KR101884022B1 - Recombinant microorganism for use in producing caproic acid and method of producing caproic acid using the same - Google Patents

Abstract

The present invention relates to a recombinant microorganism for production of caproic acid and a method for mass-producing caproic acid using the recombinant microorganism. More specifically, the present invention relates to a recombinant microorganism for production of caproic acid by introducing beta-keto-thiolases into wild- Culturing the recombinant microorganism having improved acid production ability, and the recombinant microorganism; And recovering the caproic acid from the culture. The recombinant microorganism of the present invention introduces beta-keto-thiolases activity into wild-type microorganisms and expresses caproic acid production ability, so that it can mass-produce caproic acid with high yield and high efficiency, Caproic acid can be usefully used for the production of bioplastics.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a recombinant microorganism for production of caproic acid and a method for mass production of caproic acid using the recombinant microorganism,

The present invention relates to a recombinant microorganism for production of caproic acid and a method for mass-producing caproic acid using the recombinant microorganism. More specifically, the present invention relates to a recombinant microorganism for production of caproic acid by introducing beta-keto-thiolases into wild- Culturing the recombinant microorganism having improved acid production ability, and the recombinant microorganism; And recovering the caproic acid from the culture.

World demand for biodegradable or biodegradable bioplastics in 2013 is expected to increase four-fold to 900,000 tonnes and $ 2.6 billion in price. The price competitiveness of bioplastics is improving due to increased consumer preference for eco-friendly products, increased regulations on degradable plastics, and rising prices of petroleum-based products. In addition, Western Europe already has a systematic environment for using bio-based products and a wide range of composting facilities due to the regulations related to the emission of carbon dioxide. Bioplastics will account for 1.5-4.8% of the total plastics market in 2015 and the market size is expected to reach 4 million to 12.5 million tons.

Bio plastic refers to plastic monomers such as C3, C4, C5, and C6 produced by microbial metabolism-fermentation process and polymeric materials produced therefrom by chemical or biological methods, and is decomposed by bacteria in the soil It is also called biodegradable plastic. Because it is easily biodegradable, it is applied to surgery, fracture fixer and so on. It is also suitable for the use of delayed-release pesticide because it is slowly degraded in the soil.

Some types of microorganisms store polymeric polyesters in the form of granules in the body as energy sources. They are poly-β-hydroxybutyrate (PHB), poly-β-hydrolyvalerate (PHV) and PHB / PHV This includes polyalkanoates. The bio-plastic can be produced using the polyester. On the other hand, aliphatic polyesters, polycaprolactone (PCL), and poly- (glycolic acid) (PGA) can be obtained by chemical synthesis of monomers.

In the production of conventional C3 and C4 based plastics, bioplastics using C5 and C6 monomers are growing in popularity. Bioplastics are an important part of the chemical materials industry in the world, and the marketability of C5 and C6 based bioplastics Is estimated at over KRW 4 trillion in Korea. However, production of bioplastics based on C3, C4, C5, and C6 monomers is technically mature, but mass production technologies for C5 and C6 monomers, which are raw materials, are still in a low stage and need to be strategically developed.

Previous studies have shown that intestinal bacteria such as Megasphaera Since the ability to produce C5 / C6 carboxylic acids of elsdenii has been reported, a paper that directly produced these materials using immobilized cells has been reported. However, mass production of caproic acid (hexanoic acid, C6) has not yet been successfully reported using recombinant yeast. According to previous studies, about 200 mM of hexanoic acid is produced after 200 hours, but about 50% of it is known to produce butyric acid.

Under these circumstances, the present inventors have made intensive efforts to find a method for obtaining caproic acid with high efficiency. As a result, they have found that the activity of β-keto-thiolases in conventional wild-type microorganisms, I (phbA) and II (bktB) activity, the present inventors have completed the present invention.

One object of the present invention is to introduce a β-keto-thiolases activity into wild-type microorganisms to provide a recombinant microorganism for production of caproic acid.

Another object of the present invention is to provide a method for producing a recombinant microorganism, which comprises culturing the recombinant microorganism; And recovering the caproic acid from the culture. The present invention also provides a method for mass production of caproic acid.

In one embodiment, the present invention provides a recombinant microorganism for the production of caproic acid by introducing beta-keto-thiolases activity into wild-type microorganisms.

In the present invention, caproic acid is a lower saturated fatty acid having 6 carbon atoms in the chain and no double bond in the chain, which is also referred to as hexanoic acid.

In the present invention, beta-keto-thiolases act to increase the number of carbon atoms by two by attaching acetyl-CoA to a molecule having several carbon atoms. Ralstonia eutropha Beta-ketothiolase I (phbA) and II (bktB) have been reported as beta-ketothioreases derived from bacteria (gram-negative soil bacteria).

In the present invention, E. coli , K. lactis , S. cerevisiae , K. marxianus , especially Kluyveromyces To construct a transgenic vector and transgenic system in marxianus and isolate Ralstonia PhtA and bktB derived from eutropha were introduced and cultured to produce caproic acid. Beta-ketothiolase I is encoded from the phbA gene and beta-ketothiolase II is encoded from the bktB gene.

In the present invention, an expression vector is a vector capable of expressing a target protein or a target RNA in a host cell, and a gene construct containing an essential regulatory element operatively linked to the expression of the gene insert. phb A and bkt B Genes can be obtained from a variety of microbial species, including, but not limited to, Ralstonia eutropha . < / RTI >

In the present invention, parent strains into which an expression vector is introduced include strains capable of producing butyric acid, such as E. coli , K. lactis , S. cerevisiae , and K. marxianus , in particular Kluyveromyces marxianus .

In a specific embodiment of the present invention, E. coli has two genes with phb A bkt B is Ralstonia The gene derived from eutropha was used, and pETduet-1 was used as a vector to construct the expression system (Fig. 3, Example 1). In K. lactis , two genes with phb A bkt B is Ralstonia The gene derived from eutropha was used, and the vector used for constructing the expression system was pKLAC2 (Fig. 4, Example 2). In S. cerevisiae , with phb A bkt B is Ralstonia eutropha- derived gene was used. The vector used for constructing the expression system was an expression vector pRS426 having a multiple copy plasmid, a strong promoter (GPD promoter), and each nutrient requirement (URA3, LEU2, TRP1) , Example 3). In K. marxianus Two genes with phb A bkt B is Ralstonia Eutropha- derived genes were used. The vectors used to construct the expression system were K. marxianus and E. coli PJSKM316GPD vector containing the GPD promoter which is a strong promoter in the expression shuttle vector pJSKM316 vector was used (Fig. 6, Example 4). URA3 auxotrophic strains were selected and transformed.

Since the intracellular metabolic pathway is composed of a complex network, it is not easy to predict the effect of any one enzyme activity on the entire metabolic pathway. The present inventors have found that phb A in the caproic acid biosynthetic pathway Understanding the role of the bkt B enzymatic activity and with the phb A taken in from the outside based on the biosynthetic pathway to produce the acid in the said strain bkt B enzyme activity can produce caproic acid efficiently from glucose and organic acids. Specifically, acetyl-CoA, which is converted from glucose to acetyl-CoA, is converted into 4-carbon butyric-CoA by the action of beta-ketothiolase phbA and another 6-carbon caprocyan is produced by the action of beta-ketothiolase bktB .

In a specific example of the present invention, the present inventors fermented the transformant having the recombinant expression vector inserted therein under the condition of adding sodium propionate, and confirmed the amount of produced fatty acid and the amount of sodium propionate remaining. As a result of the analysis by time, it was confirmed that about 8 g / L of C4 and about 2.4 g / L of C6 (caproic acid) were detected in the sample at 8 hours. That is, it was found that the production of caproic acid (C6), which was produced in 200 hours of the wild-type yeast and the like, in about 8 hours, increased the productivity about 25 times (FIGS. 7 and 8, Example 5).

In the present invention, " caproic acid productivity " means the concentration of caproic acid (caproic acid production rate (gg ^-1 h ^-1 )) in which the caproic acid producing microorganism biosynthes for a unit time.

In another aspect, the present invention provides a method for producing a recombinant microorganism comprising the steps of: 1) culturing the recombinant microorganism for producing caproic acid; And 2) recovering caproic acid from the culture broth.

The term " cultivation " in the present invention means cultivation of microorganisms under moderately artificially controlled environmental conditions. In the present invention, a method for culturing caproic acid using a caproic acid-producing microorganism can be carried out using a method well known in the art. Specifically, the culturing can be carried out continuously in a batch process or in an injection batch or a repeated batch batch process (but not limited thereto).

The medium used for the culture should meet the requirements of the particular strain in an appropriate manner. Culture media for E. coli , K. lactis , S. cerevisiae , and K. marxianus are known (see, for example, Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington DC, USA, 1981). Sugars which may be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, Fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Examples of nitrogen sources that may be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used individually or as a mixture. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the culture medium must contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used. In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The culture continues until the amount of caproic acid produced is maximized. Caproic acid may be released into the culture medium or contained in the cells. The medium may preferably comprise glucose or an organic acid, in particular propionic acid, which may be a substrate for caproic acid production.

A method of producing caproic acid of the present invention comprises recovering caproic acid from a cell or a culture medium. Methods for recovering caproic acid from cells or culture media are well known in the art. The caproic acid recovery method may be filtration, anion exchange chromatography, crystallization, HPLC, etc., but is not limited to these examples.

The recombinant microorganism of the present invention introduces beta-keto-thiolases activity into wild-type microorganisms and expresses caproic acid production ability, so that it can mass-produce caproic acid with high yield and high efficiency, Caproic acid can be usefully used for the production of bioplastics.

Figure 1 is a schematic diagram of the C6 caproic acid biosynthetic pathway.
Fig. 2 shows the amino acid sequence and protein weight of two enzymes belonging to beta-ketothiolase, phbA and bktB.
3 is a schematic diagram of pETduet-1 / bktB and pETduet-1 / phbA expression vectors in E. coli .
4 is a schematic diagram of bktB and phbA expression vectors in K. lactis .
5 is a schematic diagram of bktB and phbA expression vectors in S. cerevisiae .
6 is a schematic diagram of bktB and phbA expression vectors in K. marxianus .
7 is a graph showing the results of GC analysis of the produced C6 caproic acid.
FIG. 8 is a graph showing the degree of fermentation and production of C 6 caproic acid at 10 g / L of YP + sodium propionate of a recombinant microorganism of K. marxianus .

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

[Example 1] E. coli Construction of pETduet-1 / bktB and pETduet-1 / phbA expression vectors

Two genes With phbA bktB is Ralstonia The eutropha- derived gene was used and pETduet-1 was used as a vector to construct the expression system. The vector for the inserted gene is introduced into the two genes it has two MCS (Multi cloning site) and T7 promoter and expression is induced by IPTG (Isopropyl β-D-1 -thiogalacto-p yranoside) under T7 promoter at the same time System.

The two genes were amplified by Polymerase Chain Reaction (PCR) method. The primers and reaction conditions used were as follows. The primers were constructed so that the phbA gene could be inserted into Nco I and Hind III of the pETduet-1 vector, and the bktB gene could be inserted into the Nde I and Xho I restriction sites of the pETduet-1 vector.

PhbA-F: 5'-CATGCCCATGGGCACTGACGTTGTCATCGTATC-3 '(SEQ ID NO: 1)

PhbA-R: 5'-CCCGAAGCTTTTATTTGCGCTCGACTGCC-3 '(SEQ ID NO: 2)

BktB-F: 5'-GGAATTCCATATGACGCGTGAAGTGGTAGTGG-3 '(SEQ ID NO: 3)

BktB-R: 5'-CCGGCTCGAGTCAGATACGCTCGAAGATGG-3 ' (SEQ ID NO: 4)

The PCR product was electrophoresed on an agarose gel and amplified to a size of 1.2 kb. The identified amplification product was purified by PCR and extracted in pure DNA form. Extracted phbA and The pETduet-1 vector was treated with restriction enzymes Nco I and Hind III, and the bktB and pETduet-1 vectors were treated with restriction enzymes Nde I and Xho I, respectively. After restriction enzyme reaction, pETduet-1 was added with calf intestinal alkaline phosphatase (CIAP) to prevent self-ligation in the vector. The enzyme - treated DNA was purified by gel extraction method and each purified insert and vector were subjected to ligation reaction using Quick ligation kit. Each ligation reaction was transformed into E. coli TOP10 competent cells prepared by the CaCl ₂ method. The selected plasmids were subjected to DNA sequencing and finally recombinant plasmids in which phbA and bktB were properly inserted were selected. The expression vectors in which phbA and bktB were respectively introduced were named pETduet-1 / phbA and pETduet-1 / bktB. β-ketothiolase I and II (PhbA, BktB) co-expression vector construction was pETduet-1 / phbA and pETduet-1 / bktB the restriction enzyme Nde to insert bktB the pETduet-1 / phbA of the existing secure phbA insertion of I and Xho I at the same time. Each DNA was purified by gel extraction method and ligation was performed using TAKARA Quick ligation kit. The ligation reaction products were transformed into E. coli TOP10 competent cells and transformed with LB ampicillin medium. The thus-completed two genes phbA Wow bktB Were inserted into the expression vector pETduet-1 / phbA / bktB.

[ Example  2] K. lactis  of bktB Wow phbA  Construction of expression vector

Two genes With phbA bktB is Ralstonia The gene derived from eutropha was used and the vector used for constructing the expression system was pKLAC2. This vector indicates that the inserted protein is LAC4 Expression is induced by galactose and lactose under the promoter, and the gene is integrated into the K. lactis genome, which enables stable expression in the complex medium for a long time in culture. Two genes were amplified by Polymerase Chain Reaction (PCR) method. The phbA gene and the bktB gene were constructed to be inserted into the Hind III and EcoR I of the pKLAC2 vector. And amplified using the following primers.

PhbA-F: 5'-AAGCTTATGG GCACTGACGTTGTCATCGTATC-3 ', (SEQ ID NO: 5)

PhbA-R: 5'-GAATTCTTATTTGCGCTCGACTGCC-3 '(SEQ ID NO: 6)

BktB-F: 5'-AAGCTTATGAC GCGTGAAGTGGTAGTGG-3 '(SEQ ID NO: 7)

BktB-R: 5'-GAATTCTCAGATACGCTCGAAGATGG-3 '(SEQ ID NO: 8)

Each amplified gene and pKLAC2 vector were ligated with Hind III and EcoR I The purified enzyme was purified by gel extraction method. The two restriction enzymes and inserts were ligated and transformed into E. coli TOP10. Transformants were selected from LB medium containing ampicillin and the plasmids were extracted by culturing them in a liquid medium. The extracted plasmid was subjected to restriction enzyme treatment to confirm insertion of the insert into the vector. The confirmed plasmid was finally cloned by DNA sequencing. The resulting vectors were named pKLAC2 / phbA and pKLAC2 / bktB.

[ Example  3] S. cerevisiae  of phbA Wow bktB  Construction of expression vector

In S. cerevisiae , two genes with phb A bkt B is Ralstonia The expression vector pRS426, which has a multiple copy plasmid, a strong promoter (GPD promoter) and a different nutrient requirement (URA3, LEU2, TRP1), was used as a vector for constructing the expression system. Each of the introduced genes was introduced into S. cerevisiae CEN . RTI ID = 0.0 & gt; PK 2-1D . ≪ / RTI > In addition, two genes were simultaneously transfected into S. cerevisiae CEN . PK 2-1D . The resulting two genes, phbA and bktB Were inserted as pRS426 GPD bktB and pRS425 GPD phbA, respectively.

[ Example  4] K. marxianus of phbA Wow bktB  Construction of expression vector

Screening the URA3 auxotrophic strain of the URA3 gene disrupted to enable the transformation from K. marxianus and, K. marxianus, and E. coli pJSKM316GPD having a strong promoter GPD at the same expressible expression shuttle vector (pJSKM316 vector) in the vector PhbA and bktB on Ralstonia eutropha and cloned. The constructed vector was transformed into KM5? URA3 by the yeast electroporation method. The resulting two genes, phbA and bktB Were named pJSKM316GPD phbA and pJSKM316GPD bktB, respectively.

[ Example  5] K. marxianus  Recombinant microorganism FC + Sodium Propionate  Fermentation at 10 g / L and C6  Analysis of the result

The expression vector was inserted into 5 ml of YPD (1% yeast extract, 2% peptone, 2% glucose), and the transformants were inoculated into 500 ml of YPD (1% yeast extract, 2% peptone, 2% glucose) The culture broth was inoculated 1% in the medium and the bacteria were concentrated to a high concentration. The concentration of the concentrated bacteria was adjusted to about 10 at an OD 600 of 10, and was adjusted to pH 4.5 in 50 ml of YP (1% yeast extract, 2% peptone) medium and fermented at a concentration of 10 g / L of sodium propionate. The culture was incubated at 30 ° C and 100 rpm. Samples were taken every hour to confirm the amount of fatty acid and sodium propionate produced. The column was DB-FFAP (30m x 0.25mm ID, 0.25μm) and helium gas was used as the carrier. The oven was initially maintained at 100 ° C for 5 minutes, increased from 100 ° C to 250 ° C by 10 ° C per minute, and maintained at 250 ° C for 12 minutes. The injector was set to 1:50 split mode and the detector was set to 300 ° C. Butyric acid, valeric acid and caproic acid (hexanoic acid) showed retention times of 10 minutes, 11 minutes, and 12 minutes, respectively. Analysis of the samples taken at each hour showed that C4 and C6 (caproic acid) were detected at about 1.8 g / L and 2.4 g / L, respectively, at 8 hours. It was not reduced or detected. As a result, it was found that the production of C6 caproic acid, which was produced in 200 hours in the conventional wild yeast, in about 8 hours, increased the productivity about 25 times.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Recombinant microorganism for use in producing caproic acid and          method of producing caproic acid using the same <130> PA110725 / KR <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PhbA-F primer <400> 1 catgcccatg ggcactgacg ttgtcatcgt atc 33 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PhbA-R primer <400> 2 cccgaagctt ttatttgcgc tcgactgcc 29 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> BktB-F primer <400> 3 ggaattccat atgacgcgtg aagtggtagt gg 32 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> BktB-R primer <400> 4 ccggctcgag tcagatacgc tcgaagatgg 30 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PhbA-F primer <400> 5 aagcttatgg gcactgacgt tgtcatcgta tc 32 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PhbA-R primer <400> 6 gaattcttat ttgcgctcga ctgcc 25 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> BktB-F primer <400> 7 aagcttatga cgcgtgaagt ggtagtgg 28 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> BktB-R primer <400> 8 gaattctcag atacgctcga agatgg 26

Claims (7)

1) culturing a recombinant microorganism for production of caproic acid having enhanced β-keto-thiolases activity by introducing phbA gene, bktB gene, or both of the genes derived from Ralstonia eutropha ; And
2) recovering the caproic acid from the cultured cells or culture. delete delete The method according to claim 1, wherein the recombinant microorganism is E. coli, K. lactis, S. cerevisiae or K. marxianus. delete delete The method of mass production of caproic acid according to claim 1, wherein the culture is performed in a culture medium containing at least one selected from the group consisting of glucose and propionic acid.

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