KR101223816B1

KR101223816B1 - A microorganism of being able to grow and produce value-added materials in the waste-glycerol environment

Info

Publication number: KR101223816B1
Application number: KR1020100059316A
Authority: KR
Inventors: 김창준; 장용근; 김선원; 김유근; 느구엔트쿠앙
Original assignee: 김유근; 경상대학교산학협력단
Priority date: 2010-06-22
Filing date: 2010-06-22
Publication date: 2013-01-17
Also published as: WO2011162447A1; KR20110139058A

Abstract

The present invention relates to a microorganism having enhanced growth in the presence of waste glycerol, and more specifically, to a microorganism having enhanced growth even in the presence of waste glycerol, in which the otsBA gene is overexpressed, a method for enhancing the growth of the microorganism, waste glycerol The present invention relates to a microorganism capable of producing a carotenoid or a useful substance using a substrate, and a method for producing a carotenoid comprising culturing the microorganism to recover the carotenoid.

Description

A microorganism of being able to grow and produce value-added materials in the waste-glycerol environment

Globally, research on the development of environment-friendly clean alternative energy has been actively conducted due to international environmental regulations caused by soaring oil prices, depletion of fossil fuels, and carbon dioxide emissions caused by the use of fossil fuels. Among the eco-friendly alternative energy sources, biodiesel is steadily increasing at home and abroad, and biodiesel production in the United States, the world's largest biodiesel producer, has risen sharply from 2 million gallons in 2000 to 230 million gallons in 2006. The situation is expected to increase. The government's efforts to disseminate biodiesel have been made in Korea. The amount of biodiesel sold in Korea from 2002 to 2006 is about 3.3% of the US production, and the production is increasing every year.

In this biodiesel, oil (mostly edible oil), methanol, and a base catalyst (KOH or NaOH) are introduced into the reactor to generate biodiesel, which is a methyl ester of fatty acid, by transesterification reaction, and fat is combined with fatty acid and glycerol. It is broken down into fatty acids and glycerol by esterification, and glycerol is produced as a by-product. In addition, some of the fatty acids produced in the side reactions react with the base catalyst and are converted to fatty acid salts (soaps). In addition, a high temperature reaction may produce peroxides derived from unsaturated fatty acids. Therefore, waste glycerol generated during the process due to acid and base catalysts, which are mainly used in the biodiesel production process, has high concentrations of unreacted fatty acid salts and methanol, salts, peroxides and biodiesel components that have not been removed in the separation process. MONG, matter of organic non glycerol).

Glycerol may be used as a raw material for cosmetics, food preparation, and petrochemical derivatives, but in order to utilize waste glycerol in such a product family, a separation and purification process with high purity is essential. In order to separate high-purity glycerol from biodiesel, distillation, adsorption, extraction, and the like may be used. However, there is a limit in increasing the separation recovery rate in the purification process. Therefore, in order to purify and use waste glycerol, the cost of the purification process is too high, so the economic effect of the purification and sale can be said to be very low.

About 10 kg of waste glycerol is produced as a by-product per 100 kg of biodiesel produced. As the production amount of waste glycerol increases simultaneously with the production of biodiesel, the production cost of waste glycerol is continuously decreasing. For this reason, research on the utilization of waste glycerol is being carried out all over the world, but no clear solution has been proposed so that it is emerging as a new source of environmental pollution. Although various studies are being conducted, no clear solution has been suggested.

Therefore, developing a method that can use waste glycerol that has not undergone a purification process is expected to have various advantages in terms of economic benefits and minimizing environmental pollution through efficient use of waste resources.

Recently, research has been attempted to develop fermentation processes that produce expensive compounds [1,3-propanediol, etc.] or bioenergy (bio alcohols, etc.) using waste glycerol as a substrate. Impurities such as fatty acid salts (soaps) and peroxides, which act as toxic substances that inhibit the growth of microorganisms, have not been reported to be used directly in biological processes.

The production of 1,3-propandiol and 1,2-propanediol (1,2-propandiol) using biological processes is currently carried out at home and abroad, including Dupont, USA. And attempts to develop the process, but the production is extremely low, it is necessary to develop a high efficiency strain through a metabolic approach to overcome this.

In addition, in Korea, the main research field for producing biofuel for transportation from waste glycerol has been conducted in the process of producing biobutanol using Clostridium sp., An anaerobic strain. Looking at the metabolic pathway in the microorganism for the production of such bio butanol, butanol is produced through acetyl-CoA, butyryl-CoA and butylaldehyde (butyraldehyde). However, this process requires the addition of a high-cost reducing agent to inhibit the formation of by-products such as acetone and butyrate and to increase the productivity of biobutanol, and requires a high process cost due to the high oxygen sensitivity of Clostridium sp. .

Accordingly, the present inventors have made efforts to develop a bioprocess that produces high value-added compounds by solving various problems previously reported. As a result, trehalose biosynthetic gene ( otsBA ) is over-expressed, resulting in high concentrations of fatty acid salts and perlocks in waste glycerol. By developing a strain resistant to the side [hydrogen peroxide (H ₂ O ₂ ) or organic peroxide] it was confirmed that beta-carotene, a high value-added compound from waste glycerol was completed and the present invention was completed.

One object of the present invention is to provide a microorganism with enhanced growth even in the presence of waste glycerol overexpressed otsBA , a trehalose biosynthetic gene.

Another object of the present invention is to provide a method for enhancing the growth of microorganisms in the presence of waste glycerol, comprising culturing the microorganisms.

It is another object of the present invention to provide a microorganism which can use waste glycerol to which a otsBA gene and a glycerol metabolism gene are introduced as a substrate.

It is another object of the present invention to provide a microorganism capable of producing carotenoids in the presence of waste glycerol to which the otsBA gene and the carotenoid biosynthetic gene have been introduced.

Another object of the present invention is to provide a carotenoid production method comprising culturing a strain capable of producing the carotenoid, recovering the carotenoid from the culture medium of the strain.

In one aspect to achieve the above object, the present invention relates to a microorganism having enhanced growth in the presence of waste glycerol, overexpressed otsBA gene.

As used herein, the term "waste glycerol" means formed as a by-product in the production of biofuel or preferably biodiesel, and about 10 kg of waste glycerol per 100 kg of biodiesel produced is produced as a by-product. In addition to glycerol, the waste glycerol includes fatty acid salts (soaps), various salts including peroxides (K or Na, Cl), methanol, and the like, but is not limited thereto. 15% or more, most preferably refers to waste glycerol containing 15-25%. According to one embodiment of the present invention, 17.4% of the fatty acid salt may be present in the waste glycerol of the present invention.

As used herein, the term " otsBA gene is a trehalose biosynthetic operon, which is composed of otsA encoding trehalose-6-phosphate synthase , and otsB gene encoding trehalose-6-phosphate phosphatase , and is easily available to those skilled in the art from a known database. Sequence information, for example, NCBI GenBank Accession No. NC000913. When otsBA is overexpressed in bacteria, the growth of microorganisms in the presence of substances containing high concentrations of fatty acid salts and peroxides, such as waste glycerol The otsBA gene that can be used in the present invention may include without limitation genes capable of biosynthesizing trehalose, and as long as the otsBA gene maintains activity capable of biosynthesizing trehalose. And 70%, preferably 80%, more preferably Genes with 90% homology.

As used herein, the term "overexpression" means an increase in the intracellular activity of an enzyme encoded by the corresponding DNA. Overexpression of the gene of interest can enhance protein expression by modifying the promoter region of the gene and the 5'-UTR region of the gene, can be enhanced by the introduction of the gene of interest on the chromosome, vector of the gene of interest The expression level of the protein can be enhanced by introducing into a phase with a self promoter or enhanced separate promoter and transforming the strain. It can also be achieved by introducing mutations into the open reading frame (ORF) region of the gene of interest.

The method of overexpressing the otsBA gene into the microorganism is applicable to methods known in the art. In a specific embodiment of the present invention, the otsBA gene is introduced into a vector, and the microorganism is transformed using the recombinant vector.

In addition, in the present invention, the otsBA gene is transcribed to express a trehalose biosynthetic enzyme, which increases expression by a promoter which can be regulated in the transformed microorganism, and increases mRNA stability by increasing gene stability to increase expression. Can be. In addition, a part or all of the genes are deleted by specific region recombinant DNA technology, or the trehalose biosynthetic enzyme is overexpressed by the exchange of mutated fragments. However, it will be apparent to those skilled in the art that, in addition to these methods, genes may be overexpressed using various methods known in the art.

In addition, in the present invention, the otsBA gene is synthesized using a known method such as PCR, and then cleaved with a restriction enzyme to be linked to a vector, a transformant is prepared using the vector, and finally selected. Cultivation of microorganisms with enhanced growth in waste glycerol.

According to one embodiment of the present invention, the chromosomal DNA of E. coli K12 cells was used as a template, and PCR was performed by two primers (SEQ ID NOs: 1 and 2) on the side of the otsBA operon. .

In the present invention, the overexpression of the otsBA gene causes the microorganism to biosynthesize trehalose and have the ability to resist high concentrations of fatty acid salts or peroxides. It has the ability to withstand, resulting in an increase in the growth of microorganisms.

According to one embodiment of the present invention, it was confirmed that the recombinant strain transformed with the otsBA gene showed a result of enhanced growth regardless of the increase in the concentration of waste glycerol than the blank strain not transformed with the otsBA gene (FIG. 14A).

In the present invention, "transformation" includes those transformed by the gene itself and its transformation cassette. The "transformation cassette" refers to a vector having a foreign gene and having a factor that facilitates transformation of a specific host cell in addition to the foreign gene. Here, the vector is a self-replicating sequence, a genomic insertion sequence, a phage or nucleotide sequence, linear or circular, single or double stranded DNA or RNA. Generally, vectors include sequences that direct the transcription and translation of appropriate genes, selection markers, and sequences that allow self-replicating or chromosomal insertion. Specific examples of the vector include plasmid vectors (pSE, pBR, pUC, pBluscriptII, pGEM, pTZ, and pET) and phage or cosmid vectors (pWE15, M13, EMBL3, EMBL4, FIX II, DASH II). , ZAP II, gt11, Charon4A, Charon21A), but are not limited thereto.

In addition, the restriction enzyme used in the present invention may be a known restriction enzyme, preferably EcoR I and Xba I can be used, the vector is pBluescrip SK (+), pTrc99A, pBBR1MCS-2, pT-DHB, pS-NA can be used. In addition, microorganisms that can be used for transformation may be any microorganism capable of using glycerol as a substrate by introducing the otsBA gene, and may include not only natural microorganisms but also mutant microorganisms capable of producing carotenoids. Preferably Escherichia coli ), but is not limited thereto.

In addition, in the present invention, the selection of the transformant may use a vector (plasmid) having an antibiotic resistance gene, and preferably a vector (plasmid) having an ampicillin, chroramphenicol, and kanamycin resistance genes. ), And preferably, pTrc99A, pBBR1MCS-2, pT-DHB, pS-NA can be used.

As another aspect, the present invention relates to a method for enhancing the growth of microorganisms in the presence of waste glycerol, comprising culturing the microorganisms.

As described above, when using the microorganism of the present invention, it is possible to enhance the growth of microorganisms even in the presence of high glycerol waste glycerol.

In another aspect, the present invention relates to a microorganism capable of producing useful substances by introducing otsBA gene and glycerol metabolism gene using waste glycerol as a substrate.

As used herein, the term "glycerol metabolism gene" refers to a metabolic related gene capable of producing a useful substance using glycerol present in waste glycerol, but is not limited thereto, gldA (glycerol dehydrogenase, glycerol dehydrogenase gene) , glpD (glycerol-3-phosphate dehydrogenase, glycerol 3-phosphate dehydrogenase gene) can be exemplified, microorganisms to which the glycerol metabolism gene is introduced can use the glycerol contained in the waste glycerol as a substrate due to the expression of the gene In this way, useful materials can be produced.

Preferably, the useful material may be carotenoid, bio ethanol, bio butanol, 1,3-propanediol or 3-hydroxypropionic acid, but is not limited thereto. .

As another aspect, the present invention relates to a microorganism producing carotenoids in the presence of waste glycerol to which the otsBA gene and the carotenoid biosynthesis gene have been introduced.

The microorganism producing the carotenoid is transformed otsBA gene in order to have the ability to grow in the presence of waste glycerol, it can also be prepared by introducing a gene required for metabolic processes for producing carotenoids.

As used herein, the term "carotenoid" refers to a group of pigments similar to carotene, which means lycopene, beta-carotene, astaxanthin, zeaxanthin, xanthophyl, and the like. Is beta-carotene.

As used herein, the term "beta-carotene" is a precursor of fat-soluble vitamin A, has antioxidant properties, and can be used as an additive in functional foods, beverages and functional cosmetics.

Genes required for metabolism to produce the carotenoids (carotenoid biosynthetic genes) may include crtE , crtB , crtI , ipiHP1 , crtY , dxs , mvaE , mvaS , mvaK1 , mvaK2 , mvaD , ispA or idi genes, and carotenoids Depending on the type, known genes related to the biosynthesis of carotenoids may be further included. The carotenoid biosynthetic pathway by the gene is shown in FIG. 4.

Derivation of the metabolic genes required for the carotenoid biosynthesis may be derived from a known microorganism having a gene necessary for producing carotenoids, preferably Pantoea agromerence ( Pantoea agglomerans ), Haematococcus plauvialis , P. ananatis , P. ananatis , Enterococcus faecalis , Streptococcus penumoniae , and idi For genes additionally E. It can be used as the origin of coli .

Preferably, for the biosynthesis of zeaxanthin, a family of carotenoids, crtZ [Beta-carotene hydroxylase] gene may be additionally included.

The genes may be introduced into the microorganism using a known vector, preferably, pS-NA, pT-DHB, ipiHP1, pTrc99A may be used, but is not limited thereto. In addition, the carotenoid biosynthesis genes may be introduced into a microorganism using a known vector generally used for cloning or expression of genes. According to one embodiment of the present invention, the crtE, crtB, crtI, ipiHP1, crtY, dxs, mvaE, mvaS, mvaK1, mvaK2, mvaD and idi gene used in the MVA path shown in Figure 4 the strain of the otsBA transgenic In addition, by overexpression, growth was enhanced in the presence of waste glycerol, and it was confirmed that the results also maintain the biosynthesis of beta-carotene (FIGS. 12 and 14).

As another aspect, the present invention is a method for introducing otsBA , using waste glycerol as a substrate, culturing a transformed microorganism capable of producing carotenoids, and recovering the carotenoids from the culture.

In a specific example of the present invention, the culturing process of the transformed microorganism may be performed according to suitable media and culture conditions known in the art. As the type of medium, the medium used for cultivation should suitably meet the requirements of a particular strain. The medium contains various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include: carbohydrates such as glucose, fructose, sucrose, lactose, maltose, starch and cellulose, soybean oil fats such as soybean oil, regular sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid and linoleic acid Fatty acids, such as glycerol and ethanol, organic acids such as acetic acid, and the like. These carbon sources may be used alone or in combination.

In addition, examples of nitrogen sources that can be used include organic nitrogen sources and urea (CO (NH ₂ ) ₂ ), sulfuric acid such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat. Ammonium sulfate (NH ₄ ) ₂ SO ₄ ), ammonium chloride (NH ₄ Cl), ammonium phosphate (NH ₄ ) ₂ HPO ₄ ), ammonium carbonate, (NH ₄ ) Inorganic nitrogen sources such as ₂ CO ₃ ) and ammonium nitrate (NH ₄ NO ₃ ). These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogen acid (KH ₂ PO ₄ ), dipotassium hydrogen phosphate (K ₂ HPO ₄ ), and a corresponding sodium (Na) -containing salt as a phosphorus source. It may also include metal salts such as magnesium sulfate (MgSO ₄ ) or iron sulfate. In addition, amino acids, vitamins, and appropriate precursors may be included. During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, oxygen or oxygen-containing gas (eg, air) may be injected into the culture to maintain the aerobic condition of the culture.

This culture process can be used by those skilled in the art can be easily adjusted according to the type of microorganism selected. Examples of the culture method include, but are not limited to, batch culture, continuous culture or fed-batch culture, preferably, fed-batch culture or continuous culture. Cultivation is included.

These known culture methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall Interantional Editions, pp. 138-176). In addition, the culture of the microorganisms can be carried out according to well-known methods, and conditions such as the culture temperature, the incubation time and the pH of the medium can be adjusted appropriately. 2-20% of waste glycerol may be added to the medium, and the culture may be performed, preferably 2-8%.

As a method for recovering carotenoids from the culture solution, a known recovery method may be applied, and known methods include organic solvent extraction and crystallization, TLC (thin layer chromatography), gas chromatography, and high performance liquid chromatography. High-performance liquid chromatography (HPLC), gel-permeation chromatography, column chromatography, and the like may be used, but are not limited thereto.

Preferably, the carotenoid may be beta-carotene. According to one preferred embodiment, it was confirmed that the batch- and fed-batch cultures produced 130 mg / ml and 175 mg / ml, respectively, of beta-carotene (FIGS. 16 and 17).

As described above, the present invention can survive and grow in a poor environment such as waste glycerol, which has a lot of impurities toxic to microorganisms, and can produce carotenoids and biofuels including beta-carotene by using waste glycerol as a substrate. The present invention relates to a transformed microorganism in which the otsBA gene is introduced, which is generated as a by-product of biofuel, and is an environmentally friendly waste glycerol, which is a major cause of environmental pollution, and can be used to produce useful materials using the same. Has the potential for

1 shows waste glycerol, waste glycerol from which fatty acids have been removed by pretreatment (hereinafter referred to as pretreatment waste glycerol, left) and fatty acids recovered from waste glycerol (right).
2 shows the medium to which pure glycerol (left), pretreated waste glycerol (center) and waste glycerol (right) were added.
Figure 3 shows a schematic diagram of the synthesis of trehalose biosynthetic gene ( otsBA ) and the development of recombinant E. coli.
4 shows metabolic processes for producing beta-carotene and genes involved therein.
Figure 5 shows the results for the growth and beta-carotene production characteristics of the blank strain without the otsBA gene in the medium added pure glycerol (20 g / L).
Figure 6 shows the results for the growth and beta-carotene production characteristics of the recombinant strain transformed with the otsBA gene in the medium added pure glycerol (20 g / L).
Figure 7A shows the growth characteristics of blank strains and recombinant strains appearing after 86 hours of incubation in medium containing pure glycerol (20 g / L) and glycerol-derived fatty acid salt (soap), Figure 7B is pure glycerol (20 g / L) L) and waste glycerol-derived fatty acid salts (soaps) in medium containing 86 hours of blank strains and recombinant strains showing the results of beta-carotene production.
Figure 8A shows the effect on the growth of recombinant strain and blank strain depending on the concentration of methanol added, Figure 8B shows the effect on the beta-carotene production of recombinant strain and blank strain depending on the concentration of methanol added.
Figure 9A shows the effect on the growth of recombinant strains and blank strain according to the concentration of the pure glycerol added, Figure 9B shows the effect on the beta-carotene production of recombinant strains and blank strain according to the concentration of the pure glycerol added .
FIG. 10A shows the effect of the concentration of KCl added on the growth of recombinant and blank strains, and FIG. 10B shows the effect of the concentration of KCl added on the beta-carotene production of recombinant and blank strains.
FIG. 11A shows the growth of blank strains and the biosynthesis of beta-carotene in pretreated waste glycerol added medium (glycerol, 20 g / L), FIG. 11B shows the medium added with pretreated waste glycerol (glycerol, 20 g / L). ) Shows the results of growth of the recombinant strain and biosynthesis of beta-carotene.
FIG. 12A shows the results of the growth of blank strains and the biosynthesis of beta-carotene in waste glycerol added medium (glycerol, 20 g / L), FIG. 12B shows in waste glycerol added medium (glycerol, 20 g / L) Growth of recombinant strains and biosynthesis results of beta-carotene are shown.
Figure 13 shows the culture of the recombinant strain cultured for 5 days in the medium containing waste glycerol.
Figure 14A shows the results of the growth of the blank strain and recombinant strain according to the concentration of waste glycerol in the medium containing waste glycerol, Figure 14B shows the beta of the blank strain and recombinant strain according to the concentration of waste glycerol in the medium containing waste glycerol -The result of comparing the production amount of carotene is shown.
Figure 15A shows the results of comparing the growth of the blank strain and recombinant strain according to the change in the concentration of pre-treated waste glycerol, Figure 15B shows the results of comparing the beta-carotene production of the blank strain and recombinant strain according to the change in the concentration of pre-treated waste glycerol Indicates.
Figure 16 shows the concentration changes of beta-carotene, residual glycerol, acetic acid, NH ₄ over time when the recombinant strain was cultured in a batch in a bioreactor.
Figure 17 shows the change in concentration of beta-carotene, residual glycerol, acetic acid, NH ₄ over time when the recombinant strain was cultured in a bioreactor.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example One : Waste glycerol Furtherance

Waste glycerol was obtained from M Energy (Pyeongtaek, Korea), a domestic biodiesel producer. Waste glycerol, a fatty acid separated therefrom, and a sample from which fatty acids were removed by pretreatment are shown in FIG. 1. K, Na, Cl concentration in the waste glycerol was measured by the Korea Basic Science Institute, and the content of fatty acid salt and methanol (methanol) was measured by the present inventors.

The composition of the waste glycerol used in this example is shown in the following [Table 1]. The pH of the sample is 11, which is a strong base, which is expected due to the use of KOH, which is an alkali catalyst in the biodiesel production process. This can be seen that 1.1% potassium ion (K ⁺ ) per waste glycerol mass. Especially noteworthy is the large amount of fatty acid salts (17.4%). The dark color of the waste glycerol is believed to be due to fatty acid salts (soaps). The pretreated glycerol from which these fatty acid salts were removed has a clear brown color (FIG. 1).

On the other hand, Table 2 shows the composition of the fatty acid recovered from waste glycerol. Oleic acid, linoleic acid, and linolenic acid, which are unsaturated fatty acids with 18 carbon atoms, account for about 74% of the total, and palmitic acid and stearic acid, which are saturated fatty acids, stearic acid) accounts for about 22%. It can also be seen that there are also fatty acids having 15 or less carbon atoms.

Main ingredients of waste glycerol Item content Glycerol Content (%) 80.0 pH 11.0 K (%) 1.1 Na (%) 0.03 Cl (%) 0.02 Methanol (%) 14.2 Fatty acid salt (soap) 17.4

Fatty Acid Composition Recovered from Waste Glycerol Fatty Acid Carbon Number and Unsaturation Furtherance(%) C8: 0 0.03 ± 0.02 C10: 0 0.02 ± 0.00 C12: 0 0.10 0.00 C14: 0 0.45 ± 0.01 C14: 1 0.04 ± 0.00 C15: 0 0.04 ± 0.00 C15: 1 0.03 ± 0.00 C16: 0 17.30 ± 0.07 C16: 1 0.59 ± 0.01 C17: 0 0.13 ± 0.00 C17: 1 0.10 0.00 C18: 0 4.65 ± 0.11 C18: 1 30.89 ± 0.18 C18: 2 37.95 ± 0.78 C18: 3 4.85 ± 0.12 C20: 0 0.36 + 0.02 C20: 1 0.40 ± 0.00 C22: 0 0.32 ± 0.05

Example 2 medium and culture conditions

Waste glycerol obtained from M energy (Pyeongtaek, Korea) was allowed to remove or remain fatty acid salt (soap) in the culture medium, depending on the experimental conditions. The pH of the waste glycerol stock solution was adjusted to 3 using hydrochloric acid (HCl), and the free fatty acid precipitated from the solution was separated by centrifugation to prepare a medium free from fatty acid salts (soaps). Fatty acid-containing medium was added with the desired amount of fatty acid salt (soap) originally recovered from waste glycerol to the culture medium.

Pre-culture was prepared in 50 ml LB broth (5 g / l yeast extract, 10 g / l tryptone, 100 mg / l NaCl) with antibiotics (100 mg / l ampicillin, 50 mg / l chloramphenicol ( chroramphenicol) and 50 mg / l kanamycin). All flask cultures were performed at 37 ° C. and 190 rpm in a 500 ml Erlenmeyer flask. When the OD value of the culture reaches 0.6 at 600 nm (OD ₆₀₀ _nm ), the seed culture is seeded with 50 ml of main culture R medium in which pure glycerol, pretreated or untreated waste glycerol is added at different concentrations. Moved. The size of the inoculum is 10%, and the composition of the R medium is shown in the following [Table 3] and [Table 4]. Main culture was incubated at 25 ° C., 180 rpm, and when OD ₆₀₀ _nm was 5, IPTG 0.5 mM was added to induce overexpression.

All fermentations were carried out in a 3.4 L top-driven fermenter (Cobiotech, Incheon, Korea). 100 ml spawn cultures were prepared by flask culture, with 1 l R medium containing 60 g / l waste glycerol and antibiotics (100 mg / l empicillin, 50 mg / l chloramphenicol, 50 mg / l kanamycin). The reactor was inoculated. The pH was maintained at 6.8-7.0 using 2N H ₂ SO ₄ and 25% NH ₄ OH. Dissolved oxygen levels were maintained at 30% or higher by adding pure oxygen with air tubes and / or manually adjusting the stirring speed. Fermentation temperature was carried out at 25 ℃.

Composition of the R badge Configuration content K ₂ HPO ₄ 13.5 g / ℓ (NH ₄ ) ₂ HPO ₄ 4 g / ℓ MgSO ₄ .7 H ₂ 0 1.4 g / ℓ Citric acid 1.7 g / ℓ Thiamin 300 mg / l Trace metal solution 10 mg / l

Composition of Trace Metal Solution Configuration Furtherance FeSO ₄ .7H ₂ O 10.0 g / ℓ CaCl ₂ 2.0 g / ℓ ZnSO ₄ .7H ₂ O 2.2 g / ℓ MnSO ₄ .4H ₂ O 0.5 g / ℓ CuSO ₄ .5H ₂ O 1.0 g / ℓ (NH ₄ ) 6 Mo ₇ O ₂₄ .4H ₂ O 0.1 g / ℓ Na ₂ B ₄ O ₇ .10H ₂ O 0.02 g / ℓ

Example 3: E . coli otsBA Of operon Plasmid for expression ( plasmid ) Production

Genetic engineering procedures for DNA manipulation such as DNA isolation, restriction enzyme treatment, alkaline phosphatase treatment, and DNA ligation were performed by known methods.

Chromosomal DNA of cells of E. coli K12 was used as a template, and PCR was performed with two primers (SEQ ID NOs: 1 and 2) on the side of the otsBA operon (N-terminal GAA TTC GTG ACA GAA CCG TTA ACC GAA AC [SEQ ID NO: 1] and C-terminal TCT AGA CGC AAG CTT TGG AAA GGT AT [SEQ ID NO: 2]). The composition of the PCR reactions is shown in Table 5 below. PCR was denatured at 94 ° C. for 2 minutes, denatured at 94 ° C. for 40 seconds, annealed at 55 ° C. for 40 seconds, extended for 4 minutes at 72 ° C., and 30 cycles were performed.

PCR products were digested with restriction enzymes ( EcoR I and Xba I ) for 1 hour at 37 ° C. and purified by precipitation with 10 μl of 3M sodium acetate and 200 μl of 100% ethanol. PCR products were introduced into the EcoR I and Xba I sites of the pBluescript SK (+) vector. The composition of the reactants for ligation is described in Table 6 below. Ligation was performed at 16 ° C. for 12 hours in a PCR machine chamber (FIG. 3).

Composition of Reaction Mixture for PCR edifice Volume (μl) 10 Pfu buffer 5.0 10 mM dNTP mix 1.0 Primer 1 (10 pmole / ℓ) 2.0 Primer 2 (10 pmole / ℓ) 2.0 DNA template ( E. coli Chromosome) 1.0 5 × Band Doctor 5.0 Pfu (2.5 U / μl) 0.5 Add D.W. to 50.0

Composition of reactants for linkage edifice Volume (μl) T4-ligase 1.0 T4.buffer 1.0 Digested PCR Products 7.0 Digested pBluescript SK (+) vector 1.0 Total 10

1 μl of purified ligation mixture was mixed with 50 μl of E. coli DH5α competent cells on ice. The mixture was immediately transferred to an electrocuvette and a single electron pulse applied. The electrophoretic cell suspension was immediately diluted with 1 ml LB medium and incubated at 37 ° C., 200 rpm for 50 minutes. And 1 ml of culture was screened by culturing in solid LB medium to which empicillin (100 µg / ml) was added. White colonies appearing after 18 hours of incubation at 37 ° C. were selected and inoculated into test tubes containing 3 ml LB broth containing empicillin (100 μg / ml). After the culture was incubated at 37 ° C. at 200 rpm overnight, the cells were separated and the plasmid was recovered therefrom and purified. To screen the desired plasmids among the screened plasmid DNAs, the recovered plasmid using Hind III and Bg1 II enzymes were double-cut and run on agarose gels and confirmed in size by comparison with standard markers. For final confirmation of the selected plasmids, sequencing of purified plasmid DNA was performed by requesting Solgent (Daejeon, Korea).

otsBA The pBluescript SK (+) plasmid loaded with (SEQ ID NO: 3) was double-cut with EcoR I and Xba I enzymes, and the resulting otsBA fragment was purified using a Quiagene gel extraction kit (QIAGEN, Germany). The fragment was then introduced into the EcoR I and Xba I cleavage cleaning sites of the pTrc99A vector. Ligation was performed for 12 hours at 16 ° C. in the chamber of the PCR machine. Recombinant DNA was introduced into E. coli DH5α competent cells by electroporation, the same procedure as the plasmid pBluescript SK (+). The transformants after the transformation process were stored in glycerol stock containing 400 μl culture medium and 400 μl of 50% glycerol and stored in a freezer at -70 ° C.

Beta-if transformed with the E. coli strain to produce a carotene otsBA gene, the otsBA fragment using EcoRⅠ and Xba Ⅰ isolated from plasmid pTrc99A, and was inserted into the cleavage site of the same restriction enzyme of the plasmid pBBR1MCS-2. The vector contains a kanamycin resistance gene fragment. This transformation process was carried out in the same manner as described above (Fig. 3).

Example 4: preparation of a recombinant strain capable of producing beta-carotene

A strain of E. coli DH5α capable of producing beta-carotene, comprising a plasmid pT-DHB with a gene for beta-carotene biosynthesis and a plasmid pS-NA with a gene encoding an enzyme of the entire MVA metabolic pathway. Strain was defined. pT-DHB is the crtE , crtB and crtI of Pantoea agglomerans ; IpiHP 1 of Haematococcus plauvialis ; CrtY of P. ananatis ; E. coli dxs were prepared by cloning in pTrc99A (Amersham Biosciences, Piscataway, NJ). pS-NA is entrococos paecalis ATCC14508 ( Enterococcus faecalis MvaE and mvaS of ATCC14508); MvaK1 , mvaK2 , mvaK3 , mvaD of Streptococcus penumoniae ; E. coli idi was prepared by cloning in pSTV28 (TaKaRa Bio, Shiga, Japan). On the other hand, the recombinant strain additionally has a plasmid pBBR1MCS-2 including plasmid pT-DHB, pS-NA, as well as otsBA , a trehalose biosynthetic gene (FIG. 4).

Example 5: determination of the concentration of beta-carotene

To determine intracellular beta-carotene concentration, 1 ml of E. coli culture was centrifuged (10,000 rpm, 10 minutes) to obtain cells and washed once with water. The cell pellet was suspended in 1 ml of acetone, and then cultured in a dark room at 55 ° C. for 15 minutes. The suspension was centrifuged at 12,000 rpm for 10 minutes, the supernatant containing beta-carotene was measured for absorbance at 454 nm by spectrophotometer, and then standard beta-carotene (Sigma-Aldrich, St. Louis, MO) was used as an index. The concentration was determined by.

Example 6: fatty acid analysis

Folch et Fatty acids present in waste glycerol were analyzed according to the protocol developed by al . 3 to 5 g of a waste glycerol solution was taken, and 20 ml of a mixed solvent of chloroform and methanol (2: 1) were added thereto, followed by 2 hours of stirring. The mixed liquor was passed through a filter (Hyundai No. 51, Korea) to remove solid components that may be present. After adding 0.88% (w / v) sodium chloride solution to the filtrate and centrifuging at 2,000 rpm for 10 minutes, the lower organic solvent layer containing fatty acids was taken. Fatty acid was recovered by drying this in a 60 ° C. dryer for 30 minutes or flowing nitrogen gas at room temperature.

1 ml of methylene chloride and 0.5 N sodium hydroxide were added to the recovered fatty acid and heated for 10 minutes in a 90 ° C thermostat. After cooling it, 14% (w / v) BF ₃ solution dissolved in methanol was added and reacted for 10 minutes in a 90 ° C. thermostat to form fatty acid methyl esters (FAMEs) by methylation. It was extracted with nucleic acid and used for GC analysis.

Agilent technologies 6890N network gas chromatography was used for FAME analysis. Gas chromatography was performed with Supelcowax-10 fused with a flame ionization detector (FID) and a silica capillary column (60 mm × 0.32 mm × 0.25 mm). Nitrogen is a transport gas. The injector is kept at 250 ° C. and has an injection volume of 1 μl by injection mode (ratio 10: 1). The detection temperature was maintained at 180 ° C. for 6 minutes and increased to 250 ° C. by 5 per minute. The list of oven temperatures is as follows; For 2 minutes at 220 ° C., it was raised 2 times per minute to 240 ° C. and held for 20 minutes. Fatty acids were identified by comparing retention time with index fatty acids (Supelco 37 component FAME Mix) and quantified by comparing peak areas for internal indicators (C17: 0).

Example 7: methanol analysis

After sterilization using an enzyme-chemical assay, methanol concentration was measured in a medium to which waste glycerol solution and pretreated waste glycerol were added. 0.2 ml sample was mixed with 1.8 ml reaction reagent (0.05% 3-methyl-2-benzothiazoline hydrazone (MBTH-HCl), 25 mM MOPS-K + buffer, 0.5 U / ml alcohol oxidase) and reacted at 37 ° C for 15 minutes. I was. In this process, the methanol present in the sample is converted to formic acid by alcohol oxidase (AO) derived from Pichia pastoris , which is MBTH (3-methyl-2-benzothiazolione). hydrazone) and form a complex. To this was added 2 ml of colorant (0.1% FeCl ₃ solution dissolved in 30 mM HCl), which became dark blue by chemical reaction with this complex. Optical density for the final pigment mixture was measured at 670 nm using a spectrophotometer. This was quantified as a standard high purity methanol solution prepared by concentration.

Example 8: basic analysis and Ammonium ion analysis

The basic composition of pure waste glycerol and pretreated waste glycerol was determined by Korea Basic Science Institute using an inductively coupled plasma atomic emission spectroscopy (ICP-AES).

The concentration of ammonium ions was determined using the phenol method according to Solorzano et al. Briefly, 1 ml of supernatant after centrifugation of the culture broth was dissolved in 1 ml (0.8%) of phenol solution (dissolved in ethanol), 1 ml of 0.0075% sodium nitroprusside, and 2 ml of oxidation. The solution was mixed with 1.5% tri-sodium citrate, 0.08% sodium hydroxide, 0.2% sodium hypochloride. The mixture was incubated at room temperature for 4 hours and the optical density was measured at 640 nm.

Example 9: Peroxide analysis

Hydrogen peroxide (H ₂ O ₂ ) or organic perlock in medium with added glycerol solution, pretreated waste glycerol, or crude fatty acid after sterilization using PeroxiDetect ^™ kit purchased from Sigma (USA) The side was quantified. To prepare a color reagent, 25 mM ferrous ammonium sulfate solution and xylenol orange solution supplied from Sigma were mixed at a volume ratio of 1: 100. 1 ml of color developing reagent was added to the 0.1 ml sample, mixed, and left to stand at room temperature for 30 minutes. Peroxide present in the sample in the process is used to divalent 3 is oxidized to iron ions (Fe ³ ⁺⁾ to ferrous ions (Fe ² ⁺⁾ in the acidic conditions, the resulting 3 gacheol ions (Fe ³ ⁺⁾ is character Ile It forms a complex with xylenol orange and becomes reddish purple. This was measured at 560 nm using a spectrophotometer. 30% hydrogen peroxide (H ₂ O ₂ ) aqueous solution and 70% tert-butyl hydroperoxide (t-BuOOH) solution purchased from Sigma were converted into standard solutions.

Experimental Example One. otsBA Characterization of Recombinant Strains

1-1. Growth and Acetic Acid Production Characteristics

Blank strain which is a beta-carotene producing strain prepared in Example 4 on R-medium, a synthetic medium to which 20 g / l of purified glycerol (purity 98% or more) is added; otsBA was introduced to compare the characteristics of recombinant strains transformed beta-carotene producing strains. 50 ml of medium was added to a 500 ml baffled flask and incubated at 25 ° C at 180 rpm. When the cell concentration reached OD ₆₀₀ _nm 5, IPTG was added.

As a result, the blank strain reached growth of OD 17 ± 0.5 in 67 hours and stopped growing. At the same time, the beta-carotene production reached the maximum value of 122 ± 17 mg / l. Even after growth stopped, glycerol continued to decrease, and the amount of residual glycerol reached about 1.3 ± 0.1 g / l at 115 hours. Maximum acetic acid yield was very low, 0.3 ± 0.04 g / l (FIG. 5).

The recombinant strain, a beta-carotene producing strain transformed with otsBA , reached OD 23 ± 2.0 at 67 hours of cultivation and continued to grow, reaching a maximum of 28 ± 1.6 at 90 hours. The maximum production of beta-carotene was also 147 ± 3.4 mg / l higher than the blank strain. Glycerol continued to be used and was nearly depleted at 115 hours of culture. The amount of acetic acid produced was also very small, with a maximum of 0.02 g / l (FIG. 6).

1-2. Waste glycerol Fatty acid Effect on Strain Growth and Beta-Carotene Production

Fatty acids were recovered from the waste glycerol, and the effects of the fatty acids were gradually added to the growth medium of the strain prepared in Example 4 on R-medium, a synthetic medium to which 20 g / l of pure glycerol was added.

Separated fatty acids were added 0.7 wet-g, 1.4 wet-g, 2.1 wet-g, and 2.8 wet-g on the basis of wet weight. Corresponds to the amount of fatty acids in the medium added to 40 g / l, 60 g / l, 80 g / l. After 86 hours of incubation, the blank and recombinant strains were compared.

Blank strain decreased cell growth to about half level and beta-carotene production level was about 1/6 level when 0.7 wet-g fatty acid was added as compared to the case where no fatty acid was added (Fig. 7A). And 7B).

Recombinant strain increased the cell concentration until the addition of 1.4 wet-g fatty acid, the cell concentration was slightly decreased even when 2.8 wet-g was added, but there was no significant difference. Beta carotene production also did not show a big difference until the addition of 2.1 wet-g fatty acid, and when 2.8 wet-g was added, the production tended to decrease slightly (Figs. 7A and 7B).

Meanwhile, after the addition of fatty acid and autoclave, 90-105 μM of hydrogen peroxide (H ₂ O ₂ ) and 109-116 μM of organic peroxide were detected in the prepared medium.

These results indicate that fatty acids inhibit cell growth and beta-carotene biosynthesis of blank strains. The inhibitory effect of fatty acids on cell growth appears as a complex effect of direct inhibition by fatty acids and indirect inhibition due to hydrogen peroxide (H ₂ O ₂ ) or organic peroxide production. Escherichia coli has been reported to be unable to metabolize small or medium chain fatty acids (C ₆ -C ₁₂ fatty acids) and inhibit their growth (Journal of Bacteriology, 1973, vol. 115, No 3, p. 869-875; Journal of general microbiology, 1975, vol. 91, p. 233-240). Long chain fatty acids such as C18 do not show an inhibitory effect on Escherichia coli, but may be easily oxidized when high temperature heat is generated to generate hydrogen peroxide (H ₂ O ₂ ). The hydrogen peroxide (H ₂ O ₂ ) thus produced is reacted with an unsaturated fatty acid in series to form an organic peroxide. Hydrogen peroxide (H ₂ O ₂ ) or organic peroxide formed in this way, even at low concentrations, decreases the activity of enzymes involved in the respiratory chain in cells, and breaks down the structure of proteins through aggregation of cell membrane proteins through cell membrane fatty acid attack. , Or disruption of cell membranes through disruption of cell membranes, disruption of repair systems in the synthesis of intracellular DNA, or the like, causing death or inhibiting the growth of cells (Journal of Bacteriology, 1986, vol. 166, No 2, p. 519-527; Journal of Biological Chemistry, 1989, vol. 264, No. 3, p. 1729-1734; Biochimica et Biophysica Acta, 1990, 510-516; J. Agric.Food Chem., 1991, vol. 39, p. 439-442; Genetics and Molecular Biology, 2004, vol. 27. No. 2, p. 291-303).

Therefore, the recombinant strain of the present invention is hardly affected by growth and beta-carotene biosynthesis even in the presence of fatty acids, so that trehalose accumulated in cells by expression of trehalose biosynthetic gene, which was first identified by the present inventors, is hydrogen peroxide (H ₂ O). ₂ ) and results from protecting the cells from organic peroxides.

1-3. Effect of Methanol on Growth and Beta-Carotene Production of Strains

The effect of strain on growth of the strain was compared by adding methanol to the R-medium supplemented with 20 g / l of pure glycerol. Since methanol is highly volatile and highly likely to evaporate during sterilization, methanol was added after filter sterilization (pore size 0.2 μm) to investigate the effect of concentration. After 5 days of culture, the blank strain and the recombinant strain were compared.

Compared to the case where no methanol was added, the blank strain did not show a significant difference in cell growth until 7.5 g / l methanol was added, whereas the cell growth of the recombinant strain was slightly decreased. Meanwhile, when 5 g / L of methanol was added, the beta-carotene production of both strains slightly increased (FIGS. 8A and 8B).

The present results indicate that both strains have similar resistance to methanol, and despite the presence of 7.5 g / l methanol, cell growth is hardly inhibited. The addition of 5 g / l methanol is shown to promote beta-carotene biosynthesis rather. According to the existing research, E. coli has a lag phase for a certain amount of time when alcohol is added. During this period, cells can resist a certain concentration of alcohol by changing the composition of fatty acids, which are the main components of their cell membranes. You have the ability. Alcohols with lower carbon atoms have a higher maximum concentration at which cells can resist alcohol (Journal of Bacteriology, 1976, vol. 125, No. 2, p. 670-678).

1-4. Growth and Beta-Carotene Production of Strains According to Pure Glycerol Concentration

Pure glycerol was added to 20 g / l, 40 g / l, 60 g / l, and 80 g / l in R-medium, followed by incubation at each concentration for 5 days, and the growth and beta-carotene of blank and recombinant strains. Biosynthesis was compared.

Compared with the addition of 20 g / l glycerol, the blank strain did not show a significant difference in cell growth until 60 g / l glycerol was added, whereas the cell growth of the recombinant strain was slightly decreased. Beta-carotene biosynthesis showed the maximum when 40 g / l glycerol was added for the blank strain, while the recombinant strain decreased slightly with increasing glycerol concentration. (FIGS. 9A and 9B). The results show that both strains have similar resistance to glycerol but the blank strains are slightly better.

1-5. KCl Effect of concentration on the growth of recombinant strains

The blank strain and the recombinant strain prepared in Example 4 were added to the medium to which pure glycerol (20 g / L) was added with varying KCl concentration in the range of 0 to 300 mM to investigate the effect, and after 5 days of culture Cell growth was compared (FIGS. 10A and 10B). Since the K ⁺ ions (154 mM) in the form of K ₂ HPO ₄ in the basic medium present K ⁺ ions to be supplied to the KCl concentration of the form it is added to it. That is, when 100 mM KCl is added, 254 mM K ⁺ is present in the medium.

Blank strain was determined that the growth and beta-carotene production was not inhibited up to 200 mM when KCl was added, the cell growth and beta-carotene production was increased when adding 100 mM KCl.

On the other hand, recombinant strains were found to slow cell growth and beta-carotene production when KCl was added. In conclusion, recombinant strains were determined to have lower KCl resistance than blank strains (FIGS. 10A and 10B).

Experimental Example 2. otsBA Introduced recombinant strain Waste glycerol Check the possibility of using as a substrate

2-1. Preprocessed Waste glycerol And Waste glycerol Comparison of Growth and Beta-carotene Production Characteristics in Stomach

The growth and beta-carotene production characteristics of the blank strain prepared in Example 4 were investigated. As mentioned above, when pure glycerol was added, the strain started growing after showing an lag time of 18 hours, and reached a maximum cell concentration of OD 17 ± 0.5 at 67 hours of culture. At this time, beta-carotene production was also the maximum 122 ± 17 mg / l (Fig. 5).

On the other hand, in the medium to which the pre-treated glycerol was added, the blank strain showed a lag time of 48 hours, and then started to grow, and reached 114 OD at 114 hours. Beta-carotene biosynthesis amount at this time was 74 ± 12 mg / l. This corresponds to 0.65 mg / l · h, which is about one third of the productivity (1.82 mg / l · h) in pure glycerol supplemented medium (FIGS. 11A and 11B).

The lag time of the blank strain in the medium containing waste glycerol showed 48 hours similar to that in the medium containing the pretreated glycerol, and reached OD 20 ± 0.5 at 114 hours. At this point, beta-carotene production reached a maximum of 78 ± 6.8 mg / L (FIGS. 12A and 12B).

These results indicate that pretreated glycerol or waste glycerol severely inhibit cell growth and beta-carotene biosynthesis of blank strains.

In addition, as mentioned above, the recombinant strain in medium containing pure glycerol reached OD 23 ± 2.0 in 67 hours of culture, and continued to grow, reaching a maximum of 28 ± 1.6 in 90 hours. The maximum beta-carotene was 147 ± 3.4 mg / l (FIG. 6).

On the other hand, in the medium to which pretreated waste glycerol was added, the recombinant strain started to grow without lag time and reached OD 30 ± 2.1 at 114 hours. Beta-carotene biosynthesis amount at this time was 118 ± 1.1 mg / l. This corresponds to a productivity of 1.04 mg / l · h, which is 63% of the productivity (1.6 mg / l · h) in pure glycerol supplemented medium (FIGS. 11A and 11B).

On the other hand, even in the medium containing waste glycerol, the recombinant strain began to grow without lag time, and reached 114 OD at 114 hours. Beta-carotene biosynthesis did not increase further after reaching 89 ± 0.1 mg / L at 66.5 hours. This is 61% of the production concentration in the pure glycerol addition medium, but the productivity is 1.3 mg / l · h corresponds to 81% of the productivity in pure glycerol. Compared with the blank strain cultured in the medium to which waste glycerol was added, the recombinant strain showed about 2 times higher productivity than the blank strain (FIGS. 12A and 12B).

Figure 13 shows the culture after incubating the recombinant strain for 5 days in the medium to which waste glycerol is added.

These results indicate that the recombinant strain overexpressing the otsBA gene of the present invention not only increases growth in waste glycerol but also increases beta-carotene production.

2-2. Waste glycerol Effect of concentration on cell growth and growth

The concentration range of the waste glycerol examined was 20-80 g / l, and the results of growth and beta-carotene production of the strain prepared in Example 4 after 5 days of culture were compared.

Blank strain decreased cell growth with increasing glycerol concentration. In other words, when the concentration of waste glycerol was increased to 60 g / l at OD of 17 ± 1.5 in 20 g / l waste glycerol medium, OD 10 ± 0.6 of 59% was shown (FIG. 14A). Beta-carotene production also decreased from 90 ± 13.5 mg / l to its 54% level of 48 ± 9.1 mg / l (FIG. 14B).

Recombinant strains did not decrease cell concentration despite the presence of 60 g / l waste glycerol. Upon addition of 80 g / l waste glycerol, the cell concentration was reduced by about 12% (FIG. 14A). Beta-carotene production was the maximum value of 151 ± 6.2 mg / L with the addition of 40 g / L waste glycerol, and decreased to 110 ± 13.8 mg / L when adding 80 g / L (Fig. 14B).

2-3. Pretreatment Waste glycerol Effect of concentration on cell growth and growth

The content of pretreated waste glycerol was adjusted to 20 g / l, 40 g / l, 60 g / l, and 80 g / l, and the growth and beta of blank and recombinant strains according to the concentration of pretreated waste glycerol were added. -Changes in carotene production were examined.

Blank strain decreased cell growth when the concentration of pretreated waste glycerol was increased to 40 g / L, but there was little change in beta-carotene production (FIGS. 15A and 15B).

When the recombinant strain increased the concentration of pretreated waste glycerol up to 60 g / l, cell growth was reduced, but the production of beta-carotene was increased (Figs. 15A and 15B).

2-4. Pretreatment and Waste glycerol Salts present in the medium added by concentration, Fatty acid salt , Peroxide Content and its effect

The results for the salts, fatty acid salts, peroxide contents and their effects present in the media to which pretreatment and waste glycerol were added at different concentrations are shown in Table 7. Pretreated waste glycerol had a higher potassium ion content than waste glycerol at the same concentration. This is because HCl is added to remove fatty acids from waste glycerol, which is believed to be due to the release of potassium ions while fatty acids precipitate in the form of fatty acids.

The concentration of potassium in the medium upon addition of 80 g / l of waste glycerol or pretreated waste glycerol was 179 mM and 236 mM, which is lower than the concentration at which the blank strains were resistant (250 mM) (FIGS. 10A and 10B). About 8 g / l of methanol is present in the medium to which 60 g / l of waste glycerol or pretreated waste glycerol is added, which is the concentration at which the blank strain is resistant (FIGS. 8A and 8B). In addition, the blank strain showed no inhibition of cell growth and beta-carotene biosynthesis even in the medium to which high concentration (60 g / L) of pure glycerol was added (FIGS. 9A and 9B). However, the inhibition of cell growth and beta-carotene biosynthesis in medium supplemented with low concentration (20 g / l) of peglycerol was not toxic to fatty acids, hydrogen peroxides (H ₂ O ₂ ) and organic peroxides present therein. It is believed to be due. On the other hand, cell growth and beta-carotene biosynthesis of blank strains were inhibited even in the medium to which pretreated peglycerol, which almost eliminated fatty acids, was added at low concentration (20 g / L). This clearly shows that cell growth is inhibited by the toxicity of the peroxides present. However, the concentration was less affected by the addition of waste glycerol, which is considered to be less affected by its toxicity due to the lack of fatty acids.

Recombinant strain overexpressed otsBA did not inhibit cell growth and beta-carotene biosynthesis even in medium containing high concentration (60 g / L) waste glycerol. This indicates that the recombinant strain is resistant to the toxicity of high concentrations of fatty acids and peroxides. However, cell growth and beta-carotene biosynthesis in the medium containing pretreated waste glycerol were lower than those in the medium containing the same concentration of waste glycerol, indicating that the amount of potassium ions present in the pretreated peglycerol was higher than the waste glycerol. More is believed to be affected.

In general, recombinant strains overexpressing otsBA accumulate trehalose, which has been reported to allow the strain to resist high osmotic pressure induced by sodium or potassium ions (Applied Microbiology & Biotechnology, 2005, 71, No. 7, p. 3761-3769), strains of the present invention showed the opposite results of being inhibited by potassium salt. However, it is considered that the amount of potassium salt present in the concentration range of peglycerol does not have much influence on the performance of the recombinant strain. The present inventors for the first time pointed out that the biggest obstacle to be solved in using waste glycerol as a substrate for microbial culture is toxicity by fatty acids and peroxides present therein. The results suggest that the recombinant strain overexpressed otsBA developed by the present inventors can effectively use peglycerol by being resistant to toxicity thereof.

Salts, Fatty Acids, and Peroxides in Media Added Pretreated Waste Glycerol by Concentration

Waste Glycerol Added Medium Medium with pretreated waste glycerol Glycerol Concentration (g / l) Glycerol Concentration (g / l) 20 40 60 80 20 40 60 80 ¹ K + (mM) 160 ± 0.0 167 ± 0.0 173 ± 0.0 179 ± 0.0 174 ± 0.0 194 ± 0.0 213 ± 0.0 236 ± 0.0 Fatty acid salt (g / ℓ) ² 14 ² 28 ² 43 ² 57 ³ 0.2 ³ 0.4 ³ 0.6 ³ 0.8 Methanol
(g / ℓ) 2.9 ± 0.1 5.7 ± 0.3 8.6 ± 0.4 11.4 ± 0.6 2.6 ± 0.1 5.1 ± 0.2 7.7 ± 0.3 10.3 ± 0.3 Hardogen Peroxide
(uM) 8.1 ± 2.6 16.2 ± 5.2 24.3 ± 7.8 32.4 ± 10.4 1.2 ± 0.5 2.4 ± 1.0 3.6 ± 1.4 4.8 ± 1.9 Organic peroxides
(uM) 18.8 ± 1.4 37.6 ± 2.4 56.4 ± 4.1 75.2 ± 5.5 4.4 ± 1.0 8.8 ± 2.1 13.1 ± 3.1 17.5 ± 4.2

154 mM K + ions present in the form of KH ₂ PO ₄ in ¹ medium components

² Quantify by recovering fatty acid precipitated under acidic condition

³ Determination of fatty acids by extraction with organic solvent (chloroform: methanol = 2: 1)

Experimental Example 3. Using a bioreactor Batch culture

The recombinant strain prepared in Example 4 was cultured after filling a medium into which 3.4 g stirring fermenter (Cobiotech Co., Ltd.) was added 60 g / l of air supplied and controlled by temperature and pH. 16). The temperature was maintained at 25 ° C. and the pH was adjusted to neutral with 2N sulfuric acid and ammonia water. The volume of the liquid was 1.5 liters.

17 shows cell concentration, beta-carotene production over time of incubation. Dry cell weight was measured to determine the growth behavior of the cells. After 67 hours of culture, the cell concentration rapidly increased to 18 g / L at about 177 hours. This is OD ₆₀₀ _nm is a value corresponding to 73. Beta-carotene was produced after 20 hours, the production rate increased rapidly after 90 hours to reach 128 mg / l at 165 hours. The amount of acetic acid produced as a by-product was reused by the cells after reaching a maximum value of 0.1 g / l in the 57 hours at which cell growth began. The concentration of ammonium ion used as a nitrogen source decreased and began to increase after 78 hours. This was because ammonia was used for pH control, because after 57 hours, the pH of the culture medium decreased due to the production of acetic acid and ammonia was added to adjust it to neutrality (FIG. 16). Therefore, this culture condition is a condition that the nitrogen source is sufficiently supplied. Glycerol in the waste glycerol used as a carbon source was rapidly reduced after 57 hours from the start of cell growth, and was depleted at 102 hours, almost the end of cell growth. However, even after this time, slow cell growth and beta-carotene biosynthesis occur, presumably because the strain uses fatty acids present in waste glycerol, especially long chain unsaturated fatty acids such as oleic acid as carbon sources.

Experimental Example 4. Using a bioreactor Oil price culture

Cultivation of the recombinant strain prepared in Example 4 was started in batch mode at an initial 20 g / L of waste glycerol, and after the glycerol was depleted, waste glycerol was supplied by pH-stat.

17 shows cell concentration, beta-carotene production over fed-batch incubation time. Dry cell weight was measured at the same time in addition to OD to determine the exact cell growth. The maximum cell concentration was 36 g / l and the maximum beta carotene production was 175 mg / l (FIG. 17).

Experimental Example 5. Beta-carotene extraction and purification

Wet cell cake was recovered from 1.5 L of batch culture of the recombinant strain, from which beta-carotene was recovered. After washing twice with distilled water, the cells were dehydrated with 85% 2-propanol solution. 1.6 L of IBA was added thereto to extract beta-carotene from dehydrated cells. The extract was concentrated to 160 mL under reduced pressure, then 640 mL of 2-propanol was added. It was left for 4 to 12 hours to obtain beta-carotene crystals.

In addition, the recovered beta-carotene crystals were washed with 50 ml ethanol to obtain high purity beta-carotene crystals, and UV-spectrophotometry and HPLC analysis were performed to confirm the beta-carotene purity.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY KIM, YOU GEUN <120> A microorganism of being able to grow and produce value-added materials in the waste-glycerol environment <130> PA100203 / KR <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gaattcgtga cagaaccgtt aaccgaaac 29 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tctagacgca agctttggaa aggtat 26 <210> 3 <211> 2200 <212> DNA <213> Artificial Sequence <220> <223> otsBA <400> 3 ctacgcaagc tttggaaagg tagcaacttt atcgcgctgc tggctttccg cgcttcgcgg 60 aactatctgc tttaggtcgc taatgaagca ctcctgccag tggttaatat cgtttttcac 120 gataacgtcc agcatttctg catgacggga aatacgttcc gccagcgaca tagtcaatgc 180 acgatccagc gcagctgcaa cttcgtcacg atcgtagggg ttaacaatta acgccgacgt 240 taactcgttt gccgctcccg caaattgcga aagaacaaga acgcccggat tggctgggtc 300 ctgagcagca acatactctt ttgctaccag gttcatcccg tcacgcagtg gcgtcactaa 360 gcccacgtca gagtagcgga atattttcat cagtaattta cggtcaaaat gctgattcaa 420 ataataaagc ggcgtccagc ctaattgccc gtatttacca ttaattcgtc cagcttcatt 480 ttcgagctga tgacgaatat cctgataggc ttgcacatca ccacgcgacg ttggtgcaat 540 ctgggtataa cgaattttac catgatgctg cggatatttt tccagcaacg cttcataggc 600 gagaaaacgc tctggcaaac ctttggaata atccagccgt tcgacagaaa agatattttg 660 tacgtttttc agttccgctt taagttgcgc cagttttggc ggcagtggcc cggcagcctg 720 tttggctatt tctttcggtt caatgccgat cgggtagact tctgttcgaa atgctttgcc 780 ccaggctgta tggctttttg cgctacgtgt cgtgacgcgg gtcaggttag aaagacaatc 840 caggaacgcc agacgatcgt tttctgtctg gaaacccagc aaatcataat cacaaagctg 900 ttcaagcaag gtgtcatatg tcggcagcgc gttgaagatt tccggtgtcg ggaaaggaat 960 atgcagaaag aaaccaatgc gattattcac tccccgtttg cgtaattcat gcgcaaatgg 1020 caacaggtga taatcgtgga tccagataat gtcatcgtct tgcaacagcg gcagtaattt 1080 atctgccagc aacgcattta cgcgtagata gccgtcccag gcaggacgct gaaattgcac 1140 cagatcgagc cgataatgaa aagcgggcca gagaacggca ttggagaatt ggttgtagta 1200 ttcgtcaagg tcctgttcgc tgaggttaaa agaggcccac gtaatgttac cttttttcac 1260 cttttttagc ggctgatcct cattccctgt ttcaccactc cagccaaacc acagtccgcc 1320 tgcggctttc agtgccccca gtatgccaac ggcaaggcca ccggcactgg cggcgtgctc 1380 gtctggtggt gcaatccggt tagatactac gactaaacga ctcatagtca tcactcctgt 1440 tattttctct tttttgttgt aatgcggtgg ttatcatttc aagccagctc cagacatccg 1500 gcacacccgc cagtcgccat gatgcctgag ttgcacctgt gccaattttt actgacattc 1560 cgcccagtcg gttaacgact gcgaagccag attcatcggt taaatcatcg cccagaaata 1620 cgggcgttcg cccgataaag ggagcttcct gcataaaagc tgcaattgcc tcacctttac 1680 tggtacctct cggtttgatc tcgacaacac actttccctg ctgtaacgcc atttgtggcc 1740 agatctgagt aatacgttgc gctaatgtca ttaatgcgtc ttcatgctgc ggagcctgac 1800 gataatgcag cgcaaaagcc atccctttcg cctccagctc cgcgccggga tactgagcga 1860 tgactgtatg cagttgcacg ctaatatcac gcgcaatcgc atccggcaga tgaacgatat 1920 gtgttttacc attgatgtca cggcgctccg ccccatgcac gcccgctaac gggaagcgat 1980 aaggttttgc cagtgcgtca agctccacca ttgagcgccc tgatatcaat gccaatgcac 2040 catcacttgc ggttgccagt agctgtagtc cttgcagaat attgtcaggc acgacgacct 2100 gatcgggatg cggtttgatt tccgccagcg ttccatcaag atcaaaaaac caggcatatt 2160 tcgcggatag ttcaggggtt tcggttaacg gttctgtcac 2200

Claims

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A method of enhancing growth of said microorganism in the presence of waste glycerol, comprising culturing an Escherichia microorganism overexpressed with the otsBA gene.

i) the otsBA gene; And
ii) a glycerol metabolism gene selected from the group consisting of gldA (glycerol dehydrogenase) and glpD (glycerol-3-phosphate dehydrogenase),
Escherichia microorganism using waste glycerol as a substrate.

The microorganism of claim 5, wherein the microorganism is a waste glycerol producing carotenoid, bioethanol, biobutanol, 1,3-propanediol or 3-hydroxypropionic acid as a substrate. Microorganisms used.

i) the otsBA gene; And
ii) crtE (geranylgeranyl pyrophosphate synthase), crtB (phytoene synthase), crtI ( Phytoene dehydrogenase), crtY (Lycopene cyclase), dxs (1-deoxy-D-xylulose-5-phosphate synthase), mvaE (acetoacetyl-CoA synthase 3-hydroxy-3-methylglutaryl-CoA reductase), mvaS (hydroxymethylglutaryl-CoA synthase), mvaK1 (mevalonate kinase), mvaK2 (phosphomevalonate kinase), mvaD (mevalonate diphosphate decarboxylase), idi (isopentenyl diopenate isomeris ), ipiHP1 pyrophosphate carotenoid biosynthesis gene selected from the group consisting of: dimethylallyl pyrophosphate isomerase and ispA (farnesyl-diphosphate synthase)
Escherichia microorganism producing carotenoids in the presence of waste glycerol.

8. The microorganism of claim 7, wherein said carotenoid produces a carotenoid selected from the group consisting of lycopene, beta-carotene, astaxanthin, zeaxanthin, and xanthophyll.

The microorganism of claim 8, wherein the microorganism produces beta-carotene.

The microorganism of claim 9, wherein the microorganism is transformed with a vector comprising the genes crtE , crtB , crt I , ipiHP1 , crtY , dxs , mvaE , mvaS , mvaK1 , mvaK2 , mvaD and idi .

The microorganism of claim 7, wherein the microorganism is Escherichia coli.

8. The microorganism of claim 7, wherein the waste glycerol comprises at least 15% fatty acid salt.

(a) culturing the strain of any one of claims 7 to 12; And
(b) a method for producing a carotenoid comprising recovering beta-carotene from the culture of step (a).

The method of claim 13, wherein the culturing of step (a) is batch or fed-batch.