WO2019124782A2 - Method for producing acetoin, butanediol, or butanol from ethanol - Google Patents

Abstract

In a method for producing acetoin, butanediol, or butanol from ethanol according to the present invention, a cell-free catalysis method was used by designing an artificial synthetic pathway so that proteins of NOX, EtDH, FLS, BDH, and DDH and mutant proteins thereof exhibit multi-step catalytic activity as enzymes. Compared to existing fermentation methods using microorganisms, the production method according to the present invention does not require cell growth and has a short synthetic pathway, a fast reaction rate, high yield and productivity, adjustment of targeted reaction conditions is convenient, and butanol can be effectively produced. Moreover, same can be reused numerous times by fixing the proteins to nanoparticles, and are also effective for producing acetoin, butanediol, or butanol, thus being economical. Therefore, the production method may be usefully adopted in the relevant industries requiring acetoin, butanediol, or butanol.

Description

Production method of acetone, butanediol or butanol in ethanol

The present invention relates to a process for the production of acetone, butanediol or butanol in ethanol and its various applications.

Although interest in bioethanol, biodiesel, biogas, and butanol represented by bioenergy is increasing, all of the types of bioenergy mentioned above can be used as a fuel for power generation or transportation, but some disadvantages of practical application and production methods There is growing interest in hydrocarbon-based compounds, new renewable energy sources. Acetone, butanediol or butanol is very useful as an intermediate compound having a wide range of applications such as cosmetics, perfume, hormone, hygiene agent, industrial coating agent, paint additive, fiber, plastic monomer, medical supplies, vitamins, antibiotics and pesticides . In order to produce butanol at a level industrially useful by using microorganisms, a fermentation process must be carried out, and the selectivity, yield and productivity of acetone, butanediol or butanol, that is, the yield of butanol per unit time should be excellent. Excessive repeated experiments have been required to find microorganisms satisfying the conditions.

Butanediol or butanol in ethanol.

Provides a process for the production of acetone, butanediol or butanol in ethanol.

The method for producing acetone, butanediol or butanol in ethanol of the present invention is a method for producing an acetone, butanediol or butanol by designing an artificial synthetic pathway so that NOX, EtDH, FLS, BDH and DDH proteins and their mutant proteins are displayed as multi- -free catalyst method. The production method of the present invention can produce butanol efficiently because it is not necessary to grow the cells as compared with the conventional microbial fermentation method and can easily control a short synthesis route, a fast reaction rate, a high yield and productivity, and a desired reaction condition . In addition, the protein is fixed to nanoparticles and can be reused in a large number, and is also effective in producing acetone, butanediol or butanol, which is economical.

FIG. 1A shows a method for producing acetone in ethanol using a cell-free multi-catalytic system comprising the optimum enzyme of the present invention, FIG. 1B shows a method for producing 2,3-butanediol from ethanol, FIG. This is a schematic diagram of a method for producing butanol.

2 is a chiral-column GC analysis of the production of acetone, 2,3-butanediol and 2-butanol using the multistage enzyme of the present invention and its variants.

The present invention provides FLS mutant amino acids comprising at least one mutation selected from the group consisting of mutations in which the 482nd leucine is substituted with serine, arginine and glutamic acid in the FLS (formolase) amino acid represented by SEQ ID NO: 8.

The term "FLS (formolase) " in the present invention catalyzes the carbolination of three 1-carbon formaldehyde molecules into one 3-carbon dihydroxyacetone molecule. For the purpose of the present invention, FLS and its variants produce acetone using acetaldehyde produced in the course of alcohol metabolism as a substrate, and use it as a substrate to produce 2,3-butanediol and to use 2,3-butanediol as the substrate But is not limited thereto.

The term "FLS-mutated amino acid" in the present invention means substitution, insertion, deletion or modification of one or more amino acids of the wild-type FLS amino acid. (FLS: L482S) is represented by the amino acid sequence of SEQ ID NO: 10, and the 482nd leucine is replaced with arginine (FLS: L482R) is represented by the amino acid of SEQ ID NO: 11, and the mutant in which the 482nd leucine is replaced by glutamic acid is represented by amino acid of SEQ ID NO: 12 (FLS: L482E). The FLS mutant amino acid may further include at least one selected from the group consisting of mutation at position 396, mutation at position 446, mutation at position 473, mutation at position 477, mutation at position 499, But is not limited thereto, so as to achieve the purpose of converting acetaldehyde produced in the metabolism into acetone. The FLS is derived from, but is not limited to, Pseudomonas fluorescens .

In one embodiment of the present invention, the FLS mutation of the present invention has analyzed the structure of the FLS and found six remaining six hotspots (T396, T446, M473, S477, L482 and L499). Among them, molecular interactions with acetaldehyde, substrate, confirmed that W480 was the active site residue, and FLS: L482S binds more strongly to substrate than FLS, confirming hydrogen bonding.

The present invention also provides a gene coding for the FLS mutant amino acid. The present invention also relates to a gene encoding the above-mentioned FLS-mutated amino acid, a 2,3-butanediol dehydrogenase (BDH) gene, a BDH gene, an NADH oxidase gene, EtDH , A gene for a mutation of a DDH gene, a gene for a DDH (diol dehydratase), and a gene for a DDH mutation.

For the purpose of the present invention, the term "NOX (NADH oxidase)" in the present invention uses oxygen as a substrate and oxidizes NADH to regenerate NAD ⁺ . The regenerated NAD ^{< +} > can be used as coenzyme in EtDH.

The term "EtDH (ethanol dehydrogenase)" in the present invention can be used for the purpose of the present invention by dehydrogenating ethanol using NAD ⁺ and / or NADP ⁺ as a coenzyme using ethanol as a substrate. Thereafter, the production of acetaldehyde can be induced, and FLS using the acetaldehyde as a substrate can be produced to produce acetone.

For the purpose of the present invention, the term "BDH (2,3-butanediol dehydrogenase) " in the present invention catalyzes the production of 2,3-butanediol using acetophenone as a substrate and NADPH as a coenzyme. In addition, when BDH is catalytically reacted with butanone catalyzed by DDH using 2,3-butanediol as a substrate, butanol can be finally produced.

For the purpose of the present invention, the term "DDH (diol dehydratase)" in the present invention can catalyze the formation of butanone when 2,3-butanediol is used as a substrate and vitamin B12 is used as a coenzyme.

The NOX gene is derived from Lactobacillus rhamnosus and the EtDH gene or EtDH mutation gene can be derived from Cupriavidus necator . In addition, the BDH gene or the BDH mutant gene may be derived from Clostridium autoethanogenum , the DDH (diol dehydratase) gene and the DDH mutant gene may be derived from Lactobacillus brevis , But is not limited to, for the production of an enzyme for butanol production of the present invention.

The NOX gene is the nucleotide sequence of SEQ ID NO: 1, the EtDH gene is the nucleotide sequence of SEQ ID NO: 3, and the mutant of EtDH is the 46th aspartic acid of EtDH represented by the amino acid sequence of SEQ ID NO: 4 (EtDH: D46G), the nucleotide sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6.

The BDH gene may be a variant of the BDH wherein the 199th serine of BDH represented by the amino acid sequence of SEQ ID NO: 14 is substituted with alanine (BDH: S199A), the nucleotide sequence of SEQ ID NO: 15 and the amino acid of SEQ ID NO: have.

(DDH: S302A) in which the 302th serine of DDH amino acid sequence of SEQ ID NO: 18 is substituted with alanine; 337th mutant in which glutamine is replaced with alanine (DDH: Q337A); (DDH: F375I) in which the 375th phenylalanine is substituted with isoleucine, but not limited to, one or more single or multiple mutants selected from the group consisting of:

(DDH: Q337A / F375I) wherein the 337th glutamine of the DDH amino acid of SEQ ID NO: 18 is alanine and the 375th phenylalanine is replaced by isoleucine; Mutants in which the 302nd serine alanine and 37th glutamine are substituted with alanine (S302A / F375I); Or a variant (S302A / Q337A / F375I) in which the 302th serine is alanine, the 337th glutamine is alanine, and the 375th phenylalanine is isoleucine.

The mutant of DDH can express dhaR which is a reactivating factor of DDH.

In one embodiment of the present invention, plasmids were prepared to express each protein and its variants. In addition, EtDH or EtDH: D46G mutant expression plasmids and vector maps were prepared. In addition, FLS: L482S, BDH: S199A, DDH: Q337A / F375I expression plasmids and vector maps were prepared (Tables 1 and 2).

In an embodiment of the present invention, the structure of the DDH of the present invention and mutants thereof were analyzed. As a result, it was confirmed that the active site residue was E171 in the molecular interaction with the substrate 2,3-butanediol, and DDH: Q337A / F375I binds more strongly to the substrate, confirming hydrogen bonding and water bridges. In addition, the DDH of the present invention and its variants exhibit stereoselectivity and do not generate butanone with (2R, 3R) -2,3-butanediol and (2S, 3S) -2,3-butanediol was used as a substrate to produce butanone.

EtDH: D46G, EtDH: D46G, EtDH: D46G, FLS, FLS: L482S, BDH: EtDH, D46G and D46G were obtained as a result of confirming thermostability when the protein of the present invention was used as an enzyme in the present invention. S199A, BDH: S199A, DHH with dhaR: Q337A / F375 and NOX proteins exhibited thermal stability even at high 30 to 45 degrees, and particularly excellent activity at 30 degrees.

The present invention also provides a transformed microorganism into which the recombinant vector has been introduced.

The transformant of the present invention can be constructed by introducing the vector into the host cell in such a manner that the promoter can function.

Also, the present invention provides a method for producing butanol, which comprises purifying and reacting a protein produced in the transforming microorganism.

The butanol may be at least one selected from the group consisting of 2-butanol, n-butanol, isobutanol and tert-butanol, preferably 2-butanol. The production method is a method for producing butanol by using a protein produced in the transformed microorganism in a cell-free state by performing a multistage catalysis, and using ethanol as a substrate, but is not limited thereto.

The production method further includes at least one kind of coenzyme selected from the group consisting of coenzyme NAD ⁺ , NADP ⁺ , vitamin B _12, and thiamine pyrophosphate (TPP), but its treatment concentration or throughput is also not limited. The production method further comprises at least one metal ion selected from the group consisting of metal ions Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Fe ²⁺ , Ni ²⁺ , Cu ²⁺ and Zn ²⁺ , Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Fe ²⁺ and Ni ²⁺ can induce the catalytic reaction more efficiently, but this is not limited to produce butanol.

The production method produces butanol at a pH of from 5.0 to 9.0, preferably from 6.5 to 8.5, but is not limited thereto in order to achieve the objective of producing butanol. The production method produces butanol at 16 to 45 degrees, preferably 25 to 42 degrees, but is not limited thereto.

In the present invention, an artificial synthetic pathway using cascade enzymes was designed to produce C ₄ compound, butanol, from ethanol. The multi-step enzymes include NOX, EtDH, FLS, BDH and DDH and their mutants Respectively. Specifically, NOX uses oxygen as a substrate and oxidizes NADH to regenerate NAD ⁺ . The regenerated NAD ⁺ uses EtDH as a coenzyme. EtDH and its variants induce acetaldehyde production by using ethanol as a substrate and dehydrogenating ethanol using NAD ⁺ and / or NADP ⁺ as coenzyme. Thereafter, FLS and its mutants induce the production of acetone using acetaldehyde as a substrate. BDH catalyzes the formation of 2,3-butanediol using acetyl as a substrate and NADPH as a coenzyme. For the purpose of the present invention, DDH catalyzes the formation of butanone when 2,3-butanediol is used as a substrate and vitamin B12 is used as a coenzyme. Thereafter, the BDH catalyzes the catalytic reaction of BDH using catalytic butanone as a substrate to finally produce butanol.

The present invention achieves the objective of artificial synthesis of butanol in ethanol using a simplified pathway in vitro using a cell-free multi-enzyme catalysis (CFME) method. Using the above-mentioned cell-free multi-enzyme catalytic method, the problems such as low target product yield, undesired by-product production, and intracellular transport restriction of the fermentation method in which conventional metabolic engineering such as bacteria and recombinant microorganisms are cultured are solved Thus, there is no need for cell growth, and there are advantages such as short synthesis route, fast reaction rate, high yield and productivity, and control of desired reaction conditions.

In one embodiment of the present invention, the optimal method for producing butanol according to the method of Example 7-6 uses a cell-free multi-enzyme catalyst according to the artificial synthetic pathway designed in the present invention and uses ethanol as a substrate . NAD ⁺ , NADP ⁺ , TPP, vitamin B _{12 and} Mg ²⁺ were added as a coenzyme using NOX, EtDH: D46G, FLS: L482S, BDH: S199A and DDH: Q337A / F375I as enzymes. -Butanol production can be effectively induced.

The present invention also provides a method for producing butanol, which comprises immobilizing and reacting a protein produced in the transforming microorganism with nanoparticles.

The nanoparticles are attached to silicon oxide and are reacted with glutaraldehyde. However, the nanoparticles are not limited thereto, so as to achieve the purpose of producing butanol by attaching the protein of the present invention to nanoparticles. The fixed protein nanoparticles are reusable, preferably 1 to 30 times reusable, more preferably 1 to 20 times, but not limited thereto.

In one embodiment of the present invention, the optimal method for producing butanol according to the method of Example 7-6 uses a cell-free multi-enzyme catalyst according to the artificial synthetic pathway designed in the present invention and uses ethanol as a substrate . NAD ⁺ , NADP ⁺ , TPP, vitamin B _{12 and} Mg ²⁺ were added as a coenzyme using NOX, EtDH: D46G, FLS: L482S, BDH: S199A and DDH: Q337A / F375I as enzymes. -Butanol production can be effectively induced. The enzyme was attached to the nanoparticles to fix and induce butanol production.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.

≪ Example 1 > Experimental Preparation and Experimental Method

<1-1> Purchase of enzymes and reagents

DNA Polymerase High Fidelity and T4 DNA ligase were purchased from TaKaRa Biotech (Shiga, Japan) and New England Biolabs (Ipswich, MA, USA). DNA and protein markers were purchased from Tiangen Biotech (Shanghai, China). (IPTG), dithiothreitol (DTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Sinopharm (Shanghai, China). As a control, (3S / 3R) -acetone was prepared by reacting (2S, 3S) -2,3-butanediol, (2R, 3R) -2,3-butanediol, meso Butanediol, butanone and 2-butanol were purchased from Sigma-Aldrich. All other reagents were of analytical grade and commercially available unless otherwise specified.

<1-2> Bacterial strains, plasmid and bacterial growth conditions

The strains and plasmids used in the present invention are shown in Table 1 below. Escherichia coli DH5? And BL21 (DE3) were used as cloning and expression expression hosts and cultured at 37 占 폚. An expression vector was constructed using the plasmid pET28a. Luria-Bertani (LB) medium was used for strain culture and recombinant protein expression, and kanamycin was added to the medium to cultivate a recombinant strain at a final concentration of 50 μg mL ^-1 .

[Correction according to Rule 91 of the Rules 28.01.2019]

[Correction according to Rule 91 of the Rules 28.01.2019]

<1-3> Recombinant protein expression and purification of cascade enzymes

(Ethanol dehydrogenase), FLS (formolase) and BDH (ethanol) were used to produce acetone and 2,3-butanediol, which are C ₄ compounds in ethanol and finally to produce 2-butanol 2,3-butanediol dehydrogenase, DDH (diol dehydratase), and NOX (NADH oxidase) genes were isolated from each of the cupriavidus necator [TY Wu, et al., Appl. Microbiol. Biotechnol. 2016, 100, 1], Pseudomonas fluorescens [J. Siegel, et al., Proc. Natl. Acad. Sci. USA, 2015, 112, 3704], Clostridium autoethanogenum [M. Kopke, et al., Appl. Environ. Lactobacillus brevis [(Z. Chen, et al., Bioresource Technol., 2015, 197, 260), (M. Yamanishi, et al., FEBS. J , 2012, 279, 793) and Lactobacillus rhamnosus [YW Zhang, et al., Enzyme Microb. Tech., 2012, 50, 255] General Biosystems, Inc. (Anhui, China). In addition, each of the above genes was cloned into the expression plasmid pET28a. Protein expression plasmids were introduced into E. coli BL21 (DE3), and each of the pET-EtDH, pET-FLS, pET-BDH, pET-DDH, pET-dhaR, pET-DDH-dhaR, and pET- Recombinant E. coli BL21 (DE3) was cultured at 37 ° C in LB medium containing 0.5 mM IPTG at an optical density of 0.6 at 600 nm. After induction at 18 ° C for 24 hours, cells were obtained by centrifugation and disrupted by sonication in an ice bath. Cell lysates were centrifuged at 8000 × g for 10 min to remove cell debris. To obtain EtDH, FLS, BDH, dhaR and NOX enzymes, soluble fractions were purified using HisTrap HP column according to purification protocol (GE Healthcare, Little Chalfont, UK). DDH purification was performed in a conventional manner [M. Seyfried, et al., J. Bacteriol., 1996, 178, 5793]. Each of the purified enzymes was concentrated by ultrafiltration and de-chlorinated, and then detected by SDS-PAGE.

<1-4> Production of enzyme variants

Mutants for the EtDH, FLS, BDH and DDH enzymes were prepared and expressed and purified. In addition, EtDH: D46G and BDH: S199A mutants were subjected to site-directed mutagenesis using EtDH1 / EtDH2 and BDH1 / BDH2 primers shown in Table 3 below to construct EtDH or BDH variants Respectively. Recombinant plasmids pET-EtDH and pET-BDH containing wild-type EtDH and BDH genes were used as DNA templates for PCR amplification, respectively. After transformation of the recombinant plasmid containing the correct mutant gene into E. coli BL21 (DE3), the colonies were selected for kanamycin resistance and used for protein expression. After purification of each protein, the activity and kinetic parameters of EtDH and BDH variants were measured. In order to increase the catalytic efficiency of the FLS enzyme, a hot spot was analyzed by inputting the FLS structure (PDB No.: 4QPZ) using a HotSpot Wizard 2.0 server to find a new mutation region [J. Siegel, et (J. Bendl, et al., Acids Res., 2016, 44, 479). Site specific mutations were induced using the FLS1-FLS12 primer of Table 2 below with the six residuals (T396, T446, M473, S477, L482 and L499) Recombinant plasmid pET-FLS containing wild-type FLS was used as a DNA template, and a recombinant plasmid containing the mutant gene was transformed into E. coli BL21 (DE3). FLS variants were screened using the whole-cell biocatalytic method with acetaldehyde as the substrate. Specifically, when the colonies were inoculated on LB medium and the optical density reached 0.6 at 600 nm, protein expression was induced by addition of 0.5 mM IPTG at 18 degrees for 24 hours. The cells were obtained by centrifugation and incubated for 6 hours at 30 ° C. in a reaction mixture containing 50 mM phosphate buffer (pH 8.0), 100 mM acetaldehyde and 40 g L ^-1 wet cell weight (WCW) Catalytic activity was performed. Also, DDH mutants were prepared and used for the expression of dhaR, which is a reactivating factor of DDH, including S302A, Q337A, F375I, S302A / Q337A, S302A / F375I, Q337A / F375I and S302A / Q337A / The DDH variants were prepared to confirm the catalytic efficiency as compared to the wild type DDH enzyme. DDH1-DDH6 primer shown in Table 2 below to induce site-specific mutagenesis. The pET-DDH-dhaR recombinant plasmid containing wild-type DDH and its activator dhaR gene were used as DNA templates for PCR amplification. To induce each protein expression, the PCR product was transformed into E. coli BL21 (DE3) and cultured in LB medium containing 0.5 mM IPTG at 18 degrees for 24 hours. The mutants were used for the evaluation of catalytic activity using total-cell biocatalysis analysis using meso-2,3-butanediol as a substrate. The reaction mixture contained 50 mM HEPES buffer (pH 7.0), 50 mM meso-2,3-b butanediol, 20 μM coenzyme B12 and 40 g L ^-1 wet cell weight, It was carried out at 30 degrees for 6 hours. The butanone product was quantified using gas chromatography. The origins and nucleotide sequences of the respective enzymes and some variants thereof are shown in Table 4.

[Correction according to Rule 91 of the Rules 28.01.2019]

[Correction according to Rule 91 of the Rules 28.01.2019]

<1-5> Enzyme Activity Analysis

For the EtDH enzyme activity assay, the EtDH enzyme activity and its variants were incubated with the reaction mixture containing 100 mM glycine-NaOH buffer (pH 9.5), 5 mM Mg ²⁺ , 3 mM NAD ⁺ / NADP ⁺ Respectively. The activity was confirmed by NAD ⁺ / NADP ⁺ reduction at 340 nm using a spectrophotometer (UV-1800, MAPADA, Shanghai, China). One unit of EtDH activity was identified by the amount of enzyme required to reduce 1 μmol of NAD ⁺ / NADP ⁺ per minute. For FLS enzyme activity analysis, FLS enzyme activity and its variants were assayed with a reaction mixture containing 100 mM phosphate buffer (pH 8.0), 1 mM Mg ⁺ , 0.1 mM TPP and 20 mM acetaldehyde, The concentration of acetone in acetaldehyde was measured by VP reaction and calculated by the standard acetone calibration curve. For the BDH enzyme activity assay, FLS enzyme activity and its variants were assayed in 50 mM Tris-HCl buffer (pH 7.5), 0.2 mM NADPH, 1 mM DTT; And 20 mM acetone or 5 mM butanone at room temperature. The activity was confirmed by the rate of oxidation of NADPH at 340 nm using a spectrophotometer (UV-1800, MAPADA). One unit of BDH activity was defined as the amount of enzyme required to oxidize 1 μmol NADPH per minute. For DDH enzyme activity analysis, mutants containing DDH enzyme activity and dhaR activator were incubated in 50 mM phosphate buffer (pH 7.0), 1 mM coenzyme B12, 100 mM ATP, 1 mM Mg ²⁺ and 50 mM meso-2,3 &Lt; / RTI &gt; butanediol. After standing for 1 hour at room temperature under dark conditions, the reaction was stopped by adding citrate buffer (100 mM, pH 3.6) in the same volume. The butanone product was determined by gas chromatography, and one unit of DDH activity was confirmed by the amount of enzyme producing 1 μmol butanone from meso-2,3-butanediol per minute. For NOX enzyme activity assay, NOX enzyme activity was measured at room temperature with a reaction mixture containing 50 mM HEPES-NaOH buffer (pH 8.0) and 0.2 mM NADH. The activity was confirmed by the NADH oxidation rate at 340 nm using a spectrophotometer (UV-1800, MAPADA). One unit of NOX activity was determined by the amount of enzyme required to oxidize 1 μmol NADH per minute.

<1-6> VP (Voges-proskauer) reaction

Each treatment group was centrifuged at 10,000 × g for 5 minutes at 4 ° C. 0.3 mL of diluted samples, 0.3 mL of 0.5% creatine, 0.3 mL of 5% alpha-naphthol and 0.3 mL of 5% NaOH were added to each 10 mL tube to analyze the acetone concentration in each treatment group and quantify in the VP reaction. Gently shaken. The optical density of the reaction solution was measured at 520 nm using a spectrophotometer (UV-1800, MAPADA) and the concentration of acetone was calculated from the calibration curve. The calibration curve was measured between the standard acetone concentration and the optical density at 520 nm after the VP reaction in the range of 0.04-0.4 mM.

<1-7> Measurement of kinetic parameters

The kinetic parameters of EtDH and EtDH: D46G were determined at room temperature with the reaction mixture containing 100 mM glycine-NaOH buffer (pH 9.5), 5 mM Mg ²⁺ , 3 mM NAD ⁺ / NADP ⁺ and 0.5-100 mM ethanol . The kinetic parameters of FLS and its variants were confirmed at room temperature with a reaction mixture containing 100 mM phosphate buffer (pH 8.0), 1 mM Mg ⁺ , 0.1 mM TPP and 0.5-20 mM acetaldehyde. BDH and BDH: The kinetic parameters of S199A were: 50 mM Tris-HCl buffer (pH 7.5), 0.2 mM NADPH, 1 mM DTT; And 0.5-100 mM acetone or 0.5-10 mM butanone at room temperature. The Km and kcat values were confirmed by nonlinear regression fitting of Michaelis-Menten equation and were repeated three times.

<1-8> Cell-free multi-enzyme catalysis system

As shown in Figs. 1A to 1C, the synthesis of acetone, 2,3-butanediol or 2-butanol from ethanol using an amoebic cell multi-enzyme catalyst was carried out in the presence of substrate, coenzyme, metal ion and 0.5 -mL reaction mixture. The reaction conditions including temperature, pH, coenzyme and metal ion were performed under optimum conditions to increase the flux of the artificial reaction path. Acetone, 2,3-butanediol or 2-butanol. The optimum reaction conditions are as follows. Acetone production was performed in 50 mM HEPES buffer (pH 8.0), 1 mM NAD ⁺ , 0.1 mg mL ^-1 EtDH, 0.2 mg mL ^-1 FLS: L482S, 0.1 mg mL ^-1 NOX, 0.1 mM TPP, 1 mM Mg ²⁺ , 1 mM DTT, 20% DMSO and 100 mM ethanol at 30 [deg.] C. 2,3-butanediol was dissolved in 50 mM HEPES buffer (pH 8.0), 1 mM NAD ⁺ , 1 mM NADP ⁺ , 0.1 mg mL ^-1 EtDH: D46G, 0.2 mg mL ^-1 FLS: L482S, 0.1 mg mL ^-1 NOX, A 0.5-mL reaction mixture containing 0.1 mg mL ^-1 BDH: S199A, 0.1 mM TPP, 1 mM Mg ²⁺ , 1 mM DTT, 20% DMSO and 100 mM ethanol was run at 30 ° C. 2-butanol is 50 mM HEPES buffer (pH 8.0) , 1 mM NAD +, 1 mM NADP +, 0.1 mg mL -1 EtDH: D46G, 0.2 mg mL -1 FLS: L482S, 0.1 mg mL -1 NOX, 0.1 mg ^{mL -1 BDH: S199A, 0.2 mg} mL -1 DDH: Q337A / F375I, 0.2 mg mL -1 dhaR, 0.1 mM TPP, 1 mM Mg 2+, 1 mM DTT, 20% DMSO, 1 mM coenzyme B12, 100 mM ATP and 100 mM ethanol at 30 [deg.] C. All reactions were carried out for 6 hours and each product was confirmed by gas chromatography. The percentage yield for the product was calculated by the following equation: Percentage yield (%) = Product yield (mM) / Theoretical yield (mM). Theoretically, 2 moles of ethanol can produce 1 mole of acetone, 2,3-butanediol or 2-butanol.

<1-9> Recyclability of cascade reactions

The purified enzyme was mixed with active silicon oxide particles and cultured for 12 hours at 4 ° C. Before immobilization, silicon oxide particles (4830HT; Nanostructured & Amorphous Materials, Houston, TX, USA) were activated by treating nanoparticles containing glutaraldehyde (Sigma). Immobilization yield (%), and immobilization efficiency (%) was in the following immobilized enzyme as calculated with the following equation of: immobilizing efficiency _{= (α i / α f)} × 100, immobilization yield = [{P _i - ( P _w + P _s )} / P _i ] × 100. α _i is the total activity of the immobilized enzyme, α _f is the total activity of the free enzyme, Pi is the total protein content of the coenzyme preparation, and Pw and Ps are the protein concentrations of the washing solution or supernatant after fixation. For the production of acetone, 50 mM HEPES buffer (pH 8.0), 1 mM NAD ⁺ , 1.06 U mL ^-1 EtDH, 0.05 U mL ^-1 FLS: L482S, 0.98 U mL ^-1 NOX, 0.1 mM TPP, 1 mM Mg ²⁺ , 1 mM DTT, 20% DMSO, and 100 mM ethanol. In order to produce 2,3-butanediol, 50 mM HEPES buffer (pH 8.0), 1 mM NAD +, 1 mM NADP +, 0.1 mg mL -1 EtDH: D46G, 0.2 mg mL -1 FLS: L482S, 0.1 mg mL - ¹ NOX, 0.1 mg mL- ¹ BDH: S199A, 0.1 mM TPP, 1 mM Mg ²⁺ , 1 mM DTT, 20% DMSO and 100 mM ethanol at 30 degrees for 6 hours Respectively. Further, in order to confirm the reusability of the immobilized enzyme, it was carried out under the same reaction conditions as described above. After each primary reaction cycle, the immobilized enzyme was removed by centrifugation at 4000 x g for 30 minutes. The immobilized enzyme was collected and washed with deionized water and buffer. For the second-order reaction cycle, the immobilized enzyme was dissolved in a new buffer, the substrate was added, and then treated in the same manner as the first-order reaction cycle.

<1-10> Analysis method

Cell growth analysis was confirmed by measuring optical density at 600 nm using a spectrophotometer (UV-1800, MAPADA). Protein concentration was measured using the Bradford method and bovine serum albumin was used as the standard protein. GC-MS analysis was performed using a gas chromatograph system (Agilent GC9860, Santa Clara, CA, USA) equipped with a chiral column (Supelco β-DEX ™ 120, 30-m length, 0.25- Respectively. The operating conditions were as follows: N2 was used as the carrier gas at a flow rate of 1.2 mL min &lt; ^{-1 &} gt ;; The injector temperature and the detector temperature were set at 215 and 245 degrees, respectively; The column temperature was maintained at 50 ° C for 1.5 minutes and then increased to 180 ° C at a rate of 15 ° C min ^-1 .

Example 2 Confirmation of Protein Expression of Multistage Enzyme and Mutant of the Present Invention

In order to confirm the expression of the multistage enzyme of the present invention and mutant proteins thereof, the cells were induced by centrifugation at 18 ° C for 24 hours and then disrupted by ultrasonication in an ice bath. Cell lysates were centrifuged at 8000 × g for 10 min to remove cell debris. To obtain NOX, EtDH, FLS, BDH and dhaR enzymes, soluble fractions were purified using HisTrap HP column according to purification protocol (GE Healthcare, Little Chalfont, UK). DDH purification was performed in a conventional manner [M. Seyfried, et al., J. Bacteriol., 1996, 178, 5793]. Each of the purified enzymes was concentrated by ultrafiltration and de-chlorinated, and then detected by SDS-PAGE. As a result, it was confirmed that EtDH and EtDH: D46G proteins and BDH and BDH: S199A proteins were expressed at the same molecular weight, and it was confirmed that there was no difference in molecular weight expressed between wild type and mutant type. In addition, NOX proteins derived from Lactobacillus rhamnosus or Lactobacillus brevis have different molecular weights, and thus it has been confirmed that proteins expressed according to their origins are different even if they are the same genes. In addition, FLS gene was correctly expressed.

Example 3 Confirmation of Production of C ₄ Compound in Ethanol Using Multistage Enzyme of the Present Invention and its Variant

In order to induce the production of C ₄ compounds acetoin, 2,3-butanediol and 2-butanol in ethanol using the multistage enzyme of the present invention and its variants, , An artificial synthetic pathway using the above-mentioned cell-free multi-enzyme catalytic system was designed as shown in Figs. 1A to 1C. Ethanol is first dehydrogenated by NAD (P) H-dependent EtDH to produce acetaldehyde and undergoes a condensation reaction to produce acetone by the FLS of the present invention and its variants. NOX is used to regenerate NAD +. Subsequently, acetone is reduced by NADPH-dependent BDH to produce 2,3-butanediol. Finally, 2-butanol can be obtained via dehydration and hydrogenation reactions with DDH and BDH, respectively.

<3-1> Production of acetone according to the FLS enzyme of the present invention

In order to confirm the ability of the FLS enzyme of the present invention to convert acetaldehyde to acetone, a VP reaction was carried out, and 50 or 100 nM acetaldehyde as a substrate was treated, or treated with FLS enzyme for 0 or 6 hours And each condition was set. As a result, it was confirmed that acetone was produced, and the concentration of acetone obtained when the FLS enzyme was treated and the substrate acetaldehyde was changed at different concentrations was confirmed through the darkness of the color change.

<3-2> Analysis of production of acetone, 2,3-butanediol and 2-butanol using the multistage enzyme of the present invention and its variants

Using the multistage enzymes of the present invention and its variants, it was synthesized using acetylation, 2,3-butanediol and 2-butanol by artificial synthetic pathway using a cell-free multi-enzyme catalytic system. (2S, 3S) -2,3-butanediol, (2R, 3R) -2,3-dihydroxybenzoic acid, Butanediol, butanediol, butanediol, butanediol and 2-butanol were mixed and used as a control. In addition, the analysis results using the multi-step enzyme according to the present invention of the present invention were carried out by GC / GC-MS analysis. As a result, it was confirmed that when using the multi-stage enzyme-free multi-enzyme catalytic system of the present invention, acetoin, 2,3-butanediol and 2-butanol were produced at the same peaks as those of commercially available standard products 2).

Example 4 Confirmation of Catalytic Effect of Multistage Enzyme and Its Variants in the Artificial Synthetic Pathway of the Present Invention

<4-1> Comparison of catalytic efficiency by measuring kinetic parameters of the multistage enzyme of the present invention and mutants thereof

In order to compare the catalytic efficiency by measuring the kinetic parameters of the multistage enzyme of the present invention and its variants, the following experiment was conducted. Specifically, the kinetic parameters of EtDH and EtDH: D46G were determined at room temperature with a reaction mixture containing 100 mM glycine-NaOH buffer (pH 9.5), 5 mM Mg ²⁺ , 3 mM NAD ⁺ / NADP ⁺ and 0.5-100 mM ethanol Respectively. The kinetic parameters of FLS and its variants were confirmed at room temperature with a reaction mixture containing 100 mM phosphate buffer (pH 8.0), 1 mM Mg ⁺ , 0.1 mM TPP and 0.5-20 mM acetaldehyde. BDH and BDH: The kinetic parameters of S199A were: 50 mM Tris-HCl buffer (pH 7.5), 0.2 mM NADPH, 1 mM DTT; And 0.5-100 mM acetone or 0.5-10 mM butanone at room temperature. The substrate affinities, Km and kcat, were determined by nonlinear regression fitting of the Michaelis-Menten equation and repeated three times. DDH and NOX have been found to be more potent than conventional results (S. Kwak, et al., Bioresource Technol., 2013, 135, 432), (M. Kopke, et al., Appl. Environ. Microbiol., 2014, 80, 3394) . The results are shown in Table 5. As shown in Table 4, EtDH and EtDH: D46G enzymes were found to have kcat / Km values of 17.09 and 9.97 s ^-1 mM ^-1 when using ethanol as a substrate and NAD ⁺ as a coenzyme. As a result, NADP ⁺ It has been confirmed that the use of NAD ⁺ can increase the catalyst efficiency. In addition, it was confirmed that the kcat / Km value of the FLS enzyme was 7.69 × 10 ^-3 s ^-1 mM ^-1 when acetaldehyde was used as a substrate and thiamine pyrophosphate (TPP) was used as a coenzyme. FLS: L482S, FLS: L482R and FLS: L482E, which are variants thereof, confirmed that the kcat / Km values were 1.33 x 10 ^-2 , 1.06 x 10 ^-2 and 9.66 x 10 ^-3 s ^-1 mM ^-1 , It was confirmed that the catalytic efficiency was increased to 72.95%, 37.84% and 25.62%, respectively, as compared with the wild-type FLS enzyme. In addition, when BDH: S199A enzyme was compared with wild type BDH, it was confirmed that the catalyst efficiency was increased when butanone was used as a substrate and NADPH was used as a coenzyme.

[Correction according to Rule 91 of the Rules 28.01.2019]

<4-2> Confirmation of Stereoselectivity of Multistage Enzymes and Mutants of the Present Invention In order to analyze the stereoselectivity of the DDH enzyme of the present invention, cells expressing DDH enzyme and its reactivity factor dhaR (E. coli (pET-DDH-dhaR) was used to perform whole-cell biocatalysis. Three 2,3-butanediol isomers (meso-2,3-butanediol, ) -butanediol and (2S, 3S) -butanediol), the conversion of 2-butanol to butanone was confirmed. As a result, the present inventors analyzed the stereoselectivity of the DDH enzyme of the present invention and found that 20.56 mM butanone was generated from 50 mM meso-2,3-butanediol. On the other hand, it was confirmed that butanone was not detected when (2R, 3R) -2,3-butanediol and (2S, 3S) -2,3-butanediol were used as a substrate. Therefore, the DDH enzyme including dhaR exhibited a high catalytic activity in meso-2,3-butanediol in vivo, confirming excellent stereoselectivity.

<4-3> Determination of the Thermostability of the Multistage Enzyme of the Present Invention and Its Variants

(Including ethanol and NAD ⁺ ), EtDH: D46G (including ethanol and NAD ⁺ ), EtDH: D46G (ethanol, NADP ⁺ ), EtDH: D46G (containing ethanol and NAD ⁺ ), and the like were used in order to confirm the thermal stability of the multistage enzyme of the present invention and its mutants (including acetaldehyde, TPP), BDH: S199A (including acetone and NADPH), BDH: S199A (including butanone and NADPH), dhaR (including ethanol and NAD +), FLS (including acetaldehyde and TPP) : the Q337A / F375 (2,3- meso-butanediol, B12 included), NOX (O _2, comprising NADH) cells 30, 37, and incubated at 45 for 6 hours, activity was measured. As a result, it was confirmed that the thermal stability of the EtDH, FLS and NOX enzymes at 30 ° C was 87.91%, 70.43%, and 91.30%, respectively. In addition, at 37 and 45 degrees, the NOX enzyme showed continuous thermal stability.

Example 5 Production of Acetone by the Artificial Synthesis Pathway of the Present Invention

<5-1> Determination of optimal reaction conditions from ethanol to acetone

For the production of acetone in ethanol, EtDH, FLS and NOX enzymes were used. Specifically, the initial reaction is 50 mM HEPES buffer (pH 7.0), 0.1 mg mL -1 EtDH, 0.2 mg mL -1 FLS, 0.1 mg mL -1 NOX, 4 mM NAD +, 0.1 mM TPP, 1 mM Mg 2+ , 1 mM DTT, 20% DMSO, and a 0.5-mL reaction mixture containing 100 mM ethanol as an initial substrate. The reaction was carried out under the conditions of 30 ° C. for 6 hours and 17.98 mM of acetone, which is 35.96% of the theoretical yield, was produced. As a result, when using the artificial synthetic route of the present invention, It was confirmed that the reaction could be induced. The temperature was set to 20, 25, 30, 37, and 42 degrees in order to find the optimal reaction conditions, depending on the conditions of temperature, pH, coenzyme (NAD ⁺ and TPP) and metal ion. In addition, the NAD ⁺ at 1, 2, 4, 6, and 8 mM concentrations of TPP were found to be 1, 0.1, 0.2, 0.3, 0.4, and 0.5 at pH 6.0, 6.5, 7.0, 7.5, 8.0, At 0.5 mM concentration, the optimum conditions for the metal ions were determined by treating Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Fe ²⁺ , Ni ²⁺ , Cu ²⁺ and Zn ²⁺ . As a result, it was confirmed that the optimum condition for the metal ion Mg ²⁺ was the pH condition at 8.0, the temperature at 30 ° C, the NAD + concentration at 1 mM and the TPP at 0.1 mM concentration, and the reaction flux from ethanol to acetone . In order to confirm the production of acetone over time in the optimal reaction conditions, EtDH, FLS and NOX enzymes were used and treated with 100 mM ethanol as a substrate for 0, 2, 4, 6, and 8 hours. As a result, it was confirmed that at 6 hours treatment, an acetone of 22.75 mM, which is 45.50% of the theoretical yield, was produced.

<5-2> Confirmation of the rate limiting step from ethanol to acetone

In order to confirm the step of controlling the rate of ethanol to acetone, each EtDH, FLS or NOX enzyme was reduced to a concentration of 1/10, and the remaining amount of the enzyme was maintained at a constant concentration to confirm the amount of acetone. As a result, it was confirmed that when the concentration of FLS enzyme is lower than that of EtDH or NOX enzyme, it affects the production of acetone. Therefore, it was confirmed that the FLS enzyme is an enzyme important for the production of acetone.

<5-3> Identification and selection of catalytic efficiency of FLS enzyme mutants

To improve the catalytic efficiency of the FLS enzyme, a mutated hot spot of the FLS amino acid sequence was analyzed using the HotSpot Wizard 2.0 server. Using the Hotspot Wizard 2.0 server, we confirmed the saturation mutagenesis of six sites of hot spot residues (T396, T446, M473, S477, L482 and L499) and confirmed their structural models. As a result, 482 sites in FLS It is confirmed that it plays an important role. In order to select the FLS variant, FLS and its mutants L482S, L482R and L482E were subjected to the VP-method using acetaldehyde (100 mM) as a substrate using the all-cell biocatalytic method Respectively. After the purification, the activity (%) of each mutant was compared with 0.16 U / mg, which is the specific activity of FLS, and the mean ± standard deviation was calculated by repeating 3 times. The FLS mutant It was confirmed that it further produced acetone. In addition, it was confirmed that the FLS mutations L482S, L482R and L482E increased to 59.03%, 36.89% and 34.12%, respectively, from the FLS intrinsic activity. Therefore, it was confirmed that L482S was most effective for the production of acetone during the FLS mutation.

<5-4> Comparison of wild type FLS and mutation FLS: L482S structure and activity

Wild type FLS and its mutations To compare the structure and activity of FLS: L482S, a molecular dynamics simulation analysis of 100 ns was used to confirm the correlation between the structural changes of wild type FLS and its mutant FLS: L482S and enzyme activity. As a result, the wild-type FLS and its mutant FLS: L482S have a strong interaction with acetaldehyde (2.8 Å), which is a substrate mutant FLS: L482S, ). Furthermore, it was confirmed that the mutant FLS: L482S was more hydrogen-bonded than the wild-type FLS.

<5-5> Comparison of FLS: L482S activity by substrate and concentration

In order to compare the activity of FLS: L482S with ethanol as a substrate, 50 mM HEPES buffer (pH 8.0), 1 mM NAD ⁺ , 1.06 Uml ^-1 EtDH, 0.05 U mL ^-1 FLS: L482S, 0.98 The reaction was carried out with a 0.5-mL reaction mixture containing U mL ^-1 NOX, 0.1 mM TPP, 1 mM Mg ²⁺ , 1 mM DTT, 20% DMSO, and 100-500 mM ethanol. Thereafter, the formation of acetone was confirmed under the conditions of 0-6 hours and 30 degrees. As a result, when 100 mM ethanol was used as a substrate at 4 hours of reaction, 44.39 mM of acetone corresponding to 88.78% of the theoretical yield was obtained. In addition to acetone, the acetaldehyde produced by the metabolism of ethanol was measured, and it was confirmed that it accumulated to 9.58 mM for 2 hours and decreased to 5.65 mM for the next 6 hours. In addition, no other by-products were accumulated, confirming that the artificial synthetic pathway of the present invention for production of acetone using FLS: L482S is substrate specific. In addition, the effect of substrate concentration (200, 300, 400 and 500 mM) on the production of acetone was confirmed, and it was confirmed that the production of acetone and acetaldehyde was increased in a concentration dependent manner.

Example 6 Production of 2,3-butanediol through the artificial synthetic route of the present invention

<6-1> Production of EtDH: D46G mutation and BDH: S199A

(P) H to prevent NADH accumulation while simultaneously converting ethanol into acetaldehyde and simultaneously using NAD ⁺ and NADP ⁺ as coenzyme when designing the artificial synthetic pathway of the present invention to produce 2,3-butanediol in ethanol To design the purge valve regulatory node, variants of EtDH were constructed. Specifically, the amino acid sequence of EtDH (cnMDH) containing another dehydrogenase in the PDB database was aligned. As a result, a site using NAD + alone as a coenzyme and a site coexisting with NAD ⁺ / NADP ⁺ were confirmed, and EtDH: D46G containing the concurrent site was selected. In addition, BDH: S199A is a conventional method [DJ Maddock, et al., Protein Eng. Des. Sel., 2015, 28, 251]. As a result, the thermal stability of the mutant was confirmed at 30, 37 and 45 degrees. As a result, the EtDH: D46G mutant was cultured at 30 degrees for 6 hours when acetaldehyde was used as a substrate having coenzyme NAD ⁺ and NADP ⁺ And the activity levels were maintained at 86.53% and 86.67%, respectively. Also, it was confirmed that the activity level of BDH: S199A mutant was maintained at 80.81% for 6 hours at 30 ° C. when NADPH coenzyme and acetone were used as a substrate.

<6-2> Confirmation of the formation of 2,3-butanediol in ethanol by coenzyme

To confirm the formation of 2,3-butanediol in ethanol according to the coenzyme, EtDH: D46G, FLS: L482S, BDH: S199A and NOX enzyme were reacted with 100 mM ethanol in the presence of 1 mM NAD ⁺ and / or 1 mM NADP ⁺ , And the concentration (mM) of 2,3-butanediol was confirmed. As a result, it was confirmed that 2,3-butanediol was not detected when NAD ⁺ was used as a coenzyme. On the other hand, it was confirmed that 18.20 mM 2,3-butanediol was produced from ethanol in the presence of NADP ⁺ . Therefore, it is confirmed that BDH is an NADPH-dependent enzyme that converts acetone to 2,3-butanediol, and that NADP ⁺ coenzyme is required for the production of 2,3-butanediol.

<6-3> Confirmation of 2,3-butanediol formation by substrate and concentration

1 mM NADP ⁺ , 0.88 UmL- ¹ EtDH: D46G, 0.05 ^{& lt} ; RTI ID = 0.0 ^& gt ^; um ^& lt ^; / RTI ^& gt ^{; -1} FLS: L482S, 0.98 UmL ^-1 NOX, 5.11 UmL ^-1 BDH: S199A, 0.1 mM TPP, 1 mM Mg ²⁺ , 1 mM DTT, 20% DMSO and 100-500 mM ethanol Cell-free multi-enzyme catalytic reaction was performed. Thereafter, the formation of 2,3-butanediol was confirmed under the conditions of 0-6 hours and 30 degrees. As a result, it was confirmed that when the coenzyme NAD ⁺ and 1 mM NADP ⁺ were used simultaneously, 2,3-butanediol of 88.28% of the theoretical yield was produced at the reaction time of 5 hours. Therefore, it was confirmed that BDH: S199A showed high catalytic efficiency from acetone to 2,3-butanediol by inhibiting NADH accumulation and accumulating low concentration of acetone in the reaction solution throughout the reaction process. As a result, when different substrate concentrations were used, 2,3-butanediol was produced in a concentration-dependent manner, and it was confirmed that the maximum 2,3-butanediol concentration was 127.3 mM when the ethanol concentration was 500 mM.

Example 7 Confirmation of Production of 2-Butanol in Ethanol

<7-1> Confirmation of conversion of meso-2,3-butanediol to butanone by reaction conditions

In order to produce 2-butanol in ethanol, conversion of meso-2,3-butanediol to butanone was confirmed by reaction conditions. Specifically, 0.2 mg ml ^-1 DDH was used as the enzyme, and the meso- and lipid contents were measured by 0 or 1 mM coenzyme B 12, 0 or 100 mM ATP, 0 or 1 mM Mg ²⁺ and the reactivity factor 0 or 0.2 mg ml ^-1 dhaR of DDH, The effect of 2,3-butanediol on the conversion to butanone was confirmed. As shown in Table 6, it was confirmed that coenzyme B12 and ATP were required for the catalytic reaction, and dhaR and Mg ²⁺ were confirmed to effectively enhance butanol production from meso-2,3-butanediol. Therefore, it was confirmed that DDH enzyme catalyzes only (2R, 3R) -butanediol and (2S, 3S) -butanediol as a substrate of DDH but only meso-2,3-butanediol as butanone.

[Correction according to Rule 91 of the Rules 28.01.2019]

<7-2> Confirmation of production of 2-butanol by BDH: S199A mutant BDH: In order to confirm the formation of enantiomers 2-butanol in 2-butanone according to S199A variant, 50 mM HEPES buffer (pH 8.0) , A 40 g L ^-1 induced E. coli / pET28a-BDH: S199A cell (wet cell weight), and 25 mM butanone was performed and analyzed by GC. As a result, it was confirmed that when the BDH: S199A mutant was used as an enzyme, R- and S-2-butanol were produced at a ratio of 1.23: 1.

<7-3> Identification of acetone and 2,3-butanediol

In order to produce 2-butanol in ethanol, the DDH enzyme catalyzes only butanoyl meso-2,3-butanediol, which is not (2R, 3R) -butanediol and (2S, 3S) -butanediol, And the form of acetone and 2,3-butanediol produced in the reaction was confirmed. Specifically, the form of acetone and 2,3-butanediol was confirmed using a GC system equipped with a chiral column. As a result, acetaldehyde was used as a substrate, (3S) -acetone and (3R) -acetone acetone were used when the FLS: L482S mutant enzyme was used, from which meso-2,3-butanediol (65%) and (2R, 3R) -butanediol (35%) was produced.

<7-4> Identification of the rate limiting step from ethanol to 2-butanol and selection of mutants of DDH

In order to confirm the step of controlling the rate of ethanol to 2-butanol, DDH and NOX including EtDH: D46G, FLS: L482S, BDH: S199A and dhaR as a reactivity factor were used as enzymes with 100 mM as a substrate . Specifically, the concentration of DDH containing BDH: S199A or dhaR as a reactivity factor was reduced to a concentration of 1/10, and the remaining enzyme was maintained at a constant concentration and repeated three times to determine the concentration (mM) of 2-butanol . In addition, the concentration (mM) of butanone formation was confirmed using DDH and its mutants S302A, Q337A, F375I, S302A / Q337A, S302A / F375I, Q337A / F375I and S302A / Q337A / F375I. As a result, when the concentration of DDH containing dhaR, which is a reactivity factor, was lowered, the production concentration of 2-butanol was also remarkably decreased by 79.16%, indicating that DDH containing dhaR is an enzyme important for 2-butanol production Respectively. In addition, Q337A / F375I showed the highest concentration of butanone in DDH compared to other variants. In addition, protein expression of DDH: Q337A / F375I among the above DDH mutations was confirmed by SDS-PAGE, and it was confirmed that it expresses dhaR as a regulatory factor of DDH.

<7-5> Comparison of wild type DDH and mutant DDH: Q337A / F375I structure and activity

To compare the structure and activity of wild-type DDH and its mutants DDH: Q337A / F375I, we used a molecular dynamics simulation analysis of 100 ns to show the correlation between the structural changes of wild-type DDH and its mutant DDH: Q337A / F375I and enzyme activity Respectively. As a result, the molecular interaction with the substrate 2,3-butanediol was found to be the active site residue E171 in the wild-type DDH and bind to the substrate at 2.6 Å. As a result, the molecular interaction with the substrate, 2,3-butanediol, was found to be the active site residue E171 in the mutant DDH: Q337A / F375I and bound to the substrate at 1.9 Å. As a result, 100 ns molecular dynamics analysis confirmed that the active site residue E171 in the wild type DDH contained substrate and hydrogen bonds and water bridges. In the mutant DDH: Q337A / F375I, the active site residue E171 was mainly hydrogen bonded. Therefore, it was confirmed that the mutant DDH: Q337A / F375I binds more strongly to the substrate than the wild type DDH, and thus the catalytic activity is excellent.

<7-6> Confirmation of formation of 2-butanol in ethanol through the artificial synthetic route of the present invention

1 mM NAD ⁺ , 1 mM NADP ⁺ , 0.88 U mL- ¹ EtDH: D46G, pH 7.4, to confirm the formation of 2-butanol finally in ethanol via the artificial synthetic route of the present invention. 0.05 U mL ^-1 FLS: L482S, 0.98 U mL ^-1 NOX, 5.11 U mL ^-1 BDH: S199A, 0.01 U mL ^-1 DDH: Q337A / F375I, 0.2 mg mL ^-1 dhaR, 0.1 mM TPP, 1 mM Mg ^A cell-free, multi-catalytic system was prepared by treating the 0.5-mL reaction mixture containing ²⁺ , 1 mM DTT, 20% DMSO, 1 mM coenzyme B ₁₂ , 100 mM ATP and 0-100 mM ethanol for 30 min, The concentration (mM) of acetaldehyde, acetone, 2,3-butanediol, 2-butanone and 2-butanol in ethanol was determined. As a result, it was confirmed that 13.62 mM 2-butanol having a theoretical yield of 27.24% was produced. It was also confirmed that a high level of 2,3-butanediol (up to 32.55 mM) was accumulated in the reaction solution, whereas butanone was not detected during the reaction.

Example 8 Optimal Enzymes and Their Intrinsic Activity in the Artificial Synthetic Pathway of the Present Invention

The optimum enzyme in the artificial synthetic pathway of the present invention and each substrate and coenzyme affecting its activity were identified. In addition, specific activity of each enzyme was confirmed. As shown in Table 7, it was confirmed that EtDH or EtDH: D46G mutant enzyme had an intrinsic activity of 10.64 ± 0.15 and 8.81 ± 0.13 U mg ^-1 , respectively, when using ethanol as a substrate and NAD ⁺ as a coenzyme. In addition, it was observed that the FLS: L482S mutant enzyme was 0.26 ± 0.01 U mg ^-1 when acetaldehyde was used as a substrate and TPP was used as a coenzyme. In addition, BDH: S199A mutant enzymes were found to be 51.13 ± 3.74 and 57.55 ± 2.65 U mg ^-1 , respectively, when acetone or butanone was used as a substrate and NADPH was used as a coenzyme. The DDH: Q337A / F375I mutant enzyme containing dhaR was 0.05 ± 0.01 mg ^-1 when meso-2,3-butanediol was a substrate and B ₁₂ coenzyme, and the NOX coenzyme was 9.83 ± 0.12 when it was O ₂ substrate and NADH coenzyme. 0.48 mg ^-1 was observed.

[Correction according to Rule 91 of the Rules 28.01.2019]

Example 9 Recyclability of the enzyme for the production of acetone or 2,3-butanediol (Recyclability of cascade reactions)

To confirm the recycling of the enzyme for the production of acetone or 2,3-butanediol, each enzyme for the production of acetone or each enzyme for the production of 2,3-butanediol was prepared according to the method of Example 1-8 The active silicon oxide particles were mixed and cultured for 12 hours at 4 ° C. Before immobilization, silicon oxide particles (4830HT; Nanostructured & Amorphous Materials, Houston, Tex., USA) were activated by treating nanoparticles containing glutaraldehyde (Sigma) The immobilization yield (%) and immobilization efficiency (%) were confirmed. As a result, it was confirmed that when immobilized enzyme was used, acetone was effectively produced in ethanol. As a result of the first to tenth reaction cycles of the immobilized enzyme for the production of acetone, it was confirmed that even after the 10th reuse, 94% efficiency was obtained compared with the initial acetic acid production concentration. In addition, it was confirmed that 2,3-butanediol was effectively produced in ethanol when the immobilized enzyme was used. As a result of the first to tenth reaction cycles of the immobilized enzyme for the production of 2,3-butanediol, it was confirmed that the efficiency was 73% as compared with the initial primary 2,3-butanediol concentration even after the 10th reuse .

Claims (14)

1. A FLS mutant amino acid comprising at least one mutation selected from the group consisting of mutations in which the 482nd leucine is substituted with serine, arginine and glutamic acid in FLS (formolase) amino acid represented by SEQ ID NO: 8.
2. 10. The method according to claim 1, wherein the mutation in which the 482nd leucine in the FLS amino acid is substituted with serine is represented by (FLS: L482S) the amino acid in SEQ ID NO: 10 and the mutation in which the 482nd leucine is substituted with arginine is (FLS: L482R) Wherein the mutation in which the 482nd leucine is replaced with glutamic acid is represented by the amino acid of SEQ ID NO: 12 and (FLS: L482E) is represented by the amino acid of SEQ ID NO: 12.
3. The FLS mutant amino acid according to claim 1, wherein the FLS mutant amino acid further comprises at least one selected from the group consisting of mutation at position 396 th threonine, mutation at position 446 threonine, mutation at position 473 th methionine, mutation at position 477 serine, and mutation at position 499 leucine / RTI &gt; amino acid residues.
4. A gene encoding the FLS mutant amino acid of claim 1.
5. A mutant gene of EtDH, a FLS (formolase) gene, a gene coding for the FLS mutant amino acid of claim 2, a 2,3-butanediol dehydrogenase (BDH) gene, a mutation of BDH, an NADH oxidase gene, an EtDH (ethanol dehydrogenase) A recombinant vector comprising at least one gene selected from the group consisting of a gene, a DDH (diol dehydratase) gene and a mutant gene of DDH.
6. 6. The method according to claim 5, wherein the mutant of EtDH comprises a nucleotide sequence of SEQ ID NO: 5 and an amino acid sequence of SEQ ID NO: 6, wherein the 46th aspartic acid of EtDH represented by the amino acid sequence of SEQ ID NO: 4 is substituted by glycine Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
7. [Claim 15] The method according to claim 5, wherein the variant of BDH is selected from the group consisting of the nucleotide sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16, wherein the 199th serine of BDH represented by the amino acid sequence of SEQ ID NO: 14 is substituted by alanine (BDH: S199A) &Lt; / RTI &gt;
8. [6] The mutant of DDH according to claim 5, wherein the mutant DDH (S302A) in which the 302nd serine of the DDH amino acid sequence of SEQ ID NO: 18 is substituted with alanine; 337th mutant in which glutamine is replaced with alanine (DDH: Q337A); 375th mutant (DDH: F375I) in which phenylalanine is substituted with isoleucine; &Lt; / RTI &gt; wherein said recombinant vector is at least one single or multiple variant selected from the group consisting of:
9. 9. The mutant according to claim 8, wherein the mutant is a mutant (DDH: Q337A / F375I) wherein the 337th glutamine of the DDH amino acid of SEQ ID NO: 18 is alanine and the 375th phenylalanine is replaced by isoleucine; Mutants in which the 302nd serine alanine and 37th glutamine are substituted with alanine (S302A / F375I); Or a mutant (S302A / Q337A / F375I) wherein the 302st serine is alanine, the 337th glutamine is alanine, and the 375th phenylalanine is isoleucine.
10. 6. The recombinant vector according to claim 5, wherein the DDH mutant expresses dhaR, a reactivating factor of DDH.
11. A transformed microorganism into which the recombinant vector of claim 5 has been introduced.
12. 11. A method for producing at least one selected from the group consisting of acetone, butanediol, and butanol, which comprises purifying and reacting a protein produced in the transforming microorganism of claim 11.
13. A method for producing at least one selected from the group consisting of acetone, butanediol, and butanol, comprising: immobilizing and reacting a protein produced in the transgenic microorganism of claim 11 with nanoparticles.
14. 14. The method of claim 13, wherein the nanoparticles are attached to silicon oxide and reacted with glutaraldehyde.

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