KR102338436B1 - Recombinant microorganism for producing lactobionic acid and method for producing lactobionic acid using the same - Google Patents

Abstract

The present invention relates to a recombinant microorganism having increased ability to produce lactobionic acid and a method for improving productivity of lactobionic acid using the same. The recombinant microorganism provided by the present invention has excellent ability to produce lactobionic acid, and can be usefully used industrially.

Description

Recombinant microorganism and lactobionic acid production method using the same {Recombinant microorganism for producing lactobionic acid and method for producing lactobionic acid using the same}

The present invention relates to a recombinant microorganism and a method for producing lactobionic acid using the same.

Lactobionic acid, a type of polyhydroxy bionic acid as a next-generation skin whitening material, is biodegradable and biocompatible in addition to the exfoliating, anti-wrinkle, and skin whitening effects of α-hydroxy acid. , anti-aging, antioxidant, moisturizing, chelating properties, etc., and has little irritation to the skin, drawing attention as a next-generation cosmetic material that can replace glycolic acid and lactic acid. Cosmetics containing lactobionic acid It has been commercialized and is now on sale.

Lactobionic acid is a substance formed by β-1,4 bonds of galactose and gluconic acid, has a molecular weight of 358.3, and is a water-soluble white crystalline compound. Lactobionic acid can be synthesized by the oxidation reaction of the free aldehyde group in lactose, and there are currently many studies on production by chemical, electrochemical, catalytic or biological oxidation. [Satory et al., Biotechnology letters 19(12) 1205-08, 1997].

Among them, in order to respond to the interest and use of lactobionic acid as a raw material for food and cosmetics, research on biological lactobionic acid production through an environmentally friendly microbial culture method is being intensively conducted. Res ( Acetobacter orientalis ), Brookholderia cepacia ( Burkholderia cepacia ), Gluconobacter ( Gluconobacter ) genus, Gluconacetobacter ( Gluconacetobacter ) genus, Paraconiothyrium ) KD-3, Penicillium chrysoge Num ( Penicillium chrysogenum ), Pseudomonas tatrons ( Pseudomonas ) taetrolens ), Streptococcus lactis and Zymomonas mobilis mobilis ) are known.

Numerous references are referenced throughout this specification and citations thereof are indicated. The disclosures of the cited documents are incorporated herein by reference in their entirety to more clearly describe the content of the present invention and the level of the art to which the present invention pertains.

Republic of Korea Patent Publication No. 10-2085318

An object of the present invention is to provide a recombinant microorganism with improved lactobionic acid production ability and a method for improving lactobionic acid productivity using the same in order to solve the limitation of lactobionic acid production through culturing of wild-type strains.

Specifically, the present invention uses a recombinant vector overexpressing the gdh3 (Quinoprotein glucose dehydrogenase: 1.1.5.2) gene, transforming it into a wild-type microorganism to make a recombinant strain, and from this recombinant strain, lactobionic acid productivity is improved compared to the existing strain to provide a production system.

Accordingly, it is an object of the present invention to provide a recombinant vector comprising a nucleic acid sequence encoding a quinoprotein glucose dehydrogenase (gdh3) enzyme.

Another object of the present invention is to provide a recombinant microorganism overexpressing the gdh3 gene into which the recombinant vector is introduced.

Another object of the present invention is to provide a method for producing the recombinant microorganism.

Another object of the present invention is to provide a method for producing lactobionic acid using the recombinant microorganism.

Further objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

One aspect of the present invention is to provide a recombinant vector comprising a nucleic acid sequence encoding a Quinoprotein glucose dehydrogenase (gdh3) enzyme derived from a microorganism of the genus Pseudomonas.

The problem in the production of lactobionic acid is that there is a difficulty in process development, but the difficulty in securing an excellent strain plays a large role. Since the action of the produced enzyme and the oxidizing ability of the substrate are very different depending on the type of microorganism that produces lactobionic acid, which is currently known, a new enzyme with excellent lactobionic acid production capacity has been discovered, and the development of a recombinant strain and it Research on the production technology used is very important.

Previously, acetic acid bacteria were widely known as lactobionic acid-producing microorganisms, but Komagataeibacter m-GDH enzyme (membrane-bound quinoprotein glucose dehydrogenase) isolated from medellinensis has only about 5% of lactose oxidation capacity when glucose oxidation activity is 100%, so its lactose oxidation ability is insignificant, When glucose is present in the medium, there is a problem in that the ability to produce lactobionic acid through lactose oxidation is significantly inhibited. Although Gluconobacter suboxydans is an acetic acid bacterium and has various aldose oxidizing abilities, it was confirmed that the Quinoprotein glucose dehydrogenase enzyme derived therefrom does not oxidize lactose. In the case of another lactobionic acid producing strain, Acinetobacter calcoaceticus , the GDH enzyme derived therefrom had only about 65% of the lactose oxidizing activity compared to the glucose oxidizing activity.

Under this circumstance, the present inventors, in the case of gdh3 (Quinoprotein glucose dehydrogenase) enzyme isolated from Pseudomonas genus among various known lactobionic acid-producing bacteria, is selective among various substrates, unlike Quinoprotein glucose dehydrogenase enzyme isolated from other bacteria. was found to be an enzyme capable of oxidizing lactose.

In one embodiment, in the case of the gdh3 enzyme derived from Pseudomonas tetrorence of the present invention, when the glucose oxidation activity is 100%, the lactose oxidation ability is greater than 100%, preferably greater than 110%, 120% greater than, greater than 130%, greater than 140%, greater than 150%, greater than 160%, greater than 170%, greater than 180%, or greater than 190%, more preferably greater than 200%.

As described above, since the gdh3 enzyme isolated from the microorganism of the genus Pseudomonas of the present invention has the ability to selectively oxidize lactose among various substrates, the lactose oxidation ability is not inhibited even in the presence of glucose, and significant lactobionic acid productivity is achieved through lactose oxidation. There are advantages to indicate.

The Pseudomonas genus microorganism is Pseudomonas florescens ( Pseudomonas fluorescens ), Pseudomonas Moraviansis ( Pseudomonas ) moraviensis), Pseudomonas Grana den sheath (Pseudomonas granadensis), Pseudomonas and the like collection Language System (Pseudomonas Koreensis), Pseudomonas Tet Lawrence (Pseudomonas taetrolens ) is not limited.

The nucleic acid sequence encoding a Quinoprotein glucose dehydrogenase (gdh3) enzyme derived from a microorganism of the genus Pseudomonas of the present invention may be performed by synthesizing a tetrorence-derived gdh3 gene.

The expression vector including the nucleic acid sequence encoding the gdh3 enzyme may be a plasmid vector, preferably pDSK519.

Another aspect of the present invention is to provide a recombinant microorganism overexpressing the gdh3 gene into which the recombinant vector is introduced.

In one embodiment, the microorganism may be a Gram-negative bacteria, for example, including but not limited to Terrabacteria, proteobacteria, spirals, sphingobacteria, and subgroups, preferably the microorganisms are wild-type Pseudomonas microorganisms, More preferably, wild-type Pseudomonas tetrorence ( Pseudomonas taetrolens ) the recombinant vector is introduced. The recombinant microorganism may have an increased expression level of the gdh3 gene or an increased production of lactobionic acid compared to wild-type Pseudomonas tetrorence.

Another aspect of the present invention is to obtain a recombinant vector comprising a nucleic acid sequence encoding the enzyme gdh3 (Quinoprotein glucose dehydrogenase) derived from a microorganism of the genus Pseudomonas; And to provide a method for producing a recombinant microorganism having improved lactobionic acid production ability, comprising the step of introducing the recombinant vector into Gram-negative bacteria.

In one embodiment, in order to prepare the recombinant microorganism of the present invention, a gene encoding a soluble glucose dehydrogenase gdh3 (Quinoprotein glucose dehydrogenase: 1.1.5.2) derived from Pseudomonas tetrorence was amplified through a specific primer through PCR.

Thereafter, a recombinant plasmid was prepared in which the gene was inserted into the expression vector pKK223-3 plasmid for E. coli and the expression vector pDSK519 for Pseudomonas having high replication ability using restriction enzymes ( Sph I, EcoR I), which were pKK223-gdh3 and pDSK519 It was named -gdh3.

The recombinant plasmid pKK223-gdh3 was transformed into E. coli (E. coli DH5α) to prepare a recombinant E. coli, and pDSK519-gdh3 was transformed into Pseudomonas stetrorence to prepare an overexpressing recombinant strain.

Another aspect of the present invention is to provide a method for producing lactobionic acid using the aforementioned recombinant microorganism.

In one embodiment of the present invention, in the step of culturing Pseudomonas, it contains lactose at a concentration of 1 to 20% by volume, 0.2% yeast extract, 0.5% peptone, and 0.1% beef extract, and 0.5% NaCl. . The microorganism of the genus Pseudomonas is Pseudomonas tetrorence, and the culturing step may be culturing for 48 to 120 hours at 50 ml to 2.0L of NB medium, 20°C to 37°C.

If necessary, the culture can be cultured according to the lactose concentration, and in a culture medium in the presence of CaCO3 as a pH corrector, for example, the lactose concentration in the medium can be varied from 10 g/L to 200 g/L, It can be carried out with or without CaCO3 added.

In a preferred embodiment, the culture of the present invention may be performed by a fed-batch culture method, but is not limited thereto.

In addition, in an embodiment of the present invention, the method of improving productivity through culturing of the Pseudomonas tetrorence strain may be developed as disclosed in Korean Patent Application Laid-Open No. 10-2018-0047470, and Korean Patent Application Laid-Open No. 10-2018- The entire disclosure of 0047470 is incorporated herein by reference.

According to the present invention, by producing a recombinant plasmid and a recombinant Pseudomonas tetrorence strain overexpressing a gene effective for lactobionic acid conversion, a strain with superior lactobionic acid productivity than the wild-type strain can be developed, thereby improving the lactobionic acid conversion rate. Accordingly, the novel recombinant strain can be efficiently used in various industrial fields by improving the production rate in the lactobionic acid mass production process.

1 shows a schematic diagram of a recombinant plasmid for gdh3 expression in E. coli using the pKK223 vector.
Figure 2 shows a schematic diagram of a recombinant plasmid for gdh3 expression in Pseudomonas tetrorence using the pDSK519 vector.
3 is a result showing the lactobionic acid conversion of wild-type Pseudomonas tetrorence wild-type E. coli (E.coli-pKK223) and recombinant E. coli (E.coli-pKK223-gdh3) in the presence of lactose.
4 is a diagram showing the results of SDS-PAGE electrophoresis showing gdh3 overexpression by recombinant Pseudomonas tetrorence strain. (lane M: SDS-PAGE standards Marker, Broad Range, Bio-Rad, lane 1: Pseudomonas taetrolens / pDSK519 , lane 2: Pseudomonas taetrolens / pDSK519-gdh3)
5 is a graph showing the results of lactose metabolism (a) and lactobionic acid production (b) of wild-type Pseudomonas tetrorence and recombinant Pseudomonas tetrorence prepared through the present invention in a 5L fermentation tank under aerobic fermentation conditions.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples.

Example

Example 1: Preparation of recombinant plasmid and preparation of recombinant strain

Using Escherichia coli ( Ecoil ) to select the gene involved in the production of lactobionic acid, and to prepare this Pseudomonas tetrorence overexpression recombinant strain, the experiment was performed as follows.

Experimental Example 1 : Recombinant plasmid preparation

Gdh3 (Quinoprotein glucose dehydrogenase), which is expected to be essential for lactobionic acid production, from Pseudomonas tetrorence, which has excellent lactobionic acid production ability through lactose (lactose) oxidation reaction, was transferred from the Pseudomonas tetrorence genome gene template in the forward (5'-) direction. Polymerase chain reaction using primers CTGCAGAGGAATATGATATGAGTACGCAAGCGAAAGGCTCAG-3', SEQ ID NO: 1) and reverse (5'-GAATCTCATTTCTCTTTAGGATCGGGCAGCGCG-3', SEQ ID NO: 2) primers (once for 5 minutes at 94°C; once at 94°C for 30 seconds, at 55°C for 2 minutes The gdh3 gene was amplified by the presence of an EcoR ° recognition site in the forward primer and a Pst ° recognition site in the reverse primer through 30 reactions for 20 seconds and 30 seconds at 72 ° C; once for 7 minutes at 72 ° C). The amplified gene was isolated through agar gel electrophoresis, and the amplified and isolated DNA fragment was digested with EcoR ° and Pst °, and then pKK223-3 and Pseudomonas plasmids, which are conventional plasmid vectors for E. coli, cut with EcoR ° and Pst °. Recombinant plasmids shown in FIGS. 1 and 2 were prepared by introducing the vector pDSK519.

Experimental Example 2 : Recombinant strain production

Plasmids pKK223-3-gdh3 and pDSK519-gdh3 prepared in Experimental Example 1 were introduced into E. coli DH5a competent cells by heat shock (42° C.) and transformed. The pKK223-3 plasmid has an ampicillin resistance gene as a selection marker, and colonies into which pKK223-3 has been introduced were selected in LB-agar medium containing ampicillin. DNA sequencing of the PCR product through colony PCR of the selected colonies (Macrogen) to confirm the transformation of the gdh3 gene, and since the pDSK519 plasmid has a kanamycin resistance gene, E. coli transformed by the same method was selected.

In the transformation method of Pseudomonas tetrorence, a transformation method by electroporation was used in order to improve the stable production and efficiency. Cells for electrical transformation were prepared through the following process.

Pseudomonas tetrolence (P. taetrolens KCTC 12501 strain was purchased from Korea Research Institute of Bioscience and Biotechnology Biological Resources Center) on LB (Luria-Bertani) plate (1% tryptone, 0.5 % yeast extract, 1 % NaCl, 1.5 % agar) It was plated and incubated for 24 hours in a stationary incubator at 25°C. Colonies of the strain grown on the LB plate were inoculated into 200 mL of LB broth, and cultured until OD (600 nm) became 0.6 (measured with a spectrophotometer) at 150 rpm in a stirred incubator at 25 ° C. The culture medium and the strain cells were separated by centrifugation, the culture medium was discarded, and the strain cells were washed with 30 mL of 10% glycerol. 10% glycerol and strain cells were separated by centrifugation, and the separated 10% glycerol was discarded. The above process was repeated 3 times, and the washed strain cells obtained through centrifugation were released in 3 mL of 10% glycerol. When introducing the pDSK519-gdh3 plasmid into microbial cells by electric shock, 80ul of the cells for electrical transformation and 5ul of the plasmid are put into a cuvette for electric shock transformation, and after applying a voltage of 1.8kV for 5ms, SOC medium (2% tryptone , 0.5% yeast extract, 0.05% NaCl, 0.0186% KCl, 0.095% MgCl2, 0.6% glucose) 1ml) was added and cultured for 1 hour at 100rpm in a stirred incubator at 25°C to prepare a recombinant strain. The prepared recombinant strain was spread on an LB plate and cultured at 25°C. Through this, a recombinant strain was developed in which pDSK519-gdh3, a recombinant plasmid, was transformed into Pseudomonas tetrorence.

Example 2 : gdh3 Preparation of overexpressed recombinant E. coli and using the same lactobionic acid Check production capacity

Quinoprotein glucose dehydrogenase (gdh3) obtained from wild-type Pseudomonas tetrorence species is a water-soluble glucose dehydrogenase using pyrroloquinolinequinone (PQQ) as a coenzyme. Whether the gene is a gene encoding an enzyme essential for the conversion of lactobionic acid from lactose E. coli of the control (E. coli -pKK223) and the new transformed E. coli (E. coli -pKK223-gdh3) were cultured in LB medium containing PQQ and lactose to confirm cell growth and lactobionic acid conversion product Confirmed.

Escherichia coli (E.coil DH5 α/pKK223- gdh3) strain introduced with the gdh3 gene and control ( E.coil DH5 α/pKK223) were each added to 2 ml of LB medium (Tryptone 10 g, Yeast Extract 5 g, NaCl 5 g) with the antibiotic ampicillin. Including 50 μg/ml, it was cultured for 16 hours at a temperature condition of 30°C, and wild-type Pseudomonas tetrorence was incubated for 16 hours at a temperature condition of 25°C in 2ml of LB medium. After that, control E. coli and transformed E. coli were inoculated with lactose 20 g/L, CaCo ₃ 3 g/L and PQQ 20 μM in 5 ml of LB medium so that the initial OD (600 nm) of each cell was 0.2, and inoculated at 30 ° C, 200 rpm. Cultured in a stirred incubator, and 4 hours after the start of the culture, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 1 mM, and cultured for a total of 24 hours. Wild-type Pseudomonas tetrorence was also _{mixed with lactose 20g/L and CaCO 3} 3 g/L in 5ml LB medium, and cultured for 24 hours at 200rpm in a 30°C agitated incubator.

Lactose and lactobionic acid were analyzed using Agilent's high-performance liquid chromatography (HPLC 1260 model) equipped with a Refractive Index Detector (RID). A Coregel ION 300 column was used as the column, and the column temperature was maintained at 70°C. The mobile phase was 0.5mM H ₂ SO ₄ , and the flow was 0.3 ml/min. Under the above conditions, the retention times of lactose and lactobionic acid were confirmed to be 19.2 minutes and 20.4 minutes, respectively, as shown in FIG. 3 .

In this way, it was confirmed that the lactobionic acid peak not appearing in the control E. coli appeared only in E. coli overexpressing gdh3 involved in the production of Pseudomonas tetrorence-derived lactobionic acid. By metabolizing lactose, it was confirmed that it has the ability to convert lactobionic acid.

Example 3 : gdh3 overexpressed Recombinant Pseudomonas in tetrorence manufacturing and using the same lactobionic acid Check production capacity

It provides a method for determining whether the recombinant Pseudomonas tetrorence overexpression of gdh3 and the increase in lactobionic acid productivity.

Experimental Example 1 : Recombinant Pseudomonas of tetrorence Protein identification

Recombinant strain Pseudomonas reconstituted in Example 3 taetrolens / In order to check the protein expression of pDSK519-gdh3, it was cultured in NB medium at 25℃. The expression of the gdh3 gene was induced for 10 hours by adding isopropyl-1-thio-β-D-galactopyranoside (IPTG) for gene expression of the plasmid at 0.8~1.0 OD 600nm. . After centrifugation to separate the protein produced from the cultured cells, the suspension was made in PBS buffer solution. Then, the process of ultrasonication for 10 seconds and stopping for 30 seconds was repeated a total of 20 times to extract intracellular enzymes. This was suspended in 5X SDS sample buffer, treated in boiling water for 5 minutes, and then 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis was performed, and the results are shown in FIG. As shown in FIG. 4 , the expression (86 kDa) of the gdh3 protein was confirmed, and it was confirmed that the overexpression of the gdh3 gene occurred.

Experimental Example 2 : Recombinant Pseudomonas of tetrorence lactobionic acid Produce

remind Recombinant Pseudomonas taetrolens / In order to confirm the increase in lactobionic acid production according to the gdh3 overexpression of the pDSK519-gdh3 strain, 5L fermentation was performed to check the productivity compared to the metabolic conversion rate of the wild-type strain.

The strains were streaked on an NB plate and incubated at 25°C for 48 hours, then colonies formed were inoculated into 5 ml NB medium and cultured with shaking at 25°C at 200 rpm for 24 hours. The cultured seed culture was inoculated with lactose 20 g/L in 100 ml NB medium and cultured with shaking at 25 ° C. at 200 rpm for 24 hours. At the start of fermentation, the initial OD (600 nm) was inoculated into the fermentation medium to be 0.2. The medium used for fermentation was Nutrient broth (NB) (1 g/L beef extract, 2 g/L yeast extract, 5 g/L peptone, 5 g/L NaCl), and the lactose concentration change and pH correction experiment Based on the results, 200 g/L lactose and 30 g/L CaCO ₃ were added for pH correction to maintain pH 6.5 or higher during fermentation. Cultivation was carried out at a temperature of 25 ℃ by adding 2 L medium to a 5 L fermenter. Dissolved oxygen concentration (Dissolved Oxygen, DO) was designated as 30%, and fermentation was carried out by controlling the stirring speed to 200 ~ 500 rpm to maintain the dissolved oxygen concentration.

Recombinant Pseudomonas taetrolens / In the case of pDSK519-gdh3 strain, the antibiotic kanamycin was added, and IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 1 mM after 4 hours from the start of the culture, and the total fermentation was carried out for 27 hours. cell growth, pH, lactose consumption and lactobionic acid production were measured.

As a result of comparing the lactobionic acid productivity through lactose 200 g/L fermentation of the wild-type strain and the recombinant strain (FIG. 5), both strains completed metabolic conversion within 27 hours, and in the case of the recombinant strain, 4 hours faster than the wild-type 23 The conversion was completed in time. This shows a productivity of 7.77 g/L/h for the wild-type strain, while the recombinant strain shows a 21% improvement in productivity of 9.43 g/L/h.

Claims (10)

A composition for producing lactobionic acid comprising a recombinant vector comprising a nucleic acid sequence encoding a Quinoprotein glucose dehydrogenase (gdh3) enzyme derived from Pseudomonas taetrolens. delete The composition for producing lactobionic acid according to claim 1, wherein the recombinant vector is a plasmid vector. The composition for producing lactobionic acid according to claim 3, wherein the plasmid is pDSK519. Pseudomonas tetrolens ( Pseudomonas taetrolens ) A recombinant vector containing a nucleic acid sequence encoding a gdh3 (Quinoprotein glucose dehydrogenase) enzyme derived from the introduced, gdh3 gene overexpressing a recombinant microorganism for lactobionic acid production. The recombinant microorganism for producing lactobionic acid according to claim 5, wherein the recombinant microorganism is a Gram-negative bacteria. The recombinant microorganism for producing lactobionic acid according to claim 5, wherein the recombinant microorganism is a wild-type Pseudomonas taetrolens , wherein the recombinant vector is introduced. The recombinant microorganism for producing lactobionic acid according to claim 7, wherein the recombinant microorganism has an increased lactobionic acid production compared to wild-type Pseudomonas tetrorence. Obtaining a recombinant vector comprising a nucleic acid sequence encoding the enzyme gdh3 (Quinoprotein glucose dehydrogenase) derived from Pseudomonas taetrolens; and
introducing the recombinant vector into Gram-negative bacteria
A method for producing a recombinant microorganism for lactobionic acid production with improved lactobionic acid production ability, comprising: A method for producing lactobionic acid using the recombinant microorganism for producing lactobionic acid according to any one of claims 5 to 8.

Images