KR20150019086A - Novel Butanediol Producing Microorganism and Method for Preparing Butanediol Using thereof - Google Patents

Abstract

The present invention relates to a novel butanediol producing microorganism. More specifically, the present invention relates to: a novel bacillus genus butandediol producing microorganism; and a method for producing 2,3-butanediol from a fiber carbon source. When the method of the present invention is used, 2,3-butanediol can be produced by using a wood-based biomass.

Description

[0001] The present invention relates to a novel butanediol-producing microorganism and a method for producing butanediol using the same,

The present invention relates to a novel butanediol-producing microorganism, and more particularly, to a novel microorganism producing Bacillus butanediol and a method for producing 2,3-butanediol from a fibrous carbon source using the novel microorganism.

2,3-Butanediol (2,3-BDO) is a chemical raw material that is widely used in printer ink, perfume, moisturizer, softener, plasticizer, and medicinal materials. In particular, 2,3-butanediol It can be used as a raw material for rubber, so it is attracting attention as a representative biochemical raw material that can replace petrochemical raw materials. In addition, the utilization of 2,3-butanediol is expected to increase in the field of agriculture due to the physiological activity functions such as promoting crop growth.

The technology for the production of 2,3-butanediol by microorganisms has been active since the early 20th century as the demand for butadiene raw material increased sharply. As the petrochemical industry developed, microbial fermentation technology tended to decline somewhat. However, due to the recent high oil price situation and the seriousness of the environmental pollution problem in the petrochemical industry, the technology of 2,3-butanediol production by microorganisms has attracted new attention have. A representative microorganism having an excellent ability to produce 2,3-butanediol is Klebsiella pneumoniae . K. pneumoniae , a thermophilic anaerobic microorganism, is able to utilize various carbon sources including glycerol, and has excellent advantages of cell growth. The highest yields of 2,3-butanediol production to date have been reported by K. pneumoniae , producing optically inactive meso-type and optically active L- (+) - type 2,3-butanediol . However, K. pneumoniae is a mild disease, but there are disadvantages to the hospital and efforts are being made to overcome it.

As a result, 2,3-butanediol production technology by safe microorganisms is being developed at the same time. It is known that some microorganisms of the genus Bacillus , which mainly live in the soil, produce 2,3-butanediol. Unlike K. pneumoniae , microorganisms of the genus Bacillus have been reported to produce D - (-) - type optically active and meso-type 2,3-butanediol. In particular, microorganisms of the genus Bacillus have a saccharogenic enzyme activity which decomposes a fibrous carbon source and have the advantage of being able to grow at a high temperature suitable for the activity of a saccharifying enzyme.

The present inventors have made efforts to find a microorganism strain capable of producing 2,3-butanediol using a non-pathogenic and fibrous carbon source. As a result, it has been found that a new Bacillus genus microorganism is isolated, Butanediol is superior in the ability to produce butanediol, thus completing the present invention.

It is an object of the present invention to provide a novel strain of Bacillus that produces 2,3-butanediol.

Another object of the present invention is to provide a method for producing 2,3-butanediol from woody biomass using the Bacillus sp . Strain.

In order to achieve the above object, the present invention provides a Bacillus genus BRC1 strain (KCTC 12428BP) having 2,3-butanediol producing ability.

The present invention also provides a method for producing 2,3-butanediol, comprising: (a) culturing a Bacillus sp . BRC1 strain to produce 2,3-butanediol; And (b) obtaining 2,3-butanediol from the resulting 2,3-butanediol.

Using the novel Bacillus genus microorganisms according to the present invention, 2,3-butanediol can be produced using abundant woody biomass.

FIG. 1 shows the results of confirming the production of 2,3-butanediol by culturing a Bacillus sp . Microorganism using glucose as a carbon source.
Fig. 2 shows the amino acid sequence homology analysis of the 2,3-butanediol synthase of the genus Bacillus .
FIG. 3 shows the results of confirming the production ability of 2,3-butanediol of a microorganism of the genus Bacillus in a medium containing a hydrolyzate of an EFB pretreated with an alkali.
FIG. 4 shows the result of examining the correlation between saccharification enzyme concentration, strain inoculation amount and incubation temperature on the production of 2,3-butanediol by simultaneous saccharification culture of Bacillus sp . Microorganism in a medium containing EFB solids pre-treated with alkali will be.
FIG. 5 shows the results of investigation of 2,3-butanediol production ability by simultaneous saccharification culture of Bacillus sp . Microorganism in the medium containing the alkali-pretreated EFB solids.
Fig. 6 shows the results of analysis of saccharogenic activity of Bacillus sp . Microorganisms cultured in a medium containing Cellobiose as a carbon source.
FIG. 7 shows the results of analysis of the production of 2,3-butanediol by microorganisms of the genus Bacillus cultured in a medium containing Cellobiose as a carbon source.

In one aspect, the present invention relates to a novel microorganism Bacillus genus BRC1 having 2,3-butanediol producing ability.

The Bacillus genus BRC1 strain of the present invention is a strain having 2,3-butanediol-producing ability isolated from soil, and has a 16S rDNA sequence of SEQ ID NO: 1 and a 2,3-butanediol synthase gene represented by the nucleotide sequence of SEQ ID NO: .

The amino acid sequence of the 2,3-butanediol synthase of the Bacillus sp. Strain BRC1 is known to be Bacillus subtilis and Panibacillus (Bacillus), whereas the high homology of 2,3-butanediol synthase and 93.6% and 71.4% respectively of polymyxa, Brevibacterium generating a L-isomer of 2,3-butanediol synthase of saccharolyticum , and Klebsiella producing meso-type isomer pneumoniae , and 2,3-butanediol synthase of Klebsiella oxytoca .

The Bacillus genus BRC1 of the present invention has excellent exoglucanase and? -Glucosidase activity, and it was confirmed that 2,3-butanediol can be produced by saccharifying woody biomass (FIG. 6) .

The BRC1 strain of the genus Bacillus of the present invention has a saccharifying enzyme activity and is capable of growing at a high temperature, and is a strain suitable for producing 2,3-butanediol.

In another aspect, the present invention provides a method for producing 2,3-butanediol, comprising: (a) culturing a Bacillus sp . BRC1 strain to produce 2,3-butanediol; And (b) obtaining 2,3-butanediol from the resulting 2,3-butanediol.

The Bacillus genus BRC1 of the present invention was found to produce 2,3-butanediol in a glucose-containing medium, but also to produce a level of 2,3-butanediol in a medium containing woody biomass 7).

In the present invention, the carbon source of the culture medium is preferably woody biomass, more preferably palm kernel shell (EFB), cellobiose, carboxymethyl cellulose (CMC), cellulose, avicel ), Xylan, or the like is preferably used.

It is preferable that the EFB is used as a carbon source after being pre-treated with an alkali.

In one embodiment of the present invention, concurrent saccharification fermentation was carried out under optimum conditions using alkali pretreated EFB (10%, W / V), resulting in 27.42 g / L of butanediol (FIG.

The 2,3-butanediol produced according to the present invention can be utilized in agricultural fields that promote the resistance and growth of pathogens of chemical raw materials or crops.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1. 2,3- Butanediol Productive  A new branch Bacillus  Isolation of strain

In order to isolate the new 2,3-butanediol-producing Bacillus strains, a sample obtained from the soil around the innate acid was first dried on a shade for 3 days and then homogenized with a 1 mm sieve. The dried soil samples were diluted by step dilution to 10 < ^-5 > and diluted to 10 ^-5 with Tryptic Soy Agar (Diffco, Tryptone, 1.7%; Soytone, 0.3%; H ₂ HPO ₄ , 0.25%; NaCl, 0.5%; Dextrose, 0.25% %) Medium. After incubation at 30 ° C for 2 days, 16S rRNA sequence analysis (base sequence 1) of the microorganisms which are regarded as Bacillus strains according to the morphological characteristics, and a new strain of Bacillus genus were obtained. Named as Bacillus genus BRC1, (Accession No. KTCC12428BP).

Example  2. Bacillus genus BRC1  The 2,3- Butanediol  produce

(2%; Peptone, 1%; K ₂ HPO ₄ , 0.4%; Glucose, 2%) to determine if the isolated microorganisms produce 2,3-butanediol. To analyze 2,3-butanediol production. For liquid culture, the production of 2,3-butanediol was analyzed by liquid chromatography while culturing at 37 ° C and 200 rpm in LB medium containing 10 g / L of glucose. As a result, as shown in FIG. 1, most of the glucose added at 12 hours of culture was consumed, and 2,3-butanediol was produced up to 5.27 g / L and then gradually decreased.

Example  3. Bacillus genus BRC1  The 2,3- Butanediol  Synthetic enzyme gene analysis

The 2,3-butanediol synthase gene was amplified from the chromosome of Bacillus genus BRC1 using a primer prepared by comparing 2,3-butanediol synthase gene derived from Bacillus sp . Strain.

SEQ ID NO: 3: 5'-ATGAAAGCTGCAAGATGG-3 '

SEQ ID NO: 4: 5'-TTAATTCGGTTTTACTAAGAT-3

Sequence analysis of the amplified gene resulted in the nucleotide sequence of SEQ ID NO: 2 (Genebank KF358987).

As a result of the homology analysis of the amino acid sequence of the 2,3-butanediol synthase derived from the new Bacillus sp . Strain, the 2,3-butanediol synthase of Bacillus genus BRC1 mainly produced D-isomer Bacillus subtilis (Genebank CAB12443) and Panibacillus ( Bacillus ) polymyxa (Genebank HQ730089), 93.6% and 71.4%, respectively, compared with the 2,3-butanediol synthase of Brevibacterium saccharolyticum (Genebank AB009078), 8% for the 2,3-butanediol synthase, and Klebsiella pneumoniae (Genebank D86412) showed 8.5% and 9.4% similarity with 2,3-butanediol synthase, respectively.

Example  4. EFB The saccharified solution  Used Bacillus genus BRC1  The 2,3- Butanediol  produce

EFB (Palm empty fruit bunch) was reacted in 1M NaOH solution at 121 ° C for 60 minutes, and the resulting solid was hydrolyzed using a conventional saccharifying enzyme. The production of 2,3-butanediol was analyzed by culturing the Bacillus genus BRC1 in a medium containing EFB glycated solutions of various glucose concentrations in the same manner as in Example 1. [ As a result, as shown in FIG. 3, the strain BRC1 was able to grow smoothly using EFB saccharified liquid, and it was found that 2,3-butanediol was produced from this.

Example  5. EFB Using Bacillus genus BRC1  Strain Simultaneous saccharification fermentation  Establish optimal conditions

The effect of saccharification enzyme loading (cellulase, FPU), strain inoculum (g DW) and saccharification fermentation temperature (temperature) in the simultaneous saccharification fermentation of alkaline pretreatment EFB was investigated using the central composite design (Table 1).

As a result, as shown in FIG. 4, the optimum condition for simultaneous saccharification fermentation using 5% (w / v) EFB solids was 38.8 FPU / g of glycated enzyme, 2.6% (v / v) Was 44.8 ° C, and the amount of 2,3-butanediol produced was estimated to be 27 g / L (R ² = 0.9671).

Simultaneous Saccharification Fermentation Conditions of Alkali Pretreatment EFB by Central Composite Design Runs Temperature
(° C) Enzyme
(FPU / g) Inoculation
(%, v / v) 2,3-BD
(g / L) One 32 20 5.0 11.8 2 42 20 5.0 21.0 3 32 40 5.0 20.8 4 42 40 5.0 23.7 5 32 20 2.5 16.5 6 42 20 2.5 25.3 7 32 40 2.5 19.0 8 42 40 2.5 22.6 9 27 30 7.5 10.8 10 47 30 7.5 23.7 11 37 10 7.5 15.0 12 37 50 7.5 22.0 13 37 30 7.5 25.6 14 37 30 12.5 22.6 15 37 30 7.5 21.0 16 37 30 7.5 21.0 17 37 30 7.5 21.0 18 37 30 7.5 21.0 19 37 30 7.5 21.0 20 37 30 7.5 21.0

Example  6. EFB Using Bacillus genus BRC1  Strain At the simultaneous saccharification fermentation  2,3- Butanediol  produce

As a result of practicing the simultaneous saccharification fermentation experiment under the optimal conditions derived in Example 5, it was found that 27.42 g / L of 2,3-dichloro-2-propanol similar to the predicted from the alkali pretreatment EFB (10%, w / v) Butanediol was produced.

Example  7. Bacillus genus BRC1  Strain Saccharogenic enzyme  Activity analysis

The activity of the extracellularly secreted glycosyltransferase was determined by culturing the BRC1 strain of Bacillus genus in a medium containing 1% (w / v) of cellulose, CMC (carboxymethylcellulose), avicel, Respectively.

The activity of endoglucanase was measured by carboxymethyl cellulose sodium salt (CMC) as a substrate, reacted with enzyme, and the free reducing sugar was measured by 3,5-dinitrosalicylic acid (DNS) method as follows.

After 2% (w / v) CMC was dissolved in 0.05 M sodium citrate buffer (pH 4.8), 0.05 ml of culture supernatant was added and reacted in a 50 ° C thermostat for 30 minutes. Immediately after 30 minutes, 0.2 ml of 3,5-dinitrosalicylic acid (DNS) reagent was added and the reaction was stopped by heating at 100 ° C for 5 minutes. The absorbance of the resulting reducing sugar was measured at 540 nm. 1 unit was defined as the amount of enzyme producing 1 μmol of reducing sugar per minute per ml.

The activity of exoglucanase was determined by dissolving 0.25% Avicel in 0.05 M acetate buffer (pH 4.8) and reacting with 40 μl of enzyme in a 50 ° C thermostat for 2 hours. Then, 3,5-dinitrosalicylic acid (DNS) , And the reaction was stopped. The reaction was stopped by boiling in boiling water for 5 minutes, followed by color development. The resultant was cooled for 10 minutes in cold water, and the enzyme activity was assayed by measuring the absorbance at 540 nm using a spectrophotometer.

The DNS reagent was prepared by dissolving 10 g of 3,5-dinitrosalicylic acid, 300 g of Rochelle salt and 16 g of NaOH in 1 L of distilled water. 1 unit was defined as the amount of enzyme producing 1 μmol of reducing sugar per minute per ml.

β- Glucosidase activity was measured by dissolving 15 mM cellobiose in 0.05 M sodium citrate buffer (pH 4.8), mixing 200 μl of the substrate solution and 200 μl of the culture supernatant, followed by reaction at 50 ° C in a thermostat for 30 minutes. After the reaction, the mixture was boiled in boiling water for 5 minutes, cooled in cold water for 10 minutes, and glucose production was measured by HPLC analysis. One unit of enzyme activity was defined as the amount of 1 μmol of glucose produced per minute.

Reduced sugar was prepared by adding 200 μl of a solution of DNS (2,4-dinitrosalicylic acid) to 10 μl of enzyme reaction solution, boiling for 5 minutes in a constant temperature water bath at 100 ° C, cooling the reaction mixture by ice water to terminate the reaction, adding 1 ml of distilled water, Were measured and quantified.

As a result, as shown in FIG. 6, when the strain of Bacillus genus BRC1 was cultured in a culture medium containing cellobiose, the activity of glycated enzyme was detected. On the other hand, the medium containing cellulose, CMC, avicel and xylan showed very little cell growth of BRC1 strain.

Example 7 . Cellobiose Using Bacillus genus BRC1  The culture of 2,3- Butanediol  produce

The production of 2,3-butanediol by the Bacillus genus BRC1 was analyzed in the cellobiose-containing medium. When cultured in the medium containing 1% (w / v) of cellobiose as a carbon source in the same manner as in Example 1, 2,3-butanediol was produced in almost the same level as in the glucose-containing medium as shown in Fig. Respectively.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biotechnology Research Institute KCTC12428BP 20130621

<110> Korea Reseach Institute of Bioscience and Biotechnology <120> Novel Butanediol Producing Microorganism and Method for Preparing          Butanediol Using <130> P13-B149 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1250 <212> DNA <213> Bacillus sp. BRC1 <400> 1 ctgggataac tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca 60 gacataaaag gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg 120 gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gatcggccac 180 actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 240 tggacgaaag tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc 300 tctgttgtta gggaagaaca agtgccgttc aaatagggcg gcaccttgac ggtacctaac 360 cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg 420 tccggaatta ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc 480 ccggctcaac cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg 540 gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg 600 actctctggt ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat 660 accctggtag tccacgccgt aaacgatgag tgctaagtgt tagggggttt ccgcccctta 720 gtgctgcagc taacgcatta agcactccgc ctggggagta cggtcgcaag actgaaactc 780 aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 840 gaagaacctt accaggtctt gacatcctct gacaatccta gagataggac gtccccttcg 900 ggggcagagt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 960 agtcccgcaa cgagcgcaac ccttgatctt agttgccagc attcagttgg gcactctaag 1020 gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1080 tgacctgggc tacacacgtg ctacaatgga cagaacaaag ggcagcgaaa ccgcgaggtt 1140 aagccaatcc cacaaatctg ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa 1200 gctggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1250 <210> 2 <211> 1041 <212> DNA <213> Bacillus sp. BRC1 <400> 2 atgaaagcgg caagatggca caatcaaaaa gacattcgaa tcgaaaacat tgatgaacct 60 aaagcagaac cgggtaaagt caaaattaaa gtcaaatggt gcgggatttg cggaagcgac 120 cttcatgaat atttgggagg cccgatcttt attccggtcg gcaaacctca tccgttaaca 180 aacgaaatgg cgcccgtcac gatgggtcac gaattctcag gagaagtcgt tgaagtgggc 240 gaaggcgtca aaaattacag cgtcggcgac cgtgtcgtcg tggaaccgat ttttgccaca 300 cacggacatc agggagccta taaccttgat gaacaaatgg gattcttagg cttagccgga 360 ggcggcggcg gattttctga atatgtatcc gttgacgaag aattgctgtt taagcttcct 420 gaagagcttt cttacgaaca gggcgcgctt gttgagccgt cagcggtagc gctttacgcc 480 gtccgccaaa gcaaattaaa agcgggcgac aaggcggcag tttttggctg cggaccgatc 540 gggctgcttg tgattgaagc cctgaaagcg gccggcgcca cagatattta cgcggttgaa 600 ctttcaccgg aacgtcagga aaaagcgaaa gaactcggcg ctatcatcat tgatccgtca 660 aaaacggacg atgtcgttga agaaatcgct aaacgcacaa acggcggcgt tgatgtttct 720 tatgaagtaa caggcgttcc tgtcgttctc cgccaggcaa tccagtcaac aaacattgcc 780 ggtgaaacgg ttatcgtcag catctgggaa aaaggagcgg aaattcatcc gaacgatatc 840 gtcatcaaag aacggaccgt aaaaggcatt atcggatacc gtgacatctt cccttccgtt 900 cttgcactga tgaaagaggg ctacttctca gcagataagc tcgtcacgaa aaaaatcgtg 960 ctcgacgatc tgattgaaga aggctttggc gctttaatca aagagaaaaa ccaagtgaaa 1020 atcttagtaa aaccgaatta a 1041 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atgaaagctg caagatgg 18 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ttaattcggt tttactaaga t 21

Claims (6)

Bacillus sp . Strain BRC1 (KCTC 12428BP) having 2,3-butanediol producing ability.
The Bacillus sp . Strain BRC1 according to claim 1, which has the 16S rDNA sequence of SEQ ID NO: 1.
2. The Bacillus sp . Strain BRC1 according to claim 1, which has a 2,3-butanediol synthase gene having the nucleotide sequence of SEQ ID NO: 2.
A process for producing 2,3-butanediol comprising the steps of:
(a) culturing a strain of Bacillus sp . BRC1 to produce 2,3-butanediol; And
(b) obtaining the resulting 2,3-butanediol.
The method for producing 2,3-butanediol according to claim 4, wherein the carbon source of the culture medium is woody biomass.
6. The method of claim 5, wherein the woody biomass is selected from the group consisting of palm kernel shell (EFB), cellobiose, carboxymethyl cellulose (CMC), cellulose, avicel, and xylan. 3-butanediol.

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