KR20170133806A - Method of producing higher alcohol using pyridoxal phosphate based in E. coli - Google Patents

Abstract

The present invention relates to a production method of a higher alcohol using pyridoxal phosphate (PLP) based in E. Coli, which comprises a step of inoculating a culture medium containing PLP and copper (Cu) ions with a recombinant E. Coli coexpressing ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (ADH) prior to culturing. The PLP of the present invention, by forming a complex with Cu, can catalyze oxidative deamination that converts amino acids into 2-keto acid, wherein 2-keto acid can be converted into a higher alcohol by ADH. Accordingly, by converting an amino acid substrate in the culture medium into 2-keto acid by culturing the recombinant E. Coli coexpressing KivD and ADH in the culture medium containing PLP-Cu complex, and through whole-cell bioconversions capable of continuously producing a higher alcohol, effective production of a higher alcohol can be achieved.

Description

 FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a high-alcohol based on E. coli using pyridoxal phosphate,

The present invention relates to a method for producing a culture medium containing pyridoxal phosphate (PLP) and copper ion (Cu) in a medium containing ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (ADH) And a step of inoculating and culturing the recombinant Escherichia coli expressing Escherichia coli.

In nature, protein molecules have evolved in a given environment and have adapted to function optimally over time. In view of this evolutionary history, proteins have begun to require cofactors of organic matter such as metal ions or vitamin derivatives. Thus, the type of organism in which several protein-cofactor complexes play a major role in various metabolic processes is well known. In vivo metabolic processes consist of numerous chemical reactions.

Most of these reactions can be catalysed by organic-inorganic coenzymes, and among these coenzymes, PLP coenzyme, an active form of vitamin B6, has been reported as a good example. PLP is provided as an artificial molecule in a variety of enzymes, which can catalyze both substitution (amino-transferase, transaminase) and desorption (deamination enzyme, deaminase). However, the enzymatic transamination of amino acids involves amine receptors, which are often the critical molecules associated with intracellular central carbon-nitrogen metabolism. This heterologous conversion of amino groups has been reported to cause imbalance of nitrogen metabolism in cells [9]. In addition, since this correlation to amine donation / acceptance is functionally related, the reaction system also requires another amine acceptor substrate to elicit an effective peramination reaction.

Thus, in order to avoid this disadvantage associated with the reaction by the amino-transferase, it is not necessary to rely on an enzyme requiring an amine donor and to induce the depolymerization of the amino acid by an enzyme or a compound that does not require an amine donor It is said that it is efficient.

Previously reported studies have shown that PLP coenzyme itself can have catalytic activity and can convert amino acids to keto acids, regardless of the presence or absence of metal ions. It has also been reported that amino acid can be converted to keto acid through oxidative deamination in the presence of dioxygen [13].

In addition, various amino acids can be converted to the corresponding 2-keto acids and ammonia according to the side chain structure, and PLP or 5'deoxypyridoxal, a vitamin B6 co-enzyme, acts with Cu2 + It has been reported that glycine can be converted to (p-sulfophenyl) glyoxylic acid by oxidative deamination [22].

Meanwhile, butanol is an important compound widely used in various industrial fields such as a solvent used in the textile industry or an extraction solvent in the food industry, and recently, butanol such as isobutanol has been used as a substitute for fossil fuels such as petroleum It is also concentrated as an alternative fuel. Butanol fuels, unlike ethanol, do not corrode and have energy densities similar to those of gasoline, which has the advantage of alternative fuels that can replace fossil-derived petroleum in vehicle fuels. In addition to butanol, forms such as isobutanol, 2-methylbutanol and 3-methylbutanol are also of interest.

The most commonly used method for industrially producing butanol is hydroformylation of propene to produce butyraldehyde and reduction thereof to produce 1-butanol and 2-butanol . However, this process is disadvantageous in that it relies on non-reproducible resources and can release greenhouse gases to pollute the environment again. Accordingly, there is a continuing need for a method for producing butanol from a renewable material such as biomass through a biological process. In this regard, Korean Patent Registration No. 1535578 discloses a method of re-balancing a redox state to maximize the production of fermentation products in E. coli strains and an E. coli strain for producing a highly productive fermentation product developed using the method, Korean Patent No. 1473532 discloses a recombinant microorganism having enhanced butanol productivity by promoting a pathway for inhibiting the pathway for converting acetylcopheryl acetate to acetylcocoate into butyrylcholine, Patent No. 2015-0225750 discloses a method for producing a high-quality alcohol comprising the steps of converting a carbon source substrate into an amino acid in a recombinant E. coli and then converting it to a higher alcohol.

However, this method also depends on the regulation of the expression of related enzymes through recombination of intracellular metabolism, but since it may cause an imbalance of major metabolic processes in the cell in association with amine receptors in the oxidation of carbon substrate or amino acid substrate, The yield is not increased.

Accordingly, the present inventors have made efforts to develop a process for efficiently producing a high-alcohol using a biological method. As a result, the present inventors have found that the PLP-Cu complex acts as a catalyst to convert amino acids to 2-keto acids, Ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (ADH) co-expressed in E. coli convert dicarboxylic and aldehyde to higher alcohol The present invention has been completed.

Thus, the present inventors have found that, in a method of producing high-yield alcohols based on Escherichia coli, the amino acid in the medium is converted into 2-keto acid with a PLP-Cu complex, and ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase It has been confirmed that 2-keto acid can be converted into a higher alcohol through a whole cell bioconversion reaction in E. coli co-expressing an alcohol dehydrogenase (ADH), thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a method for producing higer alcohol based on E. coli using PLP.

In order to achieve the above object,

i) inducing a deamination reaction to produce pyridoxal phosphate (PLP) and copper ion (Cu) to the medium to produce 2-keto acid;

ii) inoculating recombinant E. coli overexpressing ketoisovalerate decarboxylase (KivD) in the medium from which the reaction was induced in step i); And

iii) culturing Escherichia coli inoculated in step ii) so that the 2-keto acid produced in step i) is reduced to be dicarboxylated and aldehyde-reduced, Provide a production method.

The present invention also provides a method for producing Escherichia coli-based advanced alcohols using PLP, comprising inoculating and culturing the recombinant Escherichia coli co-expressing KivD and ADH in culture medium supplemented with PLP and Cu.

In a preferred embodiment of the present invention, the medium comprises at least one amino acid selected from the group consisting of Val, Leu, Ile, Phe, and norvaline. Lt; / RTI >

In another preferred embodiment of the present invention, the medium may be an organic waste, either food waste, livestock manure or domestic wastewater.

In another preferred embodiment of the present invention, the 2-keto acid is selected from the group consisting of 2-ketoisovalerate (KIV), 2-ketoisocaproate (KIC) One selected from the group consisting of 2-keto-3-methylvalerate (KMV), phenylpyruvate (PP) and 2-ketovalerate (KV) .

In another preferred embodiment of the present invention, the E. coli may co-express an alcohol dehydrogenase (ADH) with KivD.

In another preferred embodiment of the present invention, the higher alcohol is selected from the group consisting of isobutanol, 2-methylbutanol, 3-methylbutanol, 2-phenylethanol, And butanol may be used.

Accordingly, the present invention provides a method for producing Escherichia coli-based higer alcohol using PLP.

The pyridoxal phosphate (PLP) of the present invention can catalyze an oxidative desamination reaction in which an amino acid is converted to a 2-keto acid by complexing with copper ion (Cu), and 2- Can be converted to higher alcohols by ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (ADH). Using this, recombinant Escherichia coli co-expressing KivD and ADH is cultured in a culture medium containing PLP-Cu complex to convert the amino acid substrate of the culture medium to 2-keto acid and to continuously convert the whole cell biotransformation The reaction can produce high-quality alcohol effectively.

In this case, when the amino acid mixed in a mixture medium containing various substances such as food wastes or organic wastes such as waste water is used as a substrate as well as a medium containing a single amino acid, It is possible to produce high-quality alcohol from biomass through an environment-friendly and inexpensive process, which is effective.

1 is a reaction scheme diagram of the present invention for converting an amino acid into a higher alcohol through oxidation by PLP and subsequent reduction of the enzyme.
2 is a schematic diagram of a whole cell reaction in which PLP-Cu is extracellularly applied to induce the production of high-grade alcohol.
Figure 3 shows the production of higher alcohols from amino acids in a medium containing a single amino acid as a substrate.
Figure 4 shows the production of higher alcohols from amino acids in medium containing mixed amino acids as substrates:
Figure 4a shows the conversion yield (%) of the higher alcohol produced from the amino acid; And
Figure 4b shows the yield (titer, g / l) of the higher alcohols produced from amino acids with time.

Hereinafter, the present invention will be described in detail.

As described above, the regulation of expression of related enzymes through recombination of intracellular metabolic processes may lead to imbalance of major metabolic processes in the cell in association with amine receptors in the oxidation of carbon source or amino acid substrate, There is a disadvantage that it does not increase.

Recombinant (co-expressing) ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (ADH) in a medium containing pyridoxal phosphate (PLP) -Cu complex of the present invention Escherichia coli can be cultured to convert the amino acid substrate of the culture medium to 2-keto acid, and produce high-quality alcohols through a whole-cell bioconversion reaction capable of continuously producing high-quality alcohols.

In this case, when the amino acid mixed in a mixture medium containing various substances such as food wastes or organic wastes such as waste water is used as a substrate as well as a medium containing a single amino acid, It is possible to produce high-quality alcohol from biomass through an environmentally friendly and inexpensive process.

Therefore,

i) inducing a deamination reaction to produce pyridoxal phosphate (PLP) and copper ion (Cu) to the medium to produce 2-keto acid;

ii) inoculating recombinant E. coli overexpressing ketoisovalerate decarboxylase (KivD) in the medium from which the reaction was induced in step i); And

iii) culturing Escherichia coli inoculated in step ii) so that the 2-keto acid produced in step i) is reduced to be dicarboxylated and aldehyde-reduced, Provide a production method.

The "medium" of step i) of the present invention includes any one or more amino acids selected from the group consisting of Val, Leu, Ile, Phe, and norvaline But is not limited thereto.

The " medium " of step i) of the present invention is preferably organic wastes, but is not limited to, food wastes, livestock manure or domestic wastewater.

The term " 2-keto acid " of step i) of the present invention refers to 2-ketoisovalerate (KIV), 2- ketoisocaproate (KIC) It is preferably one or more selected from the group consisting of 2-keto-3-methylvalerate (KMV), phenylpyruvate (PP) and 2-ketovalerate (KV) But is not limited thereto. More specifically, the 2-keto acid produced when the substrate amino acid is valine is 2-ketoisovaleric acid, the 2-keto acid produced when the substrate amino acid is leucine is 2-ketoisocaproic acid, The 2-keto acid produced when isoleucine is 2-keto-3-methylvaleric acid, the 2-keto acid produced when the substrate amino acid is phenylalanine is phenylpyruvic acid, and when the substrate amino acid is novaline 2-keto acid is 2-keto valeric acid.

The "E. coli" of step ii) of the present invention is preferably, but not limited to, co-expressing an alcohol dehydrogenase (ADH) with KivD. If the Escherichia coli does not express KivD, 2-keto acid can not be converted to a higher alcohol. If ADH is not over-expressed, ADH expressing wild-type Escherichia coli can be involved to produce higher alcohol, but the conversion yield is low and is not effective . When the Escherichia coli overexpresses KivD and ADH by co-expression, 2-keto acid converted from amino acid by PLP-Cu can be effectively converted to a higher alcohol, which is effective.

The term " higher alcohol " of the present invention is a mixture of isobutanol, 2-methylbutanol, 3-methylbutanol, 2-phenylethanol and butanol. , But the present invention is not limited thereto. More specifically, the higher alcohol produced when the substrate amino acid is valine is isobutanol, the higher alcohol produced when the substrate amino acid is leucine is 2-methylbutanol, and the higher alcohol produced when the substrate amino acid is isoleucine is 3-methyl Butanol, the higher alcohol produced when the substrate amino acid is phenylalanine is 2-phenylethanol, and the higher alcohol produced when the substrate amino acid is novaline is butanol.

In a specific embodiment of the present invention, the present inventors carried out a depolymerization reaction of an amino acid that converts an amino acid to a 2-keto acid using a PLP-Cu complex, and found that 5 kinds of amino acids (valine, leucine, isoleucine, phenylalanine, Valine was able to catalyze an oxidative deamination reaction of PLP-Cu to convert it to 2-keto acid (Table 1).

Further, the present inventors confirmed that the PLP-Cu complex can convert an amino acid into 2-keto acid, and then convert the converted 2-keto acid into an enzyme such that the KivD / ADH enzyme can be converted to a higher alcohol As a result of performing the reaction in connection with the reaction, it was confirmed that the final conversion yield of the higher alcohol was low because the reducing enzyme was unstable or inactivated in the in vitro environment in which there was no reductase equivalent (Table 2).

In addition, the present inventors have succeeded in continuously expressing KivD / ADH in a medium containing PLP-Cu and KivD / ADH enzyme to produce higher alcohol from amino acid, As a result of the coupling reaction of the enzyme [PLP-Cu] - [KivD / ADH], which synthesizes higher alcohols in vivo using the recombinant Escherichia coli, the medium containing a single amino acid and the mixed amino acid When PLP-Cu was added to the culture medium (4% yeast extract) and the recombinant Escherichia coli expressing KivD / ADH was cultured, the production yield of the higher alcohol was remarkably increased (FIG. 4).

Accordingly, the pyridoxal phosphate (PLP) of the present invention can catalyze an oxidative desalination reaction in which an amino acid is converted into a 2-keto acid by forming a complex with copper ion (Cu) It can be converted to higher alcohols by ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (ADH).

In this case, when the amino acid mixed in a mixture medium containing various substances such as food wastes or organic wastes such as waste water is used as a substrate as well as a medium containing a single amino acid, It is possible to produce high-quality alcohol from biomass through an environment-friendly and inexpensive process, which is effective.

The present invention also provides a method for producing Escherichia coli-based advanced alcohols using PLP, comprising inoculating and culturing the recombinant Escherichia coli co-expressing KivD and ADH in culture medium supplemented with PLP and Cu.

The "medium" of the present invention is preferably, but not limited to, any one or more amino acids selected from the group consisting of leucine, isoleucine, phenylalanine and novaline.

The " culture medium " of the present invention is preferably organic wastes such as food wastes, livestock manure, or domestic wastes, but is not limited thereto.

The "higher alcohol" of the present invention is preferably one or more selected from the group consisting of isobutanol, 2-methylbutanol, 3-methylbutanol, 2-phenylethanol and butanol, but is not limited thereto.

The pyridoxal phosphate (PLP) of the present invention can catalyze an oxidative desamination reaction in which an amino acid is converted into a 2-keto acid by complexing with copper ion (Cu), 2-keto acid is KivD and ADH It is possible to convert recombinant E. coli expressing KivD and ADH into a culture medium containing PLP-Cu complex to convert the amino acid substrate of the culture medium into 2-keto acid, The whole cell bioconversion reaction capable of producing alcohol can produce a high-quality alcohol in a single step process, so that the unit cost required for the process can be reduced and time can be saved. Also, in the case of using amino acids mixed in a mixture medium containing various substances such as food wastes or organic wastes such as wastewater, as well as a medium containing a single amino acid, the method of producing a high alcohol of the present invention may be applied It is possible to produce high-quality alcohol from biomass through an environment-friendly and inexpensive process, which is effective.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

PLP Perform the deamination of amino acids by -Cu complex

It has been reported that when the metal ion of vitamin B6 is present, PLP can catalyze an oxidative desalination reaction that converts an amino acid to 2-keto acid. Thus, the conversion of 2-keto acid was confirmed by performing a deamination reaction of an amino acid that converts amino acid to 2-keto acid using PLP-Cu complex.

Specifically, five representative amino acids (leucine, isoleucine, valine, novaline and phenylalanine) were each dissolved in distilled water at a concentration of 10 mg / ml to prepare an amino acid solution. Then, 0.1M phosphate buffer solution (pH 7.0) containing 10 mM PLP-Cu complex was prepared as a reaction solution, and the amino acid solution prepared in 1.0 ml of the reaction solution was added to initiate the depolymerization reaction. At this time, the reaction was initiated by adding the amino acid as a substrate, PLP and Cu (II) as catalysts to the reaction solution at a molar ratio of 1: 1: 1, respectively, at a concentration of 10 mM, And reacted in a constant temperature incubator. Twenty-four hours after the start of the reaction, the reaction was rapidly cooled to terminate the reaction, and the produced 2-keto acid and ammonia in the reaction were quantitatively analyzed.

Ammonia concentration was quantified using NeuLog ammonia ion-selective sensor system. The molar concentration of 2-keto acid produced by quantitative analysis was expressed as the conversion yield by dividing the initial molar concentration of substrate amino acid by the reaction (mM 2-keto acid / mM amino acid). Amino acids were derivatized using OPA and FMOC reagents and quantified by HPLC on a ZORBAX Eclipse AAA column. For the determination of 2-keto acid, fluorescence analysis was carried out after derivatizing quinoxaline to bind quinoxaline to 2-keto acid in the reaction solution through reaction using o-phenylenediamine. 410 nm emission fluorescence was measured at a stimulus wavelength of 350 nm. The fluorescence intensity measured from each reaction solution was compared with a calibration curve of 2-keto acid to determine the concentration of 2-keto acid (mM ) Were calculated.

As a result, when PLP and Cu2 + ions were added together to the reaction solution as shown in the following Table 1, an oxidative desalination reaction occurred in all five kinds of amino acids (valine, leucine, isoleucine, phenylalanine and novaline) , 2-keto acid (Table 1). For each 2-keto acid produced according to the substrate amino acid, the reaction yield is about 45-75% (mM / mM), and the production of ammonia shows a lower conversion yield than the 2-keto acid production to 20% (Table 1). It was also confirmed that the selectivity of PLP can be maintained regardless of the branched chain or aromatic structure of the side chain of the amino acid added to the substrate in the reaction solution.

Conversion yield of 2-keto acid converted by PLP-Cu complex Substrate amino acid
Product 2-keto acid
Conversion yield (%) 2-keto acid NH ₃ Balin 2-Ketoisovaleric acid
(2-ketoisovalerate, KIV) 45 19 Leucine 73 22 Isoleucine 56 20 Phenylalanine 62 20 Novaline 53 21

In vitro In vitro ) Step PLP -Cu complex and KivD / ADH  Production of higher alcohol from amino acid by enzyme

It has been confirmed that the PLP-Cu complex can convert an amino acid into 2-keto acid, so that the converted 2-keto acid can be converted into a higher alcohol through an oxidation process, 1, a high-alcohol production reaction was carried out.

Specifically, ketoisovalerate decarboxylase (KivD) was expressed and separated from Lactococcus latis according to a known method, and Saccharomyces cerevisiae was isolated and purified. (Alcohol dehydrogenase (ADH)) was isolated and isolated from the cells [14]. Then, 10 units of purified ketoisovalerate decarboxylase (KivD; Lactococcus (1 mM MgSO ₄ , 0.5 mM thiamine pyrophosphate in 0.1 M phosphate buffer, pH 10) containing 10 units of alcohol dehydrogenase (ADH; derived from Saccharomyces cerevisiae ) and 1 mM NADH coenzyme 7.0). To prepare the substrate solution, five representative amino acids (leucine, isoleucine, valine, novaline and phenylalanine) were dissolved in distilled water at a concentration of 10 mg / ml to prepare an amino acid solution. To the 0.1M phosphate buffer solution (pH 7.0) containing 10 mM PLP-Cu complex, the amino acid solution prepared above was added at a concentration of 10 mM to prepare a solution having a molar ratio of amino acid, PLP and Cu (II) of 1: 1: 1 To prepare a substrate solution. Then, the reaction mixture (including KivD, ADH and NADH) was added to the prepared substrate solution (including amino acids, PLP and Cu (II)) to initiate amino acid deamination reaction. After 1 hour, the reaction was rapidly cooled to terminate the reaction, and the produced alcohol was quantitatively analyzed by gas chromatography (GC).

As a result, as shown in the following Table 2, amino acids were converted into higher alcohols by reaction with PLP-Cu complex and KivD / ADH enzyme and converted to higher alcohol according to the side chain structure of the substrate amino acid (Table 2). When valine, leucine, isoleucine, or novaline, which are amino acids having a branched structure on the side chain, are used as a substrate in the conversion yield to the higher alcohol, the produced higher alcohol is not more than 10% The conversion yield was about 20% when phenylalanine having an aromatic ring in the side chain was used as a substrate (Table 2). In the presence of reducing equivalents that migrate in NADPH through the production of high-grade alcohols by PLP-Cu complex and KivD / ADH enzyme in vitro, 2-keto acid is produced, Reduction reaction could be observed. However, it was confirmed that the final conversion yield of the higher alcohol was lowered because the reducing enzyme was unstable or inactivated in the in vitro environment in which no reducing enzyme equivalent was present.

In vivo In vivo ) Step PLP -Cu complex and KivD / ADH  Production of higher alcohol from amino acid by enzyme

<3-1> Production of higher alcohol from amino acid in medium containing single amino acid as substrate

In the medium containing PLP-Cu and KivD / ADH enzymes, it was confirmed that the continuous conversion reaction proceeded to produce higher alcohol from amino acid, but the in vitro reaction showed low conversion yield. To overcome this, the coupling reaction of the [PLP-Cu] catalyst- [KivD / ADH] enzyme, which synthesizes a high-level alcohol in vivo using a whole-cell bioconversion reaction in E. coli BL21 (DE3) coupling reaction was performed (FIG. 2).

Specifically, Escherichia coli expressing KivD or ADH or co-expressing KivD and ADH (KivD + ADH) was prepared according to a known method [14]. Then, E. coli expressing KivD, ADH or KivD + ADH in an M9 minimal medium [15] supplemented with 10 mg / ml of amino acids (valine, leucine, isoleucine, phenylalanine or novaline) and 1 mM PLP- And inoculated and cultured with shaking at 37 ° C. When the cultured Escherichia coli had an absorbance of 0.6 at OD 600 nm, 1 mM IPTG was added to the medium and the cells were cultured at 37 ° C for 4 days. After completion of the cultivation, a medium was obtained and the high-alcohol produced in the medium was quantitatively analyzed by GC analysis.

As a result, as shown in FIG. 3, the E. coli cells expressing only ADH without overexpression of KivD did not produce high-level alcohol even though the [PLP-Cu] complex was present in the culture medium, and did not overexpress ADH, In one E. coli experimental group, it was confirmed that the amino acid could be converted to the corresponding higher alcohol, and it was confirmed that the conversion yield was about 10 to 20% (FIG. 3). E. coli cultured in culture medium without PLP-Cu complex did not produce alcohol regardless of expression of KivD or ADH, and PLP plays a major role in the conversion of amino acid to 2-keto acid and synthesis of higher alcohol The results of this study are as follows. In addition, it was confirmed that the recombinant Escherichia coli, which overexpressed KivD alone without overexpressing ADH by recombination, was able to achieve a low yield of alcohol conversion using ADH contained in Escherichia coli strains even when ADH derived from Saccharomyces cerevisiae was not overexpressed Respectively.

In addition, when Escherichia coli co-expressing ADH and KivD was cultured in a culture medium containing [PLP-Cu] to produce high-grade alcohol, the yield of high-alcohol production was significantly increased, Which is about twice as high as that of Escherichia coli expressing E. coli alone. In particular, it was confirmed that 2-phenylethanol produced using 2-phenylalanine as a substrate exhibited a conversion yield of 80% or more (FIG. 3).

<3-2> Production of higher alcohol from amino acid in medium containing mixed amino acid as substrate

In known studies for producing higher alcohols from 2-keto acids, it is known that additional addition of 2-keto acid in the medium can play an important role in increasing the conversion yield. Therefore, in order to determine whether or not a high-quality alcohol can be efficiently produced through PLP-Cu and KivD / ADH enzyme reactions when amino acids mixed in a mixture medium containing various substances such as food waste or wastewater are used as a substrate, The production of higher alcohol was confirmed from the amino acid mixture of yeast extract.

Specifically, 40 g / l BD bacto yeast extract (21.64 g / l amino acid, 4.48 g / l ash, 3.05 g / l various salts, 1.24 g / l water and 6.53 g / l carbohydrate including;) and M9 salts _{(6.3 g / ℓ NaHP 4,} 3.0 g / ℓ KH 2 PO 4, 0.5 g / ℓ NaCl, 24 ㎎ / ℓ ㎎SO 4, 2.2 ㎎ / ℓ CaCl 2 And 2.0 mg / l vitamin B1) were mixed to prepare an amino acid culture medium of 4% yeast extract. Amino acid media includes 1.20 g / l isoleucine, 1.64 g / l leucine, 1.04 g / l phenylalanine, 1.40 g / l valine, 2.40 g / l amino nitrogen and trace essential elements. Then, 1 mM PLP-Cu complex was added to the prepared amino acid medium, and E. coli expressing KivD or ADH or co-expressing KivD and ADH (KivD + ADH) was inoculated and cultured at 37 ° C with shaking. When the cultured Escherichia coli had an absorbance of 0.6 at OD 600 nm, 1 mM IPTG was added to the medium and the cells were cultured at 37 ° C for 4 days. During the main culture, the medium was obtained once every 24 hours, and the high-alcohol produced in the medium was quantitatively analyzed by GC analysis.

As a result, as shown in FIG. 4, when KivD + ADH co-expressed Escherichia coli was cultured in a medium containing PLP-Cu compared to culturing KivD + ADH coexpressing Escherichia coli in a medium without PLP-Cu, It was confirmed that the production of alcohol remarkably increased. In particular, the conversion of 2-phenylethanol from phenylalanine was found to be the highest level and it was confirmed that it exhibited a produvtivity of 1.0 g / l and a conversion yield of 55% (Figs. 4A and 4B).

Claims (10)

i) inducing a deamination reaction to produce pyridoxal phosphate (PLP) and copper ion (Cu) to the medium to produce 2-keto acid;
ii) inoculating recombinant E. coli overexpressing ketoisovalerate decarboxylase (KivD) in the medium from which the reaction was induced in step i); And
iii) culturing Escherichia coli inoculated in step ii) so that the 2-keto acid produced in step i) is reduced to be dicarboxylated and aldehyde-reduced, Production method.
The method according to claim 1, wherein the medium of step i) comprises at least one amino acid selected from the group consisting of Val, Leu, Ile, phenylalanine (Phe) and norvaline Wherein the method comprises the steps of:
The method according to claim 1, wherein the medium of step i) is organic wastes such as food wastes, livestock manure or domestic wastewater.
The method of claim 1, wherein the 2-keto acid of step i) is selected from the group consisting of 2-ketoisovalerate, KIV, 2-ketoisocaproate, Characterized in that it is at least one selected from the group consisting of 2-keto-3-methylvalerate, KMV, phenylpyruvate (PP) and 2-ketovalerate (KV) To produce a high-alcohol-based Escherichia coli using PLP.
The method according to claim 1, wherein the Escherichia coli in step ii) co-expresses an alcohol dehydrogenase (ADH) with KivD.
The method of claim 1, wherein the higher alcohol is selected from the group consisting of isobutanol, 2-methylbutanol, 3-methylbutanol, 2-phenylethanol, Wherein the PLP-based high-alcohol production method is based on E. coli.
PLP and Cu in a culture medium containing a recombinant Escherichia coli co-expressing KivD and ADH, and culturing.
The method according to claim 7, wherein the culture medium contains at least one amino acid selected from the group consisting of valine, leucine, isoleucine, phenylalanine and novaline.
[8] The method of claim 7, wherein the culture medium is an organic waste selected from the group consisting of food waste, livestock manure, and domestic wastewater.
8. The method according to claim 7, wherein the higher alcohol is at least one selected from the group consisting of isobutanol, 2-methylbutanol, 3-methylbutanol, 2-phenylethanol and butanol. Alcohol production method.

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