with ThDP and magnesium ions in E. coli GCL, presumably due to molecular packing for crystallization and activation of the D. metallilatus GCL structure. It is displaced from the site to a completely different space. These structural properties deplete two cofactors and magnesium ions at the active site of D. metallilatus GCL.
  • a comparison of FLS and two GCL structures reveals changes in the active site with significant differences in regions extending from the intrinsic C-terminus of the three enzymes (C-helix). This C-terminal region contributes to the formation of the active sites of the GCL and FLS enzymes.
  • the spatial orientation between the respective enzyme dipolymers of the three enzymes is the same as EcGCL and DmGCL, but has a large difference from FLS, which is why GCL and FLS show differences and correlations in enzymatic activity is not clear
  • the two GCLs require another cofactor FADH2 for the reduction of the second intermediate.
  • GCL accurately reflects the structural features of ThDP-dependent carboligase enzymes. Therefore, it can be seen that the catalytic mechanism for condensing two formaldehyde molecules of GCL tends to condense formaldehyde molecules of benzaldehyde lyase (FEBS J 272 (23): 6067-6076).
  • GCL can be used in the construction of synthetic methyl feeder cell factories.
  • a GCL-based whole-cell biotransformation system was investigated as follows and the results are shown in FIG. 5 .
  • the first method was to simply overexpress GCL in a soluble form in E. coli BL21 Star (DE3).
  • Another approach has been to use endocytosing E. coli as a biocatalyst that has been reported to be beneficial for biotransformation of toxic and/or hydrophobic reactive substrates (eg fatty acids). That is, for whole-cell bioconversion of formaldehyde to glycolaldehyde by recombinant E.
  • glycolaldehyde is similar to 3-hydroxy propanol.
  • ⁇ -ketoglutaric acid semialdehyde dehydrogenase KGSADH
  • Azospirillum brasilense capable of oxidizing 3-hydroxy propanol to 3-hydroxy propionic acid (KGSADH).
  • DhaT 1,3-propanediol dehydrogenase
  • a cascade reaction of formaldehyde to ethylene glycol via glycolaldehyde was carried out by adding 25 mM formaldehyde to the reaction by GCL and DhaT expressed in E. coli and recovered by affinity chromatography on a Ni-NTA gel matrix. . Since the DhaT enzyme also has a reducing activity for formaldehyde, DhaT was added 1 hour after the initiation of bioconversion by GCL.
  • KGSADH shows K M , k cat and k cat /K M values of 4 mM, 24 s -1 and 6.1 mM -1 s -1 for glycolaldehyde, respectively, so that the catalytic efficiency is GCL for formaldehyde. It can be seen that it is substantially larger than the DhaT exhibited K M , k cat and k cat / K M values of 12.5 mM, 1.6 s -1 and 0.13 mM -1 ⁇ s -1 for glycolaldehyde, respectively.
  • GCL and its derivatives can produce glycolaldehyde from formaldehyde by selectively condensing two molecules of formaldehyde with one molecule of glycolaldehyde, and also dehydrogenation of ⁇ -ketoglutarate semialdehyde from A. brasilense .
  • Enzyme (KGSADH) and coupling with 1,3-propanediol dehydrogenase (DhaT) of K. pneumoniae can be used continuously for two-step biotransformation of formaldehyde via glycolaldehyde to glycolic acid and ethylene glycol.
  • glycolic acid and ethylene glycol can be enzymatically synthesized from formaldehyde through glycolaldehyde in an environmentally friendly manner.
  • aminotransferase (Af-AT) from Agrobacterium fabrum ATU3300 and 3HMU from Ruegeria pomeroyi were selected as candidate enzymes.
  • an E. coli transformant co-expressing GCL and 3HMU can be used instead of the recombinant E. coli BL21 Star (DE3) co-expressing Af-AT with GCL.
  • E. coli transformant was used as a biocatalyst, ethanolamine was accumulated up to 8.1 mM.
  • the first step performed by adding formaldehyde to the E. coli transformant expressing only GCL and the second step adding aminotransferase, that is, Af-AT or 3HMU and an amine donor to the reaction product obtained in the first step Even if the steps are performed, more than 70% of the formaldehyde can be converted to ethanolamine.

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Abstract

La présente invention concerne la technologie de production de glycoaldéhyde, d'acide glycolique, d'éthylène glycol et d'éthanolamine, et plus particulièrement un composé pour produire du glycoaldéhyde à partir de formaldéhyde en utilisant une enzyme en tant que biocatalyseur et en utilisant le formaldéhyde comme matière première, et un procédé pour produire de l'acide glycolique, de l'éthylène glycol et de l'éthanolamine, à partir de formaldéhyde, en utilisant la composition.
PCT/KR2020/018314 2020-08-26 2020-12-15 Composition pour la production de glycolaldéhyde à partir de formaldéhyde, et procédé de production d'acide glycolique, d'éthylène glycol ou d'éthanolamine à partir de formaldéhyde à l'aide de la composition WO2022045481A1 (fr)

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Citations (2)

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KR20130093481A (ko) * 2010-04-13 2013-08-22 게노마티카 인코포레이티드 에틸렌 글리콜을 생산하기 위한 미생물 및 방법
WO2014004625A1 (fr) * 2012-06-26 2014-01-03 Genomatica, Inc. Microorganismes destinés à la production d'éthylèneglycol utilisant un gaz de synthèse

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KR102003374B1 (ko) 2017-05-17 2019-07-24 명지대학교 산학협력단 자일로스로부터 글리콜산의 생산능을 갖는 대장균, 이의 제조방법 및 이를 이용하여 글리콜산을 생산하는 방법

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KR20130093481A (ko) * 2010-04-13 2013-08-22 게노마티카 인코포레이티드 에틸렌 글리콜을 생산하기 위한 미생물 및 방법
WO2014004625A1 (fr) * 2012-06-26 2014-01-03 Genomatica, Inc. Microorganismes destinés à la production d'éthylèneglycol utilisant un gaz de synthèse

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FRANDEN MARY ANN; JAYAKODY LAHIRU N.; LI WING-JIN; WAGNER NEIL J.; CLEVELAND NICHOLAS S.; MICHENER WILLIAM E.; HAUER BERNHARD; BLA: "Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization", METABOLIC ENGINEERING, vol. 48, 7 June 2018 (2018-06-07), AMSTERDAM, NL, pages 197 - 207, XP085977744, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2018.06.003 *
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