WO2009154378A2 - Method for preparing oxides using germs in corynebacterium - Google Patents

Abstract

The present invention relates to a method for preparing oxides characterized in that strains in corynebacterium transformed with genes that code an oxygenase capable of adding oxygen to a substrate are employed in a method for preparing an oxide by inducing an oxidation wherein oxygen is added to a substrate, thus remarkably increasing the yield rate of oxides as compared to conventional methods which use existing coliform bacilli.

Description

Method for preparing oxide using bacteria of Corynebacterium

The present invention relates to a method of preparing an oxide by inducing an oxidation reaction of adding oxygen to a substrate, and to a method of preparing an oxide using a strain of Corynebacterium genus transformed with a gene encoding an oxidase. .

Oxidized compounds can be prepared by chemical methods using chemical catalysts and biological methods using biocatalysts such as enzymes or microorganisms. Usually, chemical methods are performed under high temperature and high pressure and are highly toxic organic solvents or heavy metals. Although ions are required, biological methods are carried out at room temperature and atmospheric pressure using oxidase or microorganisms producing oxidase as catalysts. In addition, it does not require organic solvents or heavy metal ions other than the substrate during the reaction, it is excellent in reaction selectivity and low amount of by-products generated.

 On the other hand, when preparing an oxidizing compound using a biocatalyst, a coenzyme such as NAD (P) H is usually required. Therefore, microbial catalysts having high coenzyme regeneration efficiency are widely used in comparison with enzyme catalysts, and are widely used as microbial catalysts, such as recombinant E. coli or Pseudomonas genus that produce oxidase.

 Recombinant Escherichia coli is easy to genetically engineer, analyze and control metabolism, and mass cultivation, but it does not have high regeneration efficiency of coenzyme, which is essential for oxidation reaction, low resistance to organic compounds, and rapidly decreases metabolic activity by producing acetic acid during reaction. There is this.

 Bacteria in the recombinant Pseudomonas are highly resistant to the toxicity of organic compounds and do not produce acetic acid during the reaction, but are less productive than recombinant E. coli due to difficult metabolic analysis and control (J. Microbiol. Biotechnol. (2007) 17: 379-392).

 Therefore, there is a great demand for researches to develop microorganisms that are easy to genetically manipulate and control metabolism, have high resistance to the toxicity of organic compounds, and produce less metabolic inhibitors such as acetic acid as biocatalysts for use in the production of oxides.

Accordingly, an object of the present invention is to develop and provide a method for producing an oxide using microorganisms having high resistance to the toxicity of organic compounds and producing less metabolic inhibitors such as acetic acid as biocatalysts.

In order to achieve the above object, the present invention provides a method for producing an oxide by inducing an oxidation reaction of adding oxygen to a substrate, the corynebacte transformed with a gene encoding an oxidase that can add oxygen to the substrate It provides a method for producing an oxide, characterized in that using the strain of the genus Leeum.

Hereinafter, the problem solving means of the present invention will be described in detail.

The method of preparing an oxide by inducing an oxidation reaction in which oxygen is added to a substrate may generate a material having a higher value than that of the added substrate. The present invention does not directly prepare an oxide by directly reacting an oxidase to the substrate. It is characterized by producing an oxide using a strain of the genus Corynebacterium transformed with a gene encoding an oxidase that can add oxygen to.

The method for producing an oxide using microorganisms has the advantage of having a small amount of by-products due to excellent reaction selectivity without the need for organic solvents or heavy metal ions other than a substrate during the reaction, and cost-effective compared to using purified enzymes. Excellent at

Oxygenases, also called oxygenated enzymes, are oxidoreductases that undergo oxidation by activating molecular oxygen. Two oxygen atoms are included in the product at the same time, only one atom of molecular oxygen enters the product, and the other produces hydrogen by obtaining hydrogen from NAPH (DPNH) or NADPH (TPNH).

The present invention transforms Corynebacterium strains to express or overexpress oxidase, and by using the strains of Corynebacterium from Example 1, Example 2, Example 3 and Comparative Example 1 than E. coli The production rate was high, and specific product formation rate, volumetric productivity, and production yield were higher than those using E. coli in the production of oxide using Corynebacterium spp.

Corynebacterium strains can be deduced about three reasons for the faster oxidation rate than E. coli.

First, Corynebacterium spp. Has higher regeneration rate of NADPH, so the oxidation rate is higher than that of Escherichia coli. In microorganisms, NADPH is regenerated by the pentose phosphate pathway and the isocitrate dehydrogenase and malic enzymes of the TCA cycle. When glucose is used as the carbon source, about 25% of glucose in E. coli is metabolized via the pentose phosphate pathway (J. Bacteriol. (1999) 181 (21): 6679-6688), Coryne In Bacterium spp., About 44% of glucose is metabolized via the pentose phosphate pathway (J. Microbiol. Biotechnol. (2006) 16 (8): 1174-1179).

Second, the Corynebacterium spp. Has a higher production rate of toxic metabolite, which may lower the activity of the biocatalyst during the oxidation reaction. For example, when oxidizing cyclohexanone, E. coli produces acetic acid at a rate of 0.05 g / g dry cells / h (Appl. Microbiol. Biotechnol. (2007) 76: 329-338) Corynebacte Leeum is produced at a rate of 0.03 g / g dry cells / h (Example 2).

Third, Corynebacterium spp. Maintains high oxidation reaction rate because it is more resistant to the toxicity of organic compounds than Escherichia coli. Corynebacterium spp. Has a very low cell wall permeability to organic compounds compared to Escherichia coli, which has a cell wall composed of mycomembrane and S-layer (J. Biotechnol. (2003) 104: 55-67). For example, E. coli catalyst cycloalkyl becomes higher as cyclohexanone concentration of 25g / l or more of cyclohexanone by a toxic effect only both losing the oxidation activity of the cyclohexanone Corynebacterium glutamicum (Corynebacterium glutamicum) catalyst cycloalkyl It has been reported that high oxidative activity was maintained even when the concentration of hexanone was 30 g / l (J. Biotechnol. (2009) 142: 164-169).

Meanwhile, the Corynebacterium spp. Bacteria used in the present invention are not limited to a specific kind, but are preferably Corynebacterium glutamicum and Corynebacterium ammoniagenes . It is good.

 On the other hand, the oxidase is not limited to a specific enzyme, but is preferably a Bayer-Villiger oxidase, but the oxidation reaction using the Bayer-Villiger oxidase is ketones. It is an oxidation reaction of ketone, which converts ketones and ketone derivatives, which are known to be relatively stable unlike aldehyde, to their oxides ester and lactone.

 The Bayer-Villiger oxidase may be, for example, cyclohexanone monooxygenase, wherein the substrate is cyclohexanone.

On the other hand, the method for producing an oxide of the present invention is preferably cultured strains of Corynebacterium strain transformed with a gene encoding an oxidase that can add oxygen to the substrate in a glucose-limited fed-batch culture, most preferably The substrate is preferably supplied after all the glucose has been consumed, because the substrate can inhibit the growth of cells.

 As described above, the present invention exhibits an effect of significantly increasing oxide productivity compared to the method using the E. coli by using a strain of Corynebacterium genus transformed to produce oxidase as a biocatalyst.

 Figure 1 shows the production of ε-caprolactone (ε-Caprolactone) from cyclohexanone (cyclohexanone).

Figure 2 shows the growth curve of recombinant Corynebacterium glutamicum and the production curve of acetic acid and ε-caprolactone. Recombinant Corynebacterium glutamicum was incubated in Riesenberg medium supplemented with biotin, and fed on a fed-batch diet using glucose when the carbon source was depleted. IPTG was added to 1.0 mM when g dry cells / l was reached to induce cyclohexanone monooxygenase production, and when the cell concentration reached 8.0 g dry cells / l, cyclohexanone was added. The lactone production reaction was initiated by addition at 6.0 g / l. The culture temperature was 30 ℃, the air supply rate was 1.0 vvm, the stirring rate was 600rpm, pH was adjusted to 6.8.

Figure 3 is a diagram showing the growth curve and acetic acid and ε-caprolactone production curve of the recombinant Corynebacterium glutamicum ( Corynebacterium glutamicum ). Recombinant Corynebacterium glutamicum was incubated in Riesenberg medium with biotin and fed with a fed-batch diet using glucose when the carbon source was depleted. The cell concentration was 15.0 g dry cells. When / l was reached, IPTG was added to 1.0 mM to induce the expression of cyclohexanone monooxygenase, and when the cell concentration reached 20.0 g dry cells / l, cyclohexanone was added. The oxidation reaction was initiated by addition at 6.0 g / l. Cyclohexanone was further added when the substrate concentration dropped below 3.0 g / l. The culture temperature was 30 ℃, the air supply rate was 1.0 vvm, the stirring rate was 1000rpm, pH was adjusted to 6.8.

4 shows a reaction in which hydroxy dyzein is produced from the substrate dyedzein.

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples, and includes modifications of equivalent technical spirit.

Example 1 Preparation of ε-Caprolactone

Example 1 is a cyclohexanone derived from Acinetobacter to produce ε-caprolactone in an oxide prepared by inducing an oxidation reaction of adding oxygen to a substrate. Corynebacterium glutamicum was transformed using a monooxygenase gene, and cyclohexanone was used as a substrate. The reaction for producing ε-caprolactone (ε-Caprolactone) from the substrate cyclohexanone is shown in FIG. 1.

1. Recombinant Corynebacterium glutamicum ( Corynebacterium glutamicum ) Produce

Recombinant plasmid was constructed so that the wild strain Corynebacterium glutamicum ATCC13032 produced cyclohexanone monooxygenase from Acinetobacter . Recombinant plasmid was derived from Acinetobacter (cyclohexanone monooxygenase) gene (Biotechnol. Prog. (2002) in the corynebacterium expression vector pEKEX-2 (Gene (1991) 102: 9398). 18: 262-268).

The bacteria transformed with the recombinant plasmid were named Corynebacterium glutamicum pEKEx2- chnB .

2. The recombinant Corynebacterium glutamicum prepared above ( Corynebacterium ε-caprolactone production using glutamicum)

(1) spawn culture

Recombinant Corynebacterium glutamicum colonies grown on 'Brain heart infusion (BHI)' agar medium containing kanamycin were cultured in BHI medium containing kanamycin for a reasonable time. And used as a spawn. The culture conditions, the temperature was 30 ℃, the stirring speed was 200rpm.

(2) ε-caprolactone production

100 ml of the recombinant Corynebacterium glutamicum culture medium was inoculated into Reesenberg medium to which 900 ml of biotin was added and then incubated with stirring at 30 ° C. at 600 rpm. Fed-fed cultivation was started when glucose was initially depleted, and when the cell concentration reached 5.8 g dry cells / l, Isopropyl β-D-1-thiogalactopyranoside (IPTG) was 1.0 mM. It was added to the culture broth to induce the expression of cyclohexanone monooxygenase (cyclhexanone monooxygenase). After that, when the cell concentration reached 7.0 g dry cells / l, cyclohexanone was added at 6.0 g / l to start the oxidation reaction.

5 hours after the start of the oxidation reaction, ε-caprolactone was produced at 5.9 g / l (FIG. 2). The specific product rate during the oxidation reaction was 0.15 g / g dry cells / h, the volumetric productivity was 1.2 g / l / h, and the yield was over 94%.

Example 2: Preparation of [epsilon] -Caprolactone

After inoculating 100 ml of the recombinant Corynebacterium glutamicum spawn culture medium prepared in the same manner as in Example 1 in a Riesenberg medium added with 900 ml of biotin, 30 Incubate with stirring at 1000 rpm at ℃.

When the carbon source was depleted, the fed-batch cultivation using glucose was started. When the cell concentration reached 15.0 g dry cells / l, the expression of cyclohexanone monooxygenase was added by adding IPTG to the culture medium at 1.0 mM. When the cell concentration reached 20.0 g dry cells / l, cyclohexanone was added at 6.0 g / l to start the oxidation reaction.

Cyclohexanone was further added when the substrate concentration fell below 3.0 g / l during the reaction. After the initiation of the oxidation reaction, ε-caprolactone was produced at 16.0 g / l after 7 hours (FIG. 3). The specific product rate during the reaction was 0.12 g / g dry cells / h, the volumetric productivity was 2.3 g / l / h, and the yield was over 94%.

Comparative Example 1: Preparation of ε-caprolactone using E. coli

E. coli was transformed into a cyclohexanone monooxygenase gene derived from Acinetobacter in the same manner as in Example 1, and a substrate was used as cyclohexanone (cyclohexanone).

Experiments under reaction conditions similar to Example 2 showed that the specific production rate of the reactants was 0.02 g / g dry cells / h and the volumetric productivity was 0.82 g / l / h (Appl. Microbiol. Biotechnol. (2007) 76: 329-338).

The production of the first and second embodiments of the Corynebacterium glutamicum ε- caprolactone with (Corynebacterium glutamicum) using the E. coli of the comparative example 1 than in Example 1, 2 and Comparative Example 1 In summary, Compared with the preparation of ε-caprolactone, the specific productivity was 5 times or more, the volumetric productivity was 2.7 times or more, and the final reaction product concentration was 45% or more.

From the above results, the present invention using the strain of Corynebacterium genus as a biocatalyst was able to confirm that the productivity of the oxide can be significantly increased than the conventional method using E. coli.

Example 3 Preparation of 3'-hydroxydaidzein (7,3 ', 4'-trihydroxyisoflavone)

The present embodiment 3 is hydroxy and daidzein (3'-hydroxydaidzein) a Bacillus (Bacillus) cytochrome P450 mono Oxygen derived to produce the oxide to be produced to induce the oxidation reaction of addition of oxygen to the substrate kinase (cytochrome monooxygenase Corynebacterium glutamicum was transformed using the gene (J. Mol. Cat. B: Enz. (2009) 59: 248-253 ), and the substrate was made of diaidzein. Was used. The reaction in which hydroxy dyzein is produced from the substrate dyedzein is shown in FIG. 4.

1. Recombinant Corynebacterium glutamicum ( Corynebacterium glutamicum ) Produce

Ferredoxin Lee; (CYP107H1 cytochrome monooxygenase) and Pseudomonas (Pseudomonas) derived reductase kinase (ferredoxin reductase wild strain of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 is Bacillus (Bacillus) P450 cytochrome mono Oxygen dehydratase derived; PdR), ferredoxin (Pdx), to construct a recombinant plasmid. Recombinant plasmids of Corynebacterium, Bacillus (Bacillus) P450 cytochrome mono Oxygen dehydratase derived from the expression vector pEKEX-2; insert (cytochrome monooxygenase CYP107H1) gene, and Corey four Pseudomonas (Pseudomonas) in tumefaciens expression vector pVWEX2 Derived ferredoxin reductase (ferredoxin reductase (PdR), ferredoxin (Pdx) genes were made by inserting.

Bacteria transformed with the recombinant plasmid were identified as Corynebacterium glutamicum (Corynebacterium glutamicum) pCYPPdRPdx.

2. The recombinant Corynebacterium glutamicum prepared above ( Corynebacterium glutamicum Hydroxy Dyzedine Production Using

(1) spawn culture

Recombinant Corynebacterium glutamicum colonies grown on 'Brain heart infusion (BHI)' agar medium containing kanamycin were cultured in BHI medium containing kanamycin for a reasonable time. And used as a spawn. The culture conditions, the temperature was 30 ℃, the stirring speed was 200rpm.

(2) Hydroxy Dyzedine Production

5 ml of recombinant Corynebacterium glutamicum culture medium was inoculated into Reesenberg medium to which 95 ml of biotin was added and then incubated with stirring at 30 ° C. at 200 rpm. When the cell concentration reached 1.0 g dry cells / l, IPTG was added to the culture medium at 1.0 mM to give P450 cytochrome monooxygenase (CYP107H1), ferredoxin reductase (PdR), and ferredoxin ( ferredoxin; Pdx). After that, when the cell concentration reached 2.0 g dry cells / l, the oxidation reaction was started by adding Dyzein at 50 μM.

Six hours after the initiation of the oxidation reaction, hydroxydydzein was produced at 2 μM. This is more than six times higher in production yield and reaction product concentration than recombinant E. coli (J. Mol. Cat. B: Enz. (2009) 59: 248-253) transformed with the same gene.

Claims (4)

1. In the method for producing an oxide by inducing an oxidation reaction of adding oxygen to the substrate,

   Method for producing an oxide, characterized in that using the strain Corynebacterium strain transformed with a gene encoding an oxidase that can add oxygen to the substrate
2. The method of claim 1,

   Corynebacterium bacteria,

   Corynebacterium glutamicum ( Coynebacterium glutamicum ), Corynebacterium ammonia genes ( Coynebacterium ammoniagenes ) characterized in that the manufacturing method of the oxide
3. The method of claim 1,

   Oxidase,

   Method for producing an oxide, characterized in that the Bayer-billigirl oxidase
4. The method of claim 1,

   The method for producing the oxide,

   Method for producing an oxide, characterized in that cultured strains of Corynebacterium transformed with a gene encoding an oxidase that can add oxygen to the substrate in a glucose-limited fed-batch culture

Images