KR102349819B1 - Method for production of glycolic acid or ethylene glycol from formaldehyde by the glycolaldehyde converted from formaldehyde using glyoxylate carboligase and its derivatives - Google Patents

Abstract

The present invention relates to a technology for producing glycolaldehyde, glycolic acid and ethylene glycol and, more specifically, to a composition for producing glycolaldehyde from formaldehyde by using formaldehyde as a starting material and an enzyme, which is a biocatalyst, and a method for preparing glycolic acid or ethylene glycol from formaldehyde using the composition.

Description

A composition for producing glycolaldehyde from formaldehyde and a method for producing glycolic acid or ethylene glycol from formaldehyde using the composition

The present invention relates to a technology for producing glycolaldehyde, glycolic acid and ethylene glycol, and more particularly, a composition for producing glycolaldehyde from formaldehyde using formaldehyde as a starting material and an enzyme as a biocatalyst, and the composition It relates to a method for preparing glycolic acid or ethylene glycol from the used formaldehyde.

Glycolaldehyde, glycolic acid and ethylene glycol are widely used as monomers for bioplastics in the textile industry, monomers for dyes and tanning agents, flavoring agents and preservatives in the food industry, skin care agents in the pharmaceutical industry, and detergents and adhesives in industry and home. However, 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). As an alternative, a bio-process for biosynthesis from biomass such as pentose through multi-step enzymatic reaction is being researched and developed, but it has problems such as low yield and generation of by-products (Appl Microbiol Biotechnol (2019) 103:2525-2535).

As another method, biosynthesis studies using enzymes from formaldehyde have been reported (Nature Chemical Biology (2019) 15:900-906), but it requires formyl-coenzyme A (CoA) as a substrate, making it economical. It is judged that high-yield production is difficult.

Therefore, it is necessary to develop a new enzymatic reaction system that produces high-value-added materials such as glycolaldehyde, glycolic acid and ethylene glycol in high yield from compounds that can be supplied in large quantities, such as formaldehyde (ACS Catal. 2019, 9, 9575-99588). is becoming

Republic of Korea Patent Publication No. 10-2018-0126322

The present inventors have completed the present invention by developing a new use of glyoxylate carboligase (GCL), which catalyzes the formation of glycolaldehyde from formaldehyde as a result of numerous studies, and a technology for producing glycolic acid and ethylene glycol using the same.

Accordingly, it is an object of the present invention to provide a 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 produce glycolaldehyde, glycolic acid and ethylene glycol in high yield from formaldehyde in an environmentally friendly manner using a glycolaldehyde composition comprising GCL and its derivatives, or novel GCL, transformant, etc. To provide a manufacturing method.

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. .

In order to achieve the above object of the present invention, the present invention provides a composition for producing glycolaldehyde comprising glyoxylate carboligase (GCL) and a derivative thereof.

In a preferred embodiment, the GCL and its derivatives catalyze a reaction in which two molecules of formaldehyde are condensed into a molecule of glycolaldehyde.

In a preferred embodiment, the GCL is derived from E. coli.

In a preferred embodiment, the K _M , k _cat and k _cat /K _M values of the GCL are 58 mM, 0.63 s ^-1 and 11 M ^-1 o s ^-1, respectively.

The present invention also provides a novel glyoxylate carboligase (GCL) composed of the amino acid sequence set forth in SEQ ID NO: 1.

In a preferred embodiment, the novel GCL catalyzes the condensation of two molecules of formaldehyde into glycolaldehyde molecules to produce glycolaldehyde.

In a preferred embodiment, the K _M , k _cat and k _cat /K _M values of the novel GCL are 22 mM, 0.5 s ^-1 and 23 M ^-1 os ^-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.

In addition, the present invention provides a transformant comprising the vector described above.

In a preferred embodiment, the transformant is Escherichia coli.

In a preferred embodiment, the E. coli is caveolin-1 (CAV1)-mediated endocytosing E. coli (CAV1-mediated endocytosing E. coli ).

The present invention also provides a method for preparing glycolaldehyde from formaldehyde comprising contacting formaldehyde with any one of the above-mentioned compositions, any one of the above-mentioned novel GCLs, and one or more of the above-mentioned transformants.

In a preferred embodiment, the step is carried out at 30°C to 65°C.

_{In a preferred embodiment, 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 above step, are 58 mM, 0.63 s ^-1 and 11, respectively. M ^-1 ㅇs ^-1 or less.

In addition, the present invention provides a first step of forming glycolaldehyde by 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; and a second step of adding α-ketoglutaric semialdehyde dehydrogenase (KGSADH) or ALDH to the reactant obtained in the first step.

In a preferred embodiment, the KGSADH is derived from Azospirillum brasilense , and the K _M , k _cat and k _cat /K _M values are 4 mM, 24 s ^-1 and 6.1 mM ^-1 os ^-1, respectively.

In a preferred embodiment, when KGSADH is added in the second step, more than 70% of the formaldehyde is converted to glycolic acid.

In addition, the present invention relates to a first step of contacting formaldehyde with any one of the compositions described above, any one of the novel GCLs described above, and one or more of the aforementioned transformants to form glycolaldehyde, and the reactant 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 the ethylene glycol.

In a preferred embodiment, 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 os ^-1, respectively.

In a preferred embodiment, when DhaT is added in the second step, 70% or more of the formaldehyde is converted to ethylene glycol.

The glycolaldehyde composition of the present invention described above can produce glycolaldehyde from formaldehyde in an environmentally friendly manner, including GCL and its derivatives.

In addition, the 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.

In addition, the production method of the present invention can produce glycolaldehyde, glycolic acid and ethylene glycol in high yield from formaldehyde in an environmentally friendly manner using a glycolaldehyde composition including GCL and its derivatives, or novel GCL, transformant, etc. can

These technical effects of the present invention are not limited only to the above-mentioned range, and even if not explicitly mentioned, the effects of the invention that can be recognized by those of ordinary skill in the art from the description of the specific content for the implementation of the invention to be described later of course included

1a is a photograph of the result of purification of EcGCL expressed in E. coli as a single band by SDS-PAGE, and FIG. 1b is Hydrogenobacter thermophilus- derived acetolactate synthase (HtALS), Vibrio vulnificus- derived acetolactate synthase (VvALS), Escherichia coli- derived oxalyl- (EcOCA), Deinococcus metallilatus-derived GCL (DmGCL), and E. coli- derived GCL (EcGCL) are graphs comparing the carbonization activity.
2 is a graph showing the effect of reaction temperature on the carbonization action of EcGCL.
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, and 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 results 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.
7 shows a reaction scheme for producing glycolic acid and ethylene glycol after producing glycolaldehyde from formaldehyde using GCL and its derivatives according to the present invention.
Figure 8a shows the reaction scheme of glyoxylate, a natural substrate catalyzed by GCL, and Figure 8b shows the scheme of the natural substrate, 3-hydroxypropanol, catalyzed by KGSADH and DhaT.

The terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the description of the invention exists, but is not limited to one or more other It is to be understood that this does not preclude the possibility of addition or presence of features or numbers, steps, operations, components, parts, or combinations thereof.

Terms such as 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.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present invention, it should not be interpreted in an ideal or excessively formal meaning. does not

In interpreting the components, it is construed as including an error range even if there is no separate explicit description. In particular, when the terms "about", "substantially", etc. of degree are used, they may be construed as being used in a sense at or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented. .

In the case of a description of a temporal relationship, for example, 'immediately' or 'directly' when a temporal precedence is described as 'after', 'following', 'after', 'before', etc. This includes cases that are not continuous unless this is used.

Hereinafter, the technical configuration of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Like reference numbers used to describe the invention throughout the specification refer to like elements.

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. It is to produce glycolic acid and ethylene glycol after generating the aldehyde.

Accordingly, the composition for producing glycolaldehyde of the present invention includes glyoxylate carboligase (GCL) and its derivatives.

That is, glyoxylate carboligase is an enzyme that catalyzes the condensation of two molecules of glyoxylate as shown in the following reaction scheme to provide 2-hydroxy-3-oxopropanoate (also called tartronic half aldehyde),

It can be obtained from various microorganisms. For the first time in the present invention, it was revealed that GCL and its derivatives catalyze the reaction of condensing two molecules of formaldehyde into glycolaldehyde molecules. to be.

Here, 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. In particular, as described below, it was found that the wild-type enzyme glyoxylate carboligase (GCL) obtained from E. coli K-12 produces the largest amount of glycolaldehyde from formaldehyde.

_{In addition, 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 58 mM, 0.63 s ^-1 and 11 M ^-1 o s ^-1, respectively.

Next, the 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 22 mM, 0.5 s ^-1 and Since 23 M ^-1 ㅇs ^-1 , it can be seen that it has about twice as much excellent properties as compared to the above-described wild-type GCL.

Here, 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" refers to the ability to catalyze the condensation of two molecules of formaldehyde to perform a reaction to convert it to glycolaldehyde, as shown in FIG. 7, at least 50% or more, preferably 70%, More preferably, it refers to having an amino acid sequence homology of 90% or more.

Also, "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. The term "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. Therefore, it preferably has, but is not limited to, the 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.

Therefore, the greatest feature of the new GCL of the present invention is carried out during the reaction K _M, k _cat and k _cat / K _M value is 22 mM, respectively 0.5 s ^-1 and 23 s ^-1 M ^-1 o has the ability to perform 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. As such, 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. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. The nucleic acid molecule of the present invention is derived from a prokaryotic cell, and it is preferable to use a prokaryotic cell as a host in consideration of the convenience of culture and the like.

In particular, expression of E. coli, for example 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. A number of suitable vectors that can be used for expression in E. coli and yeast are known, and in the present invention, a novel GCL gene was inserted into a hexahistidine-labeled expression vector pET-21a derivative and used.

In addition, in the present invention, 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 by using Saito Singh E. coli (CAV1-mediated endocytosing E. coli) , it was confirmed that the endo Saito Singh E. coli can be a excellent biocatalyst.

In the present invention, as described below, the microbial transformant transformed by the vector, that is, the host microorganism containing the desired expression 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.

Next, 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. _{In one embodiment, 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 58 mM, respectively. , 0.63 s ⁻¹ and 11 M ⁻¹ o 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.

Next, in the method for producing glycolic acid of the present invention, 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. In the first step of forming α-ketoglutaric semialdehyde dehydrogenase (KGSADH) or Aldehyde dehydrogenase (eg, AldA from E. coli) or Aldehyde oxidase (eg, Aldehyde oxidase from Gluconobacter oxydan s) to the reactant obtained in the first step, the second step of adding In one embodiment, 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 o s ^{-1, respectively.} and, when KGSADH is added in the second step, more than 70% of the formaldehyde is converted to glycolic acid.

Next, in the ethylene glycol production method of the present invention, 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. A first step of forming a; and a second step of adding 1,3-propanediol dehydrogenase (DhaT), alcohol dehydrogenase, or glycolaldehyde reductase (eg, E. coli-derived FucO, YqhD) to the reactant obtained in the first step. 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.

Example 1

Two GCLs were selected through the following carboligase selection strategy.

7, in order to find a candidate enzyme for ligating two molecules of formaldehyde to one molecule of glycolaldehyde (Scheme 1), DALI server (http://www.http://ekhidna2.biocenter.helsinki) Structural similarity search using synthetic formaldehyde-converted FLS (PDB ID 4qq8) as reference structures for proteins listed in .fi/dali/) and Protein 3D Structure Data Bank (http://www.rcsb.org) was performed.

This search resulted in several structurally related enzymes (Z score greater than 30 and less than or equal to 3 Å), including acetolactate synthase (ALS), oxalyl-CoA decarboxylase (OCD), and glyoxylate carboligase (GCL). approximate squared deviation). Most of these enzymes require other cofactors for their reported catalytic activity, although some enzymes (such as FAD) require other cofactors, as shown in Table 1 below, which shows the results of the relative activity of carboligase to formaldehyde and acetaldehyde. ThDP dependent. Considering the microscopic structural features of the active site and substrate specificity, two ALSs from Hydrogenobacter thermophilus and Vibrio vulnificus , and two GCLs from E. coli and Deinococcus metallilatus , respectively, were selected.

Name Source Activity for formaldehyde Activity for acetaldehyde note EcsucA E. coli - ++ the present invention TtMend T. terrenum - - the present invention MbsucA M. bovis - + the present invention VvsucA V. vulnificus - +++ the present invention HtALS H. thermophilus + - the present invention VvALS V. vulnificus + - the present invention TtTKS T. thermophilus - - the present invention HtTKS H. thermophilus - - the present invention ECOCD E. coli + - the present invention DmGCL D. metallilatus ++ - the present invention EcGCL E. coli ++++ - the present invention EcGCL _R484M/N283Q E. coli ++++++ - the present invention GALS P. putida +++ Nat. Commun. (2019)*

* Nat. Commun. (2019) 26:1378

Example 2

To obtain 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.

Among the several derivatives construction, double mutant replacing the Arg484 and Asn283 to Met484 and Gln283 are each 22 mM, 0.5 s ^-1 and 23 s ^-1 M ^-1 o of K _M, k _cat and k _cat / K _M Enzyme activity with a value was shown. By selecting the double mutant, a novel GCL consisting of the amino acid sequence described in SEQ ID NO: 1 was completed.

Since 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.

Experimental Example 1

To estimate the catalytic activity of a candidate enzyme for ligating two molecules of formaldehyde to one molecule of glycolaldehyde, the enzyme was prepared by expression in E. coli and purified by affinity chromatography on a Ni-NTA gel matrix, and the results are plotted. 1a, acetolactate synthase (HtALS) from Hydrogenobacter thermophilus , acetolactate synthase (VvALS) from Vibrio vulnificus , oxalyl-CoA decarboxylase (EcOCA) from Escherichia coli , GCL from Deinococcus metallilatus (DmGCL) and E. coli- derived GCL (EcGCL) to compare the carbon-carbon linkage (carbonization) activity of the enzyme was added to the reaction medium at 1 mg / mL, the results are shown in Figure 1b. Initial bioconversion rates were determined based on product concentrations identified on HPLC.

As shown in Figure 1a, most enzymes were expressed in soluble form in E. coli and purified as a single band by SDS-PAGE. Among the enzymes tested as shown in Fig. 1b, glyoxylatecarbolligase (GCL) of E. coli K-12 produced the highest amount of glycolaldehyde from formaldehyde 10 hours after biotransformation, and GCL was It was shown to have the strongest activity in the carboligation of formaldehyde (carbon-carbon linkage).

Experimental Example 2

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.

As shown in Figure 2, the specific glycolaldehyde formation rate and final product concentration increased with the reaction temperature of 30 to 50 ℃. This result indicates that the enzyme has significant thermostability and the optimum reaction temperature can exceed 50°C.

Experimental Example 3

Whether 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 results of Figure 3 show that the enzyme was dissolved in a reaction medium of 50 mM potassium phosphate buffer (pH 6) containing 25 mM formaldehyde and sufficient amounts of magnesium (II) ions, thiaminpyrophosphate and flavinadenine dinucleotide at 1 mg/kg. This is the result obtained by maintaining the temperature from 30°C to 60°C after adding at a concentration of mL.

As described above, the results of FIG. 3 show that the GCL and its derivatives of the present invention maintain activity even at 50° C.

Experimental Example 4

To understand the reaction properties of GCL to ligation of formaldehyde, the reaction kinetics were investigated and the results are presented in Table 2.

^* Data were adapted from a previous study (Nat. Commun. (2019) 26:1378).

As shown in Table 2, the steady-state dynamics in wild-type enzyme, GCL study showed respectively 58 mM, 0.63 M ^-1 s ^-1 and 11 o K _M, k _cat and k _cat / K _M values in s ^-1 , 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)). In addition, 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 22 mM, 0.5 s ^-1 and 23 M ^-1 os ^-1 , respectively.

This is a very important feature because formaldehyde is highly toxic to both enzymes and microbial cells. Moreover, formaldehyde can be converted to formic acid by background enzymes when microbial cells are used as biocatalysts. These results show that GCL and its derivatives can be used as novel and promising carboligases for the production of glycolaldehyde from formaldehyde in high conversion yields.

Experimental Example 4

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. 8a). 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.

The entire structural feature observed in the E. coli GCL as shown in Figure 4a is D. It is well conserved in the metallilatus GCL structure (mean-squared deviation of 0.805 ㅕ out of 526 aligned residues). α- helix (E. coli Ala474-Leu494 of GCL) provides a residue for interaction with the E. coli of the GCL ThDP and magnesium ions, which is probably due to the molecular packing for crystallization D. metallilatus activity of GCL structure It is separated from the site into a completely different space. These structural properties deplete two cofactors and magnesium ions at the active site of D. metallilatus GCL.

As shown in Fig. 4B, 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. In addition, as shown in FIG. 4B , 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 In particular, the two GCLs require another cofactor FADH2 for the reduction of the second intermediate. Nevertheless, 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).

Experimental Example 5

As demonstrated recently with GALS, GCL can be used in the construction of synthetic methyl feeder cell factories. Thereby, 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. coli BL21 Star (DE3) co-expressed with caveolin-1 (Cav1) with GCL, 25 mM formaldehyde was added to potassium phosphate buffer (pH 6.0). ) to initiate biotransformation in the quiescent growth phase (cell density: 12 g dry cells/L).

As shown in Fig. 5, when E. coli BL21 Star (DE3) pET-EcGCL was used as a biocatalyst, only 6 mM glycolaldehyde was produced from 25 mM formaldehyde and the conversion yield was maintained below 50%. However, surprisingly, co-expression of caveolin-1 (CAV1) with EcGCL in E. coli BL21 Star (DE3) resulted in CAV1-mediated endocytosis of E. coli producing about 8.4 mM glycolaldehyde with a conversion rate of 67%. made to do In addition, the formaldehyde condensation rate reached 7.5 mM/h, and these experimental results showed that endocytosing E. coli would be an excellent biocatalyst.

Example 3

The chemical structure of glycolaldehyde is similar to 3-hydroxy propanol. Thereby, as shown in Figure 8b, α-ketoglutaric acid semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense capable of oxidizing 3-hydroxy propanol to 3-hydroxy propionic acid ( KGSADH). ) was selected as a candidate.

Thus, a cascade reaction of formaldehyde to glycolic acid via glycolaldehyde was carried out by adding 25 mM formaldehyde to the reaction with GCL and KGSADH expressed in E. coli and recovered by affinity chromatography on Ni-NTA gel matrix. . KGSADH was added after bioconversion by GCL for 1 h because the enzyme also has oxidative activity for formaldehyde.

As shown in Figure 6a, the condensation of formaldehyde to glycolaldehyde by EcGCL resulted in the formation of glycolaldehyde to 9 mM at t = 1 h.

This addition of KGSADH to the reaction medium resulted in an accumulation of glycolic acid up to 8.8 mM, indicating that 70% of the formaldehyde was converted to the target product.

Example 4

1,3-propanediol dehydrogenase (DhaT) from Klebsi ella pneumoniae, known to reduce 3-hydroxypropanol to 1,3-propanediol, was selected as a candidate for generating ethylene glycol from glycolaldehyde.

Thus, 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.

As shown in Fig. 6b, when DhaT was added to the reaction medium, ethylene glycol was accumulated to 8.8 mM, indicating that 70% of the formaldehyde was converted to the target product.

Experimental Example 6

The reaction kinetics of KGSADH and DhaT used in Examples 3 and 4 to formaldehyde and glycolaldehyde were investigated, and the results are shown in Table 3.

From Table 3, respectively KGSADH 4 mM for the glycol aldehyde, 24 s ^-1 and 6.1 s ^-1 mM ^-1 o of K _M, k _cat and k _cat / K _M value represented by the catalyst efficiency for the GCL formaldehyde It can be seen that it is substantially larger than the DhaT exhibited respectively 12.5 mM, 1.6 s ^-1, and 0.13 mM ^-1 o K _M, k _cat and k _cat / K _M values in s ^-1 for the glycol aldehyde.

The above experimental results show that 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 α-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 through glycolaldehyde to glycolic acid and ethylene glycol. show Therefore, according to the present invention, glycolic acid and ethylene glycol can be enzymatically synthesized from formaldehyde through glycolaldehyde in an environmentally friendly manner.

As described above, the present invention has been illustrated and described with reference to preferred embodiments, but it is not limited to the above-described embodiments, and within the scope of not departing from the spirit of the present invention, those of ordinary skill in the art to which the present invention pertains Various changes and modifications will be possible.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Method for production of glycolic acid or ethylene glycol from formaldehyde by the glycolaldehyde converted from formaldehyde using glyoxylate carboligase and its derivatives <130> DPP-2020-0100-KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 623 <212> PRT <213> Artificial Sequence <220> <223> Point mutation sequence of glyoxylate carboligase <400> 1 Met Ser Val Ala Ser Arg Ser Ala Glu Arg Val Phe Leu Thr Lys Phe 1 5 10 15 Glu Ser Trp Lys Asn Phe Pro Ile Asn Arg Gly Arg Asn Lys Met Ala 20 25 30 Lys Met Arg Ala Val Asp Ala Ala Met Tyr Val Leu Glu Lys Glu Gly 35 40 45 Ile Thr Thr Ala Phe Gly Val Pro Gly Ala Ala Ile Asn Pro Phe Tyr 50 55 60 Ser Ala Met Arg Lys His Gly Gly Ile Arg His Ile Leu Ala Arg His 65 70 75 80 Val Glu Gly Ala Ser His Met Ala Glu Gly Tyr Thr Arg Ala Thr Ala 85 90 95 Gly Asn Ile Gly Val Cys Leu Gly Thr Ser Gly Pro Ala Gly Thr Asp 100 105 110 Met Ile Thr Ala Leu Tyr Ser Ala Ser Ala Asp Ser Ile Pro Ile Leu 115 120 125 Cys Ile Thr Gly Gln Ala Pro Arg Ala Arg Leu His Lys Glu Asp Phe 130 135 140 Gln Ala Val Asp Ile Glu Ala Ile Ala Lys Pro Val Ser Lys Met Ala 145 150 155 160 Val Thr Val Arg Glu Ala Ala Leu Val Pro Arg Val Leu Gln Gln Ala 165 170 175 Phe His Leu Met Arg Ser Gly Arg Pro Gly Pro Val Leu Val Asp Leu 180 185 190 Pro Phe Asp Val Gln Val Ala Glu Ile Glu Phe Asp Pro Asp Met Tyr 195 200 205 Glu Pro Leu Pro Val Tyr Lys Pro Ala Ala Ser Arg Met Gln Ile Glu 210 215 220 Lys Ala Val Glu Met Leu Ile Gln Ala Glu Arg Pro Val Ile Val Ala 225 230 235 240 Gly Gly Gly Val Ile Asn Ala Asp Ala Ala Ala Leu Leu Gln Gln Phe 245 250 255 Ala Glu Leu Thr Ser Val Pro Val Ile Pro Thr Leu Met Gly Trp Gly 260 265 270 Cys Ile Pro Asp Asp His Glu Leu Met Ala Gly Met Val Gly Leu Gln 275 280 285 Thr Ala His Arg Tyr Gly Asn Ala Thr Leu Leu Ala Ser Asp Met Val 290 295 300 Phe Gly Ile Gly Asn Arg Phe Ala Gln Arg His Thr Gly Ser Val Glu 305 310 315 320 Lys Tyr Thr Glu Gly Arg Lys Ile Val His Ile Asp Ile Glu Pro Thr 325 330 335 Gln Ile Gly Arg Val Leu Cys Pro Asp Leu Gly Ile Val Ser Asp Ala 340 345 350 Lys Ala Ala Leu Thr Leu Leu Val Glu Val Ala Gln Glu Met Gln Lys 355 360 365 Ala Gly Arg Leu Pro Cys Arg Lys Glu Trp Val Ala Asp Cys Gln Gln 370 375 380 Arg Lys Arg Thr Leu Leu Arg Lys Thr His Phe Asp Asn Val Pro Val 385 390 395 400 Lys Pro Gln Arg Val Tyr Glu Glu Met Asn Lys Ala Phe Gly Arg Asp 405 410 415 Val Cys Tyr Val Thr Thr Ile Gly Leu Ser Gln Ile Ala Ala Ala Gln 420 425 430 Met Leu His Val Phe Lys Asp Arg His Trp Ile Asn Cys Gly Gln Ala 435 440 445 Gly Pro Leu Gly Trp Thr Ile Pro Ala Ala Leu Gly Val Cys Ala Ala 450 455 460 Asp Pro Lys Arg Asn Val Val Ala Ile Ser Gly Asp Phe Asp Phe Gln 465 470 475 480 Phe Leu Ile Glu Glu Leu Ala Val Gly Ala Gln Phe Asn Ile Pro Tyr 485 490 495 Ile His Val Leu Val Asn Asn Ala Tyr Leu Gly Leu Ile Arg Gln Ser 500 505 510 Gln Met Ala Phe Asp Met Asp Tyr Cys Val Gln Leu Ala Phe Glu Asn 515 520 525 Ile Asn Ser Ser Glu Val Asn Gly Tyr Gly Val Asp His Val Lys Val 530 535 540 Ala Glu Gly Leu Gly Cys Lys Ala Ile Arg Val Phe Lys Pro Glu Asp 545 550 555 560 Ile Ala Pro Ala Phe Glu Gln Ala Lys Ala Leu Met Ala Gln Tyr Arg 565 570 575 Val Pro Val Val Val Glu Val Ile Leu Glu Arg Val Thr Asn Ile Ser 580 585 590 Met Gly Ser Glu Leu Asp Asn Val Met Glu Phe Glu Asp Ile Ala Asp 595 600 605 Asn Ala Ala Asp Ala Pro Thr Glu Thr Cys Phe Met His Tyr Glu 610 615 620

Claims (21)

delete delete delete delete A novel glyoxylate carboligase (GCL) comprising the amino acid sequence shown in SEQ ID NO: 1 .
6. The method of claim 5,
The novel GCL is a novel glyoxylate carboligase, characterized in that it catalyzes the condensation of two molecules of formaldehyde into glycolaldehyde molecules to produce glycolaldehyde.
7. The method of claim 6,
Novel glyoxylate carboligase, characterized in that _{the K M} , k _cat and k _cat /K _M values of the novel GCL are 22 mM, 0.5 s ^-1 and 23 M ^-1 os ^{-1, respectively.}
A nucleic acid molecule having a nucleotide sequence encoding the novel GCL of claim 5.
A vector comprising a nucleic acid molecule encoding the novel GCL of claim 8.
delete delete delete A process for preparing glycolaldehyde from formaldehyde comprising the step of contacting formaldehyde with at least one of the novel GCLs of any one of claims 5 to 7.
14. The method of claim 13,
The step is a glycolaldehyde production method, characterized in that carried out at 30 ℃ to 60 ℃.
15. The method of claim 14,
_{In the above step, 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, are 58 mM, 0.63 s ^-1 and 11 M ^-1 s ^{− A} method for producing glycolaldehyde, characterized in that 1 or less.
A first step of contacting formaldehyde with at least one of the novel GCLs of any one of claims 5 to 7 to form glycolaldehyde; and
A method for producing glycolic acid comprising a; a second step of adding α-ketoglutaric semialdehyde dehydrogenase (KGSADH) or ALDH to the reactant obtained in the first step.
delete 17. The method of claim 16,
When KGSADH is added in the second step, more than 70% of the formaldehyde is converted to glycolic acid.
A first step of contacting formaldehyde with at least one of the novel GCLs of any one of claims 5 to 7 to form glycolaldehyde; and
A method for producing ethylene glycol comprising a; a second step of adding 1,3-propanediol dehydrogenase (DhaT) or ADH to the reactant obtained in the first step.
delete 20. The method of claim 19,
When DhaT is added in the second step, 70% or more of the formaldehyde is converted to ethylene glycol.

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