KR101149882B1 - Method of high productivity butanol production using microorganism with elevated butanol titer - Google Patents

Abstract

The present invention relates to a method for producing butanol with high productivity using a high butanol producing strain, specifically Clostridium Beerlinki beijerinckii ) NCIMB 8052 After mutating a wild-type strain, and screening strains capable of producing butanol at a high concentration, the present invention relates to a method for producing butanol with high productivity using butanol high production strains. Butanol high-producing strain prepared by the method of the present invention can be very useful in the bio-butanol commercialization technology.

Description

Method of high productivity butanol production using microorganism with elevated butanol titer

The present invention relates to a microorganism and a method for producing butanol using the same.

The recent rise in international crude oil prices has accelerated the development of alternative energy sources that can replace gasoline, a transportation fuel. In the United States and Brazil, bioethanol production using starch sugar or raw sugar has exceeded 100 trillion gallons as of the end of 2007, and the market is gradually expanding. In addition, biodiesel based on vegetable oils is rapidly growing to replace existing crude oils derived from crude oil, mainly in Europe and Southeast Asia.

In addition to bioethanol, interest in various alternative energy sources has recently increased, with butanol being the most representative of them. Butanol has many advantages over ethanol as a fuel. First, the energy content is much higher than that of ethanol, which is almost close to gasoline level, low vapor pressure, low volatility, low hygroscopicity, and low corrosiveness, allowing transmission using the existing gasoline transport infrastructure pipeline. Butanol as a fuel has been inferior to ethanol in terms of technology until now, but it has attracted much attention as a next-generation alternative energy. Butanol is one of the oldest microbial fermentation products, along with ethanol, and has been referred to as ABE fermentation because acetone, butanol and ethanol are simultaneously produced by microorganisms of the genus Clostridium, which was metabolized by Pasteur in 1861. Butanol fermentation had secured economic feasibility until petrochemicals until the 1950s, but since the rapid development of petrochemicals, it is no longer competitive. However, since the 1990s, the interest has been re-emerged as an alternative energy source in the future, and a lot of research and development is progressing.

In terms of the results of the development of high-butanol-producing strains, the most research using the Clostridium microorganism, which is well known as the conventional ABE fermentation, is the Hans Blaschek group of the University of Illinois in the United States. Through the process of finding a strain resistant to a chemical called deoxyglucose), a high-butanol-producing strain with high amylose resolution was developed (Hans Blaschek et. al . , Appl . Environ . Microbiol . 57 (9): 2544-2548, 1991). The strain having excellent amylose resolution is Clostridium Beirusky ( Clostridium beijerinckii ) was designated as BA101, it has been reported as the strain showing the best butanol fermentation to produce butanol of 20 g / l level (US Patent No. 6358717). In addition to the strain development research, the research group has been carrying out various research and development related to butanol production, such as the development of extraction fermentation process using gas stripping, P2 medium development. In addition, the Michigan Biotechnology Institute group has also developed and reported a new strain by chemical variation using Clostridium acetobutyricum ATCC 824 strain (US Patent No. 5192673). . Recently, through the process of finding strains resistant to chemical mutations and cell membrane inhibitors at the Korea Research Institute of Chemical Technology, butanol-resistant and highly productive strains were developed (Korean Patent Application No. 10-2008-0078005).

In 2001, the complete sequence of the representative wild strain Clostridium acetobutylicum ATCC 824 was revealed (Nolling J et. al . , J. Bacteriol . 183 (6): 4823-4838, 2001), many new strains produced using recombinant DNA technology have also been reported. A representative research group is the Papoutsakis group at Northwestern University in the United States, which has shown new possibilities by discovering genes favorable for butanol production using antisense RNA and genomic DNA libraries. In addition, the Bennett group of Rice University has conducted a number of studies on the correlation between the production of organic acids and butanol using recombinant DNA technology (Scotcher MC et. al . , Appl. Environ . Microbiol . 71 (4): 1987-1995, 2005).

Recently, many butanol-producing strains are aware of the limitation of still producing low concentrations of butanol, and many studies have been conducted to utilize E. coli or other strains as butanol-producing strains. Representatively, DuPont clones all genes of the butanol biosynthetic metabolic pathway of Clostridium strains, which are highly resistant to other butanols such as E. coli (International Patent Publication No. 2007/041269) or Lactobacillus (Lactobacillus). Patent No. 2008-0124774) to try to develop a new recombinant strain, but the concentration of fermentation is still very small. In addition, the Liao group of UCLA recently designed a novel butanol metabolic pathway by combining amino acid metabolism of E. coli, 2-ketoacid decarboxylase, and alcohol dehydrogenase. Reported (Atsumi et al . , Nature 451 (3): 86-90, 2008). But the actual level of butanol production is also negligible.

In the 1980s, much research was conducted on the effect of butanol on microorganisms in Clostridium. The main effect is that the increase in butanol increases the saturated acyl groups in the cell membrane components compared to unsaturated acyl groups (Vollherbst-Schneck K). et al ., Appl . Environ . Microbiol . 47 (1): 193-194, 1984), and for similar reasons, it has been reported that the resistance to butanol is increased by the addition of oleic acid and laidic acid from the outside (Baer SH). et al ., Appl . Environ . Microbiol. 53 (12): 2854-2861, 1987). In addition, the production of butanol increases at a concentration that inhibits growth in Clostridium, making it difficult to maintain the homeostasis of intracellular pH, lowering the level of ATP as an energy source for cells, and lowering the glucose absorption rate as the main carbon source. (Bowles LK & Ellefson WL, Appl . Environ . Microbiol . 50 (5): 1165-1170, 1985). However, despite various studies on the cell membrane and physiological effects of the Clostridium microorganisms of butanol, there have been no reports of successful cases of improving these physiological characteristics and applying them to the development of new strains.

As described in the above research and development trends, butanol production is still below 20 g / L through various approaches of strain development. In order to overcome this problem, various researches are being conducted in terms of processes such as gas extraction or permeation evaporation using membranes. However, many difficulties are expected until commercialization, so developing a fermentation method with high butanol productivity is the most efficient method for commercial production of butanol. This can be called.

Thus, the present inventors screened a high-butanol-producing strain after chemical mutation to the microorganism, in order to increase the productivity of butanol of the genus Clostridium microorganism well known by conventional ABE fermentation, and cultured for butanol production of the strain The present invention was completed by optimizing the process to confirm that butanol can be produced with high productivity.

It is an object of the present invention to provide a high butanol producing strain and a method for producing butanol with high productivity using the same.

In order to achieve the above object, the present invention is a butanol high-producing mutant Clostridium Beinglinki ( Clostridium) deposited with accession number KCTC11610BP beijerinckii ) provides a CBE-2-68 strain.

In addition,

1) species culture the strain of claim 1;

2) main culture of the strain cultured in step 1);

3) It provides a butanol mass production method comprising the step of obtaining the supernatant by centrifuging the main culture solution of step 2).

Mutant Clostridium Bassilinki of the present invention beijerinckii ) The CBE-2-68 strain produces butanol at a higher efficiency of 60% or more than the wild-type Clostridium Bayerlinki NCIMB 8052 strain, and when cultured according to the method of the present invention, the butanol production efficiency is confirmed to be higher. Therefore, the strain of the present invention and the butanol production method using the same can be very usefully used in the bio-butanol commercialization technology.

1 is a graph showing the butanol fermentation pattern of the parent strain and the deposited strain using the optimized butanol production process of the present invention.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention is a butanol high-producing mutant Clostridium Beinglinki ( Clostridium) deposited with accession number KCTC11610BP beijerinckii ) provides a CBE-2-68 strain.

The strain of the present invention is an increase in butanol production of at least 30%, preferably at least 60%, than the Clostridium Beirinki NCIMB 8052 wild type strain.

In a specific embodiment of the present invention, a variant strain library was prepared by treating the Clostridium Beaverinky NCIMB 8052 cells with an alkylating chemical EMS (Ethylmethane sulfonate) for mutagenesis. After culturing the strains of the library under anaerobic conditions, 56 strains with 30% or more improvement in butanol production compared to the parent strain were selected and named CBE1 to CBE56, respectively. The 56 strains were treated with EMS again to induce mutations, and CBE-2-68 was selected as the best strain with 60% or more improvement in butanol production compared to the parent strain. It was deposited on 24 November, and was given accession number KCTC11610BP.

Butanol-resistant and high butanol-producing strains of the present invention can produce a high concentration of butanol, it can be usefully used for the mass production of butanol.

In addition,

1) species culture the strain of claim 1;

2) main culture of the strain cultured in step 1);

3) It provides a butanol mass production method comprising the step of obtaining the supernatant by centrifuging the main culture solution of step 2).

After step 3) may further comprise the step of purifying butanol. Purifying the butanol is preferably performed through chromatography using a column, but any method used in the art for separating gas including butanol may be used without particular limitation.

When the absorbance at 600 nm of the seed culture solution of step 1 reaches 1.5 to 8, preferably 2 to 5, and more preferably 2, the main culture is inoculated into the main culture solution. Preferred but not limited to.

In step 2), the main culture is inoculated at 10 to 15% (v / v) of the seed culture solution in the main culture, and the main culture is more preferably inoculated at 10% (v / v), but not limited thereto. It doesn't happen.

The main culture of step 2)

Iii) culturing the seed culture solution of step 1) in the main culture medium for 5 to 15 hours, preferably 5 hours in anaerobic conditions; And,

Ii) after adjusting the pH of the main culture solution of step iii) to 5.0 to 6.0, preferably 5.0 to 5.5, the culture temperature of 31 to 36 ℃, preferably 32 to 34 ℃, more preferably 34 ℃ And incubating for 25 to 50 hours, preferably 40 hours in anaerobic conditions, but is not limited thereto.

In order to establish the optimum culture conditions for mass production of butanol using the strain of the present invention in a specific embodiment of the present invention, when the strain of the present invention inoculated into the main culture medium and the seed culture liquid for the main culture medium As a result of confirming the optimal volume ratio of, it was confirmed that inoculation so that the volume ratio of the seed culture medium to the culture medium at the initial absorbance of the logarithmic growth period is 10%, the most effective for the butanol fermentation of the resistant variant strain of the present invention ( See Tables 1 and 2).

During butanol fermentation, the initial pH control of the culture is a very important process variable. In general, in the case of Clostridium microorganisms, when fermentation is performed using glucose, organic acids accumulate at the neutral pH level, and the acidity in the medium increases as the culture proceeds to produce an organic solvent including butanol. Has been converted to (Papoutsakis et al . , Bioenergy Chapter 25: 323-334, 2008, ASM Press). Therefore, in the case of butanol producing strains, in order to maximize the production of butanol as the precursor of butanol and the efficient conversion to butanol, the initial pH control of the culture medium during fermentation and the pH control in the main culture are necessary. Thus, as a result of confirming the pH adjustment time and the proper pH of the main culture solution in the main culture, when the pH was adjusted to 5.0 after 10 hours in the main culture, it was confirmed that the most effective in the butanol fermentation of the resistant variant strain of the present invention (Table 3 And Table 4).

In addition, after culturing the strain and the parent strain Clostridium Bayerkinki NCIMB 8052 under the optimized culture conditions, butanol production was compared, it was confirmed that the productivity is improved by about 84% or more (see FIG. 1). )

The butanol production method according to the present invention using the strain of the present invention can be useful for mass production of butanol because it can produce butanol with high efficiency compared to the parent strain.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Preparation of Mutant Strain Libraries by Chemical Mutation

<1-1> Cultivation

Clostridium beijerinckii NCIMB 8052 cells, which were stored in spores at 4 ° C., were heat-treated at 80 ° C. for 10 minutes, and then inoculated in 5 ml RCM medium (DIFCO, USA) for 16 in anaerobic conditions. Primary seed cultures were performed for hours. Thereafter, 1 ml of the culture solution was inoculated again into 100 ml RCM medium, and then secondary culture was performed for 4 hours until the absorbance at 600 nm reached a logarithmic growth phase.

<1-2> Main culture

Add 1 liter of modified P2 medium (Annous A & Blaschek HP, Appl . Environ . Microbiol . 56: 2559-2561, 1990) containing 60 g / l of glucose at pH 6.0 and 2 g / l of yeast extract . The seed culture solution was inoculated into a fermenter adjusted to anaerobic conditions by supplying 0.5 vvm level, followed by main culture at 37 ° C. and 200 rpm.

<1-3> Determination of Concentrations of Mutagenic Chemicals

After the main culture for about 4 to 5 hours to reach the beginning of the logarithmic growth phase, the cells were recovered by centrifugation of about 10 ml of the main culture solution and then resuspended in 1 ml of TYA medium. 1 ml of each of the chemicals for the mutagenesis EMS (Ethylmethane sulfonate) (Sigma, USA) at the concentration of 0, 1, 2, 5, 10 and 15 μl / ㎖ respectively, 37 ℃, under anaerobic conditions, Mutations were induced for 60 minutes at 200 rpm. Then, washed three times with 1 ml of PT buffer (Annous A & Blaschek HP, Appl . Environ . Microbiol . 56: 2559-2561, 1990), and then suspended in 1 ml of TYA medium, 37 ° C. under anaerobic conditions. , And incubated for about 7 hours at 200 rpm. After diluting the culture solution 1,000 times with sterile water, and then counting the number of surviving cells by plating in RCM solid medium, the killing rate per treatment concentration was calculated using Equation 1 below.

EMS treatment concentration (ul / ml)  Cell count (colonies)  % Mortality 0 (control) To 10,000 0 One To 10,000 0 2 To 10,000 0 5 To 7,000 30 10 53 99.47 15 0 100

As a result, as shown in Table 1, the optimal EMS treatment concentration at which the killing rate was about 99% or more was determined to be 10 ul / ml. In addition, the cells that survived the EMS treatment at 10 ul / ml was used as a mutant strain library.

Butanol  High Production Strain Selection

<2-1> Culture of Mutant Strains

The mutant strain library obtained in Example 1-3 was plated in RCM solid medium in the same manner as in Example 1-3, and then incubated at 37 ° C. and 200 rpm under anaerobic conditions for about 4 days. As a result, a total of 1323 mutant strains were obtained, and the mutant strains were simultaneously incubated in 14 96-well microplates (volume: 2.5 ml / well) in order to shortly select high butanol-producing strains from the strains. Specifically, a total of 1323 resistant mutant strains using a colony picking device QPIX2 (GENETIX, USA) using a 96-well plate containing 1 ml of P2 medium containing 1% antifoam (Antifoam A; Sigma, USA) per well After inoculation to, it was incubated for 4 days at 37 ° C. under anaerobic conditions.

<2-2> Butanol  dose

The 96-well plate of Example 2-1 was centrifuged at 3000 rpm for 10 minutes using a dedicated centrifuge (Hanil, Korea) to separate cells and fermentation broth, and then HPLC (1100 series Agilent, USA) was performed by a known method. Bunday et al . , Enzyme Microbiol . Technol . 12 (1): 24-27, 1990), butanol concentration was analyzed using an Aminex HPX-87H column.

As a result, 56 strains in which butanol production was improved by at least 30% compared to the parent strain were obtained, which were named CBE1 to CBE56.

<2-3> Regeneration of Mutant Strains

56 strains obtained in Example 2-2 were induced again by the same method as in Example 1-3, and then plated in RCM medium, respectively, and then subjected to about 4 at 37 ° C. and 200 rpm under anaerobic conditions. Incubated daily. Thereafter, the culture was performed in the same manner as in Example 2-1, and the butanol production was quantified by the method of Example 2-2.

As a result, among the 56 strains, CBE-2-68 strains with improved butanol production of over 60% compared to the parent strain were selected as the best butanol-producing strains. It was deposited on the 24th and was given accession number KCTC11610BP.

Butanol  Production fermentation process optimization

<3-1> Main culture  Inoculation time

After culturing the CBE-2-68 strain by the method of Example 1-1, when the absorbance at 600 nm of the seed culture solution was 2, 5, 8 and 10, 100 ml of the seed culture solution was performed. After inoculation into 1 L of the main culture medium of Example 1-2, the main culture was carried out in the same manner as in the above 1-2. After the main culture was completed, butanol production amount of the main culture solution was measured in the same manner as in Example 2-2.

As a result, as shown in Table 2, it was confirmed that inoculation into the main culture medium when the absorbance at the beginning of the logarithmic growth phase was 2 was most effective for the butanol fermentation of the resistant mutant strain of the present invention.

Seed Culture OD (600 nm)  Maximum Butanol Concentration (g / ℓ) One 14.7 2 16.4 5 15.3 8 13.5 10 10.9

<3-2> Main culture  At the time of inoculation, Cultivation Volume ratio

After straining the CBE-2-68 strain by the method of Example 1-1, 50, 100, 150 and 200 ml of the seed culture solution were inoculated into 1 L of the main culture medium of Example 1-2, respectively. The culture was carried out in the same manner as in Example 1-2. After the main culture was completed, butanol production amount of the main culture solution was measured in the same manner as in Example 2-2.

As a result, as shown in Table 3, it was confirmed that inoculating so that the volume ratio of the seed culture medium to the main culture medium is 10%, the most effective for the butanol fermentation of the resistant variant strain of the present invention.

Species Culture Size (Volume Ratio,%)  Maximum Butanol Concentration (g / ℓ) 5 14.5 10 16.4 15 15.7 20 12.4

<3-3> Main culture pH  adjustment

After straining the CBE-2-68 strain by the method of Example 1-1, 100 ml of the seed culture solution was inoculated into 1 L of the main culture medium whose pH of the main culture medium of Example 1-2 was adjusted to 6.5. By the same method. After 10 hours, the pH of the main culture solution was adjusted to 4.5, 5.0, 5.5, and 6.0 or higher. The difference in the time to reach the highest butanol production according to the adjusted pH was analyzed by measuring the concentration of butanol in the same manner as in Example 2-2.

As a result, as shown in Table 4, butanol fermentation of the strain of the present invention was confirmed that the most effective when the pH of the culture medium is 5.0 or more.

Adjustable pH  Butanol maximum concentration (g / ℓ)  Reach time (hr) 4.5 or more 3.7 12 5.0 or more 16.5 32 5.5 or more 14.5 30 6.0 and above 14.1 30

<3-4> Main culture pH  Adjustment point

The CBE-2-68 strain was cultured in the same manner as in Example 1-1, and then 100 ml of the seed culture solution was inoculated in 1 L of the main culture medium of Example 1-2, and then cultured in the same manner. . After incubation for 5, 10, 15 and 20 hours, the pH of the main culture solution was adjusted to 5.0. The difference in time to reach the peak butanol production according to the pH adjustment time was analyzed by measuring the concentration of butanol in the same manner as in Example 2-2.

As a result, after the main culture for 5 hours as shown in Table 5, when the pH of the main culture solution was adjusted to 5.0 or more, it was confirmed that butanol fermentation of the mutant strain of the present invention is most effective.

Adjust time (hr)  Butanol maximum concentration (g / ℓ)  Reach time (hr) 2 10.7 47 5 17.1 32 10 16.8 32 15 16.7 32 20 8.0 30

<3-5> Culture temperature adjustment

After culturing the CBE-2-68 strain by the method of Example 1-1, 100 ml of the seed culture solution was inoculated into 1 L of the main culture medium of Example 1-2, and Example 1-2 and The main culture was carried out in the same manner. The temperature of the culture solution was adjusted to 30, 32, 34 and 37 ° C, and then the fermentation phase was measured by measuring the concentration of butanol in the same manner as in Example 2-2. Analyzed.

As a result, as shown in Table 6, butanol fermentation of the strain of the present invention was confirmed to be most effective when the temperature of the main culture solution was adjusted to 34 ° C.

Fixed temperature (℃)  Butanol maximum concentration (g / ℓ)  Reach time (hr) 30 14.1 32 32 16.5 32 34 17.1 30 37 14.1 30

Optimization of culture conditions

The CBE-2-68 strain and the culture strain Clostridium Bayeringki NCIMB 8052 was optimized in Examples 3-1 to 3-5 (inoculation time is OD 600 nm 2; species at inoculation Culture volume ratio is 10%; initial pH of the main batch is 6.2; the temperature of the main batch is 34 ° C; pH control time and pH of the main culture solution: adjusted to 5.0 or more after 5 hours of the main culture; and, pH in the main culture After the adjustment, the incubation time was 40 hours) and the concentration of butanol was measured in the same manner as in Example 2-2.

As a result, the parent strain produced about 10.7 g / l butanol, whereas the CBE-2-68 strain of the present invention produced 19.7 g / l butanol. As a result, the CBE-2-68 strain of the present invention confirmed that the butanol productivity was improved by approximately 84% or more than the parent strain.

Korea Biotechnology Research Institute KCTC11610 20091221

Claims (15)

delete 1) species culture of the butanol high production mutant Clostridium beijerinckii CBE-2-68 strain deposited with accession number KCTC11610BP;
2) When the absorbance at 600 nm of the seed culture medium of step 1) reached 1.5 to 8, 5 to 17% (v / v) of the strain cultured in the culture medium was inoculated at 5 to 17% under anaerobic conditions. Main culture for 15 hours;
3) adjusting the pH of the main culture solution of step 2) to 5.0 to 6.0 and then incubating for 25 to 50 hours at an incubation temperature of 31 to 36 ° C. and anaerobic conditions; And,
4) Butanol mass production method comprising the step of obtaining the supernatant by centrifuging the main culture solution of step 3).
The method for mass production of butanol according to claim 2, further comprising purifying butanol after step 4).
delete The method for mass production of butanol according to claim 2, wherein in step 2), the absorbance at 600 nm of the seed culture solution reaches 2 to 5, and the main culture is inoculated into the culture medium.
The method for mass production of butanol according to claim 5, wherein the culture medium is inoculated in the main culture medium when the absorbance at 600 nm of the seed culture solution reaches 2, and the main culture is carried out.
delete The method for mass production of butanol according to claim 2, wherein the main culture is inoculated at 10 to 15% (v / v) in the main culture medium in step 2).
The method for mass production of butanol according to claim 8, wherein the main culture is inoculated at 10% (v / v) with the seed culture solution.
delete The method for mass production of butanol according to claim 2, wherein the step 2) is carried out for 5 hours under anaerobic conditions.
The method for mass production of butanol according to claim 2, wherein the pH of the main culture solution is adjusted to 5.0 to 5.5 in step 3).
The method for mass production of butanol according to claim 2, wherein the culture medium is cultured at a culture temperature of 32 to 34 ° C in step 3).
The method for mass production of butanol according to claim 13, wherein the culture medium is incubated at a culture temperature of 34 ° C.
The method for mass production of butanol according to claim 2, wherein the step 3) is incubated for 40 hours under anaerobic conditions.

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