WO2012030195A2

WO2012030195A2 - Mutant microorganism having high ethanol generation ability from glycerol or crude glycerol, and method for producing ethanol using same

Info

Publication number: WO2012030195A2
Application number: PCT/KR2011/006536
Authority: WO
Inventors: 김철호; 서정우; 오백록; 허선연
Original assignee: 한국생명공학연구원
Priority date: 2010-09-03
Filing date: 2011-09-02
Publication date: 2012-03-08
Also published as: WO2012030195A3; WO2012030195A9; KR20120024019A; KR101777211B1

Abstract

The present invention relates to a mutant microorganism having enhanced capacity for generating ethanol using glycerol or crude glycerol as a carbon source, and to a method for producing ethanol using same. More particularly, the present invention relates to a mutant microorganism having high ethanol generation ability from glycerol or crude glycerol, wherein the enzymatic activity of alcohol dehydrogenase (AdhII) is increased and the enzymatic activity of 1,3-propanediol oxidoreductase (DhaT) is decreased in the mutant organism. The present invention also relates to a microorganism improved by means of the aforementioned mutation, and to a method for producing ethanol using the mutant microorganism.

Description

【Specification】

[Name of invention]

Mutant microorganisms having high ethanol production from glycerol or waste glycerol and a method for producing ethanol using the same

Technical Field

<1> The present invention relates to a mutant microorganism having an increased ability to produce ethane using glycerol or waste glycerol as a carbon source, and a method for producing ethanol using the same, more specifically, alcohol dehydrogenase S (alcohol dehydrogenase, Adhn) Mutant microorganism having high ethanol performance from glycerol or waste glycerol with increased enzymatic activity and reduced enzymatic activity of 1,3-propanedi oxidoreductase (1,3 ᅳ propanol oxidoreductase, DhaT) Improved mutant microorganisms of microorganisms and a method for producing ethanol using the mutant microorganisms.

<2>

Background Art

At present, as a major by-product of the biodiesel manufacturing process, waste glycerol is generated in an amount equivalent to about lOT (w / w) of the total production (Johnson and Taconi, 2007). Such waste glycerol not only affects the market price of conventional glycerol, but also can be released directly to the environment, which is becoming an important environmental problem such as treatment cost (da Silva eia / .2009). The development of a method for converting low-cost waste glycerides into industrially valuable materials, including fuels and bioactive substances, is well underway.

<4> Glycerol is genus Lactobacillus (genus ^ ac obac / / s), genus Clostridium (genus)

C / os r / i // uffl), genus fe e / Obac er and genus Klebsiella have been used as fermentative nutrients by several bacteria, including propane di, ethanol and other It can be converted into various compounds. Recently, the Gonzales research group produced ethane from glycerol by anaerobic fermentation of E. coli, which was traditionally known as non-fermentable microorganism (Dharmadi ea / .2006; Gonzalezei.a / .2008; Murarkaeia / .2 (X) 8). Compared to the cost of producing ethanol from corn starch-derived glucose, the price from glycerol is about 40% lower (Yazdani and Gonzalez, 2007). Therefore, attention has been paid to the metabolic engineering of microbial strains for the effective fermentation of ethane and by-products from glycerol (Yazdani and Gonzalez 2008; Durnin eta J.2009). <5> The highest yield of fermented ethane from glycerol reported to date is 19.5 g / L at 35 hours incubation time in fed-batch culture process of Klebsiella oxytoca and productivity at that time Is 0.56 g / L per hour (Yang G ea / .2007). In fermentation cultures with recombinant E. coli, the maximum fermentation yield of glycerol to ethanol was 20.7 g / L for 96 hours, but the productivity per hour It was 0.22 g / L, much lower than Toka (Durnin eia / .2009).

Therefore, the present inventors have made diligent efforts to develop microorganisms that produce industrially useful ethanol with high efficiency using glycerol or waste glycerol as a carbon source. It was confirmed that the production of mutant microorganisms having a performance, and using the microorganisms, can produce ethane from glycerol or waste glycerol with high efficiency, thereby completing the present invention.

<7>

<8> Summary of the Invention

An object of the present invention is to provide a mutant microorganism having high ethanol production performance from glycerol or waste glycerol and a method for producing the same.

Another object of the present invention is to provide a method for producing ethanol from glycerol or waste glycerol using the mutant microorganisms.

In order to achieve the above object, the present invention is to increase the enzyme activity of alcohol dehydrogenase (Adhn), 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) The present invention provides a mutant microorganism having high ethanol production from glycerol or waste glycerin with reduced enzymatic activity.

In addition, the present invention increases the enzyme activity of alcohol dehydrogenase (Adhn) and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT). In a mutant microorganism having high performance from reduced glycerol or waste glycerol, a mutant microorganism having a gene encoding Lactate dehydrogenase (LdhA) is deleted.

In addition, the present invention increases the enzyme activity of alcohol dehydrogenase (Adhn) and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT). From reduced glycerol or waste glycerol, pyruvate dehydrogenase in mutant microorganisms with high ethanol production A gene encoding (pyruvate dehydrogenase, Pdc) and a gene encoding aldehyde dehydrogenase (aldehdyde dehydrogenase, Adhll) are introduced to provide a mutant microorganism overexpressed.

In addition, the present invention is to increase the enzyme activity of alcohol dehydrogenase (Adhll), pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (alddhdyde dehydrogenase (Adhll)) High ethanol production from glycerol or glycerol with reduced enzymatic activity of 1,3-propanol oxidoreductase (DhaT) and lactate dehydrogenase (LdhA) It provides a variant microorganism having.

In addition, the present invention (a) by irradiating gamma rays to microorganisms alcohol dihydrogenase

(acohol dehydrogenase (Adhll)) to increase the enzyme activity and 1,3-propanediol oxidase ductase (1,3-propanol oxidoreductase, DhaT) to reduce the enzymatic activity; And (b) mutations such that the enzyme activity of the alcohol dehydrogenase (Adhll) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) is reduced. It provides a method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol comprising the step of obtaining a predetermined microorganism.

(acohol dehydrogenase (Adhll)) to increase the enzyme activity and 1,3-propanediol oxidase ductase (1,3-propanol oxidoreductase, DhaT) to reduce the enzymatic activity; (b) the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is mutated to decrease the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase (DhaT)). Obtaining a live microorganism; And (c) deleting the gene encoding the lactate hydrogenase from the obtained microorganism. Provided is a method for producing a mutant ^' microorganism having high production of ethane from glycerol or waste glycerol, including. .

In addition, the present invention ( _a ) by irradiating gamma rays to microorganisms alcohol dihydrogenase

(alcohol dehydrogenase, Adhll) to increase the enzyme activity and 1,3-propanediol oxidase ductase (1,3-propanol oxidoreductase, DhaT) to reduce the enzymatic activity; (b) Increased enzyme activity of alcohol dehydrogenase (Adhll) and reduced enzyme activity of 1,3—propanedi oxidoreductase (1,3-propanol oxidoreductase (DhaT) Obtaining a microorganism; And (c) phase Glycerol or waste glycerol comprising overexpressing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (Adhll) in the obtained microorganism. It provides a method for producing a mutant microorganism having high ethanol production.

(acohol dehydrogenase, Adhll) to increase the enzyme activity and 1,3—propanediol oxidase ductase (1,3-propanol oxidoreductase, DhaT) to reduce the enzymatic activity; (b) mutated to increase the enzyme activity of the alcohol dehydrogenase (Adhll) and to reduce the enzyme activity of 1,3-propanediol oxidoreductase (DhaT). Obtaining a live microorganism; And (c) a gene encoding the lactate hydrogenase in the microorganism obtained above, the gene encoding the pyruvate dehydrogenase (Pdc) and an aldehyde dehydrogenase (aldehdyde dehydrogenase). Adhll) provides a method for producing a mutant microorganism having high performance in ethane from glycerol or waste glycerol comprising the step of overexpressing the gene encoding.

In addition, the present invention provides a method for producing ethane comprising the step of culturing the mutant microorganisms in a medium containing glycerol or waste glycerol as a carbon source to produce ethane, and then recovering ethane from the culture solution. to provide.

<20>

[Brief Description of Drawings]

1 is a schematic diagram showing the metabolic pathway of glycerol fermentation of Krebssiella pneumoniae.

Figure 2 shows the ethane production of mutant microorganisms according to the control and the present invention ((a) K. pneumoniae Cui ^, (b) K. pneumoniae GEM167¾- ^).

Figure 3 shows the production of ethane according to the pH of K. pneumoniae GEM167 variant strains.

Figure 4 shows the production of ethane according to the air injection amount of K. pneumoniae GEM167 mutant strain.

5 shows ethanol production by fed-batch culture using pure glycerol of K. pneumoniae GEM167 mutant strain.

FIG. 6 is a fed-batch culture using waste glycerol of K. pneumoniae GEM167 mutant strain. Ethanol production by

Figure 7 shows the production method (A) of the K. pneumoniae W Δ IdM mutant strain and Southern hybridization results (B) confirming the ldhA mutation.

Figure 8 shows the results of fermentation of pure glycerol and fed ethane production by fed-batch culture of K. pneumoniae GEM167 ldhA mutant strains.

Figure 9 shows the results of fermentation and production of ethanol biodiesel waste glycerol by fed-batch culture of K. pneumoniae GEM167 Δ ldhA mutant strains.

FIG. 10 shows a route for producing improved ethane of K. pneumoniae GEM167 strain.

11 shows pure glycerol (a) black waste glycerol (b) fed-batch culture and ethanol production of K. pneumoniae GEM167 / pBR-pdc-adhII strain.

12 shows K. pneumoniae GEM167.

Strain Pure Glycerol

(a) Black shows waste glycerol (b) fed-batch culture and ethanol production.

<33>

<34> Detailed Description of the Invention and Specific Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, crab ciella

Gamma-irradiation results in mutations that increase the enzyme activity of alcohol dehydrogenase (Adhll) and decrease the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) It was confirmed that the microorganisms prepared and the mutant microorganisms produced were able to produce ethane efficiently using glycerol or waste glycerol as a single carbon source.

Therefore, in one aspect, the present invention is to increase the enzyme activity of alcohol dehydrogenase (Adhll) and 1,3-propanedi to oxidoreductase (1,3-propanol oxidoreductase, DhaT) The present invention relates to a mutant microorganism having high ethanol production from glycerol or waste glycerin with reduced enzyme activity.

In one embodiment of the present invention, gamma-rays were irradiated with lkGy dose to induce microorganisms, and then cultured in a medium containing glycerol as a single carbon source to isolate mutant microorganisms with improved ethanol production. Metabolite production of mutant microorganisms As a result, compared with the control group, the production of glycerol oxidative metabolite was significantly increased with little production of glycerol reduced metabolite. Especially, ethanol production was increased by 8 times compared with the control group. It was confirmed.

The mutant microorganism according to the present invention is cultured in a medium containing glycerol or waste glycerol as a carbon source to produce ethanol with high efficiency. As a result of irradiating gamma rays to fermentable microorganisms, glycerol is used as alcohol dehydrogenase. mutation of a gene encoding (alcohol dehydrogenase, Adh Π) or a gene encoding 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)) resulting in a change in the enzyme activity encoded by the gene. Changes in some or a substantial part of the biosynthetic pathways that cause changes or enzymes can be attributed to the conversion of glycerol into highly efficient ethanol.

In another embodiment of the present invention, the enzymatic activity of the mutant microorganism irradiated with gamma rays

As a result, the enzyme activity of alcohol dehydrogenase (Adhn) did not increase significantly when cultured in a medium containing glucose as a single carbon source, whereas in medium containing glycerol as a single carbon source. In culture, the enzyme activity of alcohol dehydrogenase (Adhn) was increased by three times compared to the control group, and 1,3—propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)). It was confirmed that the enzyme activity of the control was reduced. From these results, it could be confirmed that the change in the enzyme activity of the mutant microorganisms causes the glycerol to change the fermentation metabolic pathways, thereby increasing the production of glycerol oxidative metabolic pathways and, in particular, the production of ethanol.

In the present invention, the microorganism is not particularly limited, for example, bacteria,

Yeast, bearish or the like. Preferably, the bacterium is genus Krebsiella (genus j57ebs / e // a), genus Lactobacillus (genus ^ ac obac /// £ / s), genus Clostridium (genus, genus Enterobacter) Etc., and more preferably, it may be of the genus Crab-siella, which is preferably Crab-siella pneumoniae 057ebs / e // a 77 ffl7iw / ae) or glycerol or waste glycerol. Microorganisms capable of using as a carbon source are not limited thereto.

The present invention also relates to a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC 11742BP) having high performance in ethane from glycerol or waste glycerol.

In the present invention, the alcohol dehydrogenase (alcohol dehydrogenase, Deletion of additional lactate hydrogenase genes in microorganisms in which a mutation occurs in which Adhll) increases the enzyme activity and decreases the enzyme activity of 1,3-propanol oxidoreductase (DhaT). It was confirmed that by improving the ethanol production capacity.

In another aspect, the present invention provides an alcohol dehydrogenase (alcohol dehydrogenase,

Adhll) has increased enzyme activity and reduced enzyme activity of 1,3-propanol oxidoreductase (DhaT). In a microorganism, the present invention relates to a mutant microorganism which has deleted a gene encoding Lactate dehydrogenase (LdhA).

In the present invention, a mutation in which the enzyme activity of alcohol dehydrogenase (Adhll) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3—propanol oxidoreductase, DhaT) is decreased. Lactate dehydrogenase is used in the mutant microorganisms in order to confirm that excess lactate is produced when glycerol is produced as ethanol using carbon as a carbon source, and to suppress the production of lactate. Reduced the activity of the enzyme.

In one embodiment of the present invention, the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase, DhaT). As a result of the homologous recombination of the lactate dehydrogenase gene in the microorganism Klebsiella / euw ^ / ae GEM167, which had this decreased mutation, ethane was increased from 21.5 g / L to 28.9 g / L. It was confirmed that.

Accordingly, the present invention also relates to lactate dehydrogenase (Lactate dehydrogenase) in a mutant microorganism Klebsiella / weu / »o / 7 / ae GEM167C Accession No. KCTC11742BP having high ethanol production from glycerol or waste glycerol. It relates to a mutant microorganism which has deleted a gene encoding LdhA).

<48> In the present invention, alcohol dehydrogenase (Adh)

Pyruvate dehydrogenase in microorganisms with increased enzyme activity of Π) and reduced enzymatic activity of 1,3-propanediol oxidoreductase (DhaT). It was confirmed that ethanol production ability could be improved by overexpressing the gene encoding Pdc) and aldehyde dehydrogenase (Adhll). Therefore, in another aspect, the present invention is to increase the enzyme activity of alcohol dehydrogenase (Adhll) and 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) Genes encoding pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldehdyde dehydrogenase) in mutant microorganisms with high performance in glycerol or waste glycerol with reduced enzyme activity , Adhll) relates to a mutant microorganism overexpressed by introducing a gene encoding.

In the present invention, a mutation in which the enzyme activity of alcohol dehydrogenase (Adhll) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)) is decreased. Pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldehdyde dehydrogenase) to enhance the pathway of producing ethanol through aldehydes as a carbon source by using microorganisms Adhll) increased the enzyme activity.

In one embodiment of the present invention, the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase, DhaT). Genes encoding pyruvate dehydrogenase (Pdc) and genes encoding aldehyde dehydrogenase (aldhdyde dehydrogenase, Adhll) in the microorganism Klebsiella pneumoniae GEM167, in which this reduced mutation has occurred, have been described. As a result of using the transformant, the amount of ethane produced was confirmed to increase from 21.5g / L to 25g / L.

The present invention also relates to genes and aldehydes that encode pyruvate dehydrogenase (Pdc) in a mutant microorganism Klebsiella GEM167 (Accession No. KCTC11742BP) having high ethanol production from glycerol or waste glycerol. It relates to a mutant microorganism overexpressed by introducing a gene encoding dehydrogenase (aldehdyde dehydrogenase, Adhll).

According to another aspect of the present invention, the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) In mutant microorganisms with high ethanol production from glycerol or waste glycerol with reduced enzymatic activity, the gene encoding Lactate dehydrogenase (LdhA) is deleted. It relates to a mutant microorganism overexpressed by introducing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (alddhdyde dehydrogenase, Adhll).

In the present invention, a mutation in which the enzyme activity of alcohol dehydrogenase (Adhll) is increased and the enzyme activity of 1,3-propanedi is reduced to 1,3-propanol oxidoreductase (DhaT) In order to suppress the production of lactate by using the microorganisms in which the microorganism has been generated, it is necessary to reduce the activity of lactate dehydrogenase enzyme in the mutant microorganisms, and to produce ethane via aldehyde as a carbon source. To enhance, the enzyme activity of pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldehdyde dehydrogenase, Adhll) was increased.

In one embodiment of the present invention, the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase, DhaT). Lactate dehydrogenase gene was deleted by homologous recombination in the microorganism Klebsiella w «/ TO / 2 / ae GEM167 where this decreased mutation occurred, and pyruvate dehydrogenase (Pdc) was deleted. Gene encoding and aldehyde dehydrogenase (aldehdyde dehydrogenase, Adhll) coding gene transformed using a recombinant vector, the ethanol production was confirmed to increase from 21.5g / L to 31.0g / L.

The present invention also deletes a gene encoding lactate dehydrogenase (LdhA) in a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP) having high ethanol production from glycerol or waste glycerol. , pyruvate dehydrogenase (pyruvate dehydrogenase, Pdc) of the coding gene and the aldehyde dehydrogenase ^"relates to a mutant microorganism in which over-expression by introducing a gene encoding a (aldehdyde dehydrogenase, Adhll) to.

In another aspect, the present invention (a) by irradiating gamma rays to microorganisms increases the enzyme activity of alcohol dehydrogenase (Adhll) and 1,3-propanediol oxidase ductase ( Mutating the enzyme activity of 1,3-propanol oxidoreductase (DhaT) to decrease; And (b) the enzymatic activity of the alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is mutated to decrease the enzymatic activity of 1,3-propanol oxidoreductase (DhaT). Beauty It relates to a method for producing a mutant microorganism having high ethanol performance from glycerol or waste glycerol comprising the step of obtaining the organism.

In another aspect, the present invention (a) by irradiating gamma rays to the microorganism increases the enzyme activity of alcohol dehydrogenase (Adhll) and 1,3-propanediol oxidoreductase (1, Mutating to reduce the enzyme activity of 3-propanol oxidoreductase (DhaT); (b) the enzyme activity of the alcohol dehydrogenase (Adhll) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is decreased. Obtaining a live microorganism; And (c) relates to a method for producing a mutant microorganism having ethanol high production ability from glycerol or waste glycerol comprising the step of deleting the gene encoding the lactate hydrogenase in the obtained microorganism.

According to another aspect of the present invention, (a) by irradiating gamma rays to microorganisms, the enzymatic activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanediol oxidoreductase (1, Mutating to reduce the enzyme activity of 3-propanol oxidoreductase (DhaT); (b) the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is mutated to decrease the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase (DhaT) Obtaining a microorganism; And (c) overexpressing the gene encoding pyruvate dehydrogenase (Pdc) and the gene encoding aldehyde dehydrogenase (Adhll) in the obtained microorganism. Or it relates to a method for producing a mutant microorganism having a high production of ethane from waste glycerol.

In another aspect, the present invention (a) by irradiating gamma rays to microorganisms increases the enzymatic activity of alcohol dehydrogenase (Adhll) and 1,3-propanediol oxidoreductase (1, Mutating to reduce the enzyme activity of 3-propanol oxidoreductase (DhaT); (b) the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is mutated to decrease the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase (DhaT) Obtaining a microorganism; And (c) a gene encoding lactate hydrogenase in the obtained microorganism, a gene encoding pyruvate dehydrogenase (Pdc) and an aldehyde dehydrogenase (aldehdyde dehydrogenase, Adhll). Overexpressing a gene encoding a glycerol or lung The present invention relates to a method for preparing a mutant microorganism having high ethanol production from cerol.

In the present invention, the gamma ray may be irradiated with an appropriate dose depending on the type of the mutant microorganism, for example, 0.1kGy ~ 10kGy dose. Preferably from 0.5 kGy to 5 kGy, more preferably from 0.5 kGy to 3 kGy, most preferably lkGy dose.

In another aspect, the present invention includes the step of culturing the mutant microorganism according to the present invention in a medium containing glycerol or waste glycerol as a carbon source to produce ethanol, and then recovering ethane from the culture medium. It relates to a process for producing ethane.

In one embodiment of the present invention, the ethanol production yields of the mutant strains of the present invention including K. / weifflOT / ae GEM167 were compared with previously reported results. As a result, as shown in Table 10, K. pneumoniae GEM167 Δ IdhA strain overexpressing the Pdc-Adhll enzyme gene showed a maximum yield of 31 g / L ethanol, which was 20.7 g / L (the existing maximum yield). Compared to recombinant Escherichia coli).

In the industrial process of producing ethanol from glycerol, the production yield is 40-50 g / L.

It is predicted that there will be economic feasibility. Considering the production yield of the mutant strains of the present invention, if the cultivation process is optimized, the commercial value is considered to be very high.

In another embodiment of the present invention, in order to determine the effect of pH on the production of ethane of the mutant microorganisms, culturing the mutant microorganisms while varying the pH of the culture medium to compare the ethanol production, pH 6 and It was confirmed that high ethane showed high productivity under neutral condition of pH 7, and ethanol production of mutant microorganism according to air injection amount was high. It was.

In the present invention, the culturing and recovery of mutant microorganisms can be carried out using a known culture method, and in addition to the specific medium and the specific culture method used in the embodiment of the present invention, whey , Saccharification fluid such as CSUcorn steep liquor) and other media can be used.

<67>

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, the scope of the present invention these implementations It will be apparent to one of ordinary skill in the art not to be construed as limited by the examples.

<69>

<70> Example

<71>

Example 1 Preparation of Mutant Strains with Improved Ethanol Production from Glycerol

<73> 50 ml flask culture of Uebebsiel lapneumoniae) C \ x strain was cultured at 0D _600nm of 0.4-0.6, followed by diluting the cells with PBS buffer (8g NaCl, 0.2g C1, 1.44g).

Na ₂ HP0 ₄ , 0.24gKH ₂ P0 ₄ in lLo fdisti 11 edH ₂ 0, pH7.4) Suspended and irradiated under gamma-sun lkGy condition, when the mortality is about 99%, the suspended cells are diluted with suitable sterile water. Plated on LB medium. A total of 26,000 colonies were obtained, followed by adding 20 g / L glycerol to 1 L of medium containing glycerol as the only carbon source [0.1 M potassium phosphate buffer (pH 7.0), 2 g / L (NH ₄ ) ₂ S0 ₄ , 0.2g / LMgS0 ₄ , 0.002g / LCaCl ₂ 2H ₂ 0, lg / L yeast extract, 1 ml iron solution (5 g / L FeS0 ₄ 7H ₂ 0), 4mIHCl (37%, w / v), lml Trace element solution (70 mg / L

ZnC 1 ₂ , lOOmg / LMnC 1 ₂ 4H ₂ 0, 60mg / LH ₃ B0 ₃ , 200mg / LCoC 1 ₂ 4H ₂ 0, 20mg / LCuC 1 ₂ H ₂ 0, 25mg / LNiCl ₂ 6H ₂

0,35mg / LNa2Mo0 ₄ 2H20, ½mCl (37%, w / v))] and cultured at 120 rpm at 37 ^° C to examine the growth of the strain. At the same time, the residual amount of glycerol and The amount of metabolites, including mutants, was analyzed by liquid chromatography to isolate mutant strains with improved ethanol production. The mutant strains selected through in vitro culturing were cultured at a culture temperature of 37 ^° C., agitation speed of 200 rpm, and air injection rate of 0.5 m using a 5L fermenter, and compared to the control strain Cu.

As a result, in the mutant strains compared to the control, almost no 1,3-propanedi and 3-hydroxypropionic acid, which are metabolites of glycerol reduction metabolic pathways, were produced. The production was shown to increase significantly compared to the control. In particular, the production of ethanol showed an extremely high yield of ethane (high performance) at 8.6 g / L, an increase of about 8 times from 1.1 g / L (Fig. 2, Table 1).

<75> <76> [Table 1]

Metabolite Production of K. pneumoniae Cu and GEM 167 Mutant Strains (g / L)

compare

<77>

On the other hand, experiments conducted under the same conditions in a medium containing glucose instead of glycer as the only carbon source showed that the mutant strains showed metabolite production characteristics similar to those of the control. The mutant strain with the improved amount of ethanol specific to glycerol was named GEM167, and was deposited with the KCTC 11742BP accession number to the Korea Biotechnology Research Institute Gene Bank.

<79>

Example 2: Enzyme activity of ^. / Eiffl? O;? / A ^ EM167 mutant strain

<8i> In order to measure the enzymatic activity of the mutant strain K. w / roiw / ae GEM167 prepared in Example 1, glycerol black-as a unique carbon source-was cultured in the same medium containing glucose- After the cells were recovered, the cells were washed with 50 mM potassium phosphate buffer (pH 7.5), dissolved again, and ground by sonication.

Enzyme Activity Assay for Determination of Enzyme Activity of Alcohol Dehydrogenase (Adhll) The mixed solution was prepared by glycine-KOH buffer (pH 9.0) (50 mM) and NAD + (1 mM). The reaction mixture was prepared by adding 100 mM ethanol to the prepared mixture. The reaction was carried out for 30 minutes at 37 ^° C and then measured the hop intensity at 340 nm (Postma E. eta J, Appl Environ Microbiol., 55: 468-477,1989).

<83> Enzyme Activity Assay for Measuring Enzyme Activity of Glycerol Dehydratase (DhaB) The mixed solution was reacted for 10 minutes at 37 ^° C by adding glycerol to the final concentration of 20mM in the culture strain grinding solution. Then the final concentration is lOmM Add vitamin B ₁₂ and react at 37 ° C for 5 minutes. Then, 1M citrate buffer (pH 3.6) was added, MBTH 0.1% was added, and reacted at 100 ^° C. for 10 minutes, and the absorbance was measured at 315 nm.

Enzyme Activity Assay for Measuring Enzyme Activity of Glycerol Dehydrogenase (DhaD) Glycerol was mixed with glycine-KOH buffer (pH 9.0) (50 mM) and NAD + (1 mM) in culture strains. ) Was added, and the reaction was started by adding 100 mM glycerol to the prepared mixture. The reaction was carried out for 30 minutes at 37 ^° C and then measured for absorbance at 340 nm.

<85> Enzyme Activity Assay for Determination of Enzyme Activity of 1,3-propanediol oxidoreductase (DhaT) The mixed solution was formulated with glycine-KOH buffer (pH 9.0) ( 50 mM) and NAD + (1 mM) were added. The reaction was started by adding 100 mM 1,3-propanedi to the prepared mixture. The reaction was measured for 30 minutes at 37 ^° C and then measured for absorbance at 340 nm.

As a result, as shown in Table 2, when Glycer was incubated in the medium, GEM

The activity of alcohol dehydrogenase (Adhll) enzyme of 167 strains was shown to be more than three times higher than that of the control Cu strain. On the other hand, when cultured in a medium containing glucose as a carbon source, unlike glycerol medium, the increase in alcohol dehydrogenase (Adhll) enzyme activity was not significant. Significant increases in alcohol dehydrogenase (Adhll) enzyme activity in glycerol medium are consistent with the metabolite production assays above. Meanwhile, 1,3-propanediol oxidoreductase (DhaT) enzyme activity was decreased in GEM 167 mutant strains, which reduced production of 1,3-propanedi and Is a matching result.

<87>

K. pneu niae Cu strain and the enzyme ^"sex (enzyme units) of GEM 167 mutant

Enzyme ^1, St. units are expressed as unit / g protein.

Example 3: Analysis of Ethanol Productivity of Mutant Strains According to pH <9i> In order to determine the effect of pH on the production of ethane of the mutant strain K. pneumoniae GEM167, by using the same method as in Example 1 to change the pH of the culture medium to 5, 6, 7 and 8 The ethanol production of the mutant strain was measured while maintaining the culture. As a result, as shown in Figure 3, it showed a high ethanol productivity in the neutral conditions of pH 6 and 7, especially in the acidic condition pH 5 it was found that the cell growth and ethanol productivity is very poor.

<92>

Example 4: Ethanol Productivity Analysis of Mutant Strains According to Aeration Level

In order to examine the effect of aeration on the ethanol production of the mutant strain K. / eu / roi / ae GEM167, using the same method as in Example 1, areation conditions were 0.0 wm, 0.1 wm and 0.5 wm, respectively. , 1.0 wm, 2.0 wm and 3.0 wm. As a result, as shown in Fig. 4, it was confirmed that ethanol production was higher in the case of aeration with Ol m and 0.5wm than with 0.0 m without any aeration. It showed that ethane is advantageous for the production of ethane.

<95>

Example 5: Ethanol Productivity Analysis from Glycer of Mutant Strains

In the same manner as in Example 1, GEM 167 was fermented and cultured by a fed-batch method while maintaining the areation at 0.5 wm and the pH at 6.8 to 7.2. As a result, as shown in FIG. 5 and Table 3, the maximum yield of ethane was 21.5 g / L at 23 hours of incubation, and the yield per hour.

The highest level of ethanol production of 0.93 g / L is known to date.

<98>

<99> [Table 3]

Fermentation of K. pneumoniae GEH 167 mutant strains by fed-batch

<100> Example 6 Productivity Analysis of Ethanol from Waste Glyceres of Mutant Strains

In the same manner as in Example 1, fermentation of GEM 167 was performed using waste glycerol, a biodiesel by-product, as the only carbon source, while maintaining areation at 0.5 wm and pH 6 · 8 to 7 · 2. As a result, as shown in Fig. 6 and Table 4, the maximum yield of ethane was 19.9 g / L and the hourly yield was 0.87 g / L at 23 hours of cultivation, which showed ethanol production similar to that of pure glycerol.

Table 4

Fermentation Culture of K. pneumoniae GEM 167 Mutant Strain with Waste Glycerin

<105>

Example 7 Improvement of ^. / e z 2 / aeGEM167 Mutant Strain: Preparation of Lactate-def icient derivative

<i07> (1) Preparation of GEM167 o 4 mutant strain

As shown in Tables 3 and 4, 21.5 g / L of ethanol was produced during glycerol fermentation culture of ^. / ¾ iffl /? / a ^ EM167 mutant strains, and lactate was 11.5 g / L. L is produced, and the major competitive metabolite of ethanol is lactate.

Therefore, if the production of lactate is inhibited in the GEM167 strain, the production of ethanol can be expected to increase, and to confirm this, the production of lactate-deleting mutant strains from GEM167 mutant strains was analyzed for the characteristics of fermentation and ethanol production of glycerol It was.

The lactate dehydrogenase gene (/ α 4) catalyzes the production of lactate on the chromosome of the GEM167 strain. In order to remove the IdhA gene, the downstream sequence 0.9 kb was amplified and linked using PI, P2 primer (upstream) or P3, P4 primer (downstream), respectively, and the antibiotic apramycin resistance gene was inserted between the homologous recombinat. Ion DNA DNA Cassette Constructed DNA Cassette (/ Q 4 Gene Upstream Sequence-Apramycin Resistance Gene Gene Downstream sequences) were introduced into the K. pneumoniae GEM167 strain by electrolayering, followed by homologous recombinat ion to insert a DNA cassette into chromosomal DNA to isolate transformants that exhibit apramycin resistance.

<111>

<112> [Table 5]

Primer sequence used to prepare GEM 167 AldhA strain

<113>

<ιΐ4> Southern hybridization was performed on the isolated transformants to confirm that the DNA cassette was correctly inserted into the gene. As a result of processing the chromosomal DNA of the parent strain GEM167 and the resulting transformants with restriction enzymes and performing hybridization using the downstream sequence of the ΜιΑ gene as a probe, 3.2 kb of DNA was expected in the GEM167 strain. Fragment It has been shown that the 2.3 kb DNA fragment binds to the transformants (FIG. 7). On the other hand, when the apramycin resistance gene was used as a probe, in contrast to the binding of the same DNA fragment in the transformants, the transformant obtained was found to have no binding DNA fragment in the GEM167 strain. Was correctly substituted by the apramycin resistance gene.

<115>

<2> (2) Lactate dehydrogenase Enzyme Activity Verification

<Π7> GEM167 in a medium used in Example 1 containing glycerol or glucose

IdhA strain was cultured to analyze the activity of lactate dehydrogenase enzyme protein. The recovered culture cells were washed with 50 mM potassium phosphate buffer (pH 7.5), resuspended, crushed by the sonication method, and used as an enzyme source. Enzyme activity assay mixture to measure LdhA enzyme activity was prepared by adding glycine-KOH buffer ( _P H 9.0) (50 mM) and NAD + (ImM) to culture strain pulverized solution, and 100 mM lac in the prepared mixture. The reaction was started by adding tate. The reaction was reacted for 30 minutes at 37 ^° C. and then absorbance was measured at 340 nm. As a result, as shown in Table 6, GEM167! Lactein / Q½4 strain It was confirmed that the yit dehydrogenase enzyme activity was significantly reduced.

<118>

<119> [Table 6]

Lactate Dehydrogenase Activity of K. pneumoniae GEM167 and GEM167zl / o 4 Mutant Strains

Strain LdM activity (U / ms)

GEM167 0 99 士 0 02

GEM167 Δ ldhA 0 08 士 0 01

<120>

<i2i> Example 8: Ethanol production by fed-batch culture of K.pneumon / a 167 Δ JdhA mutant strain

<i22> K. pneumoniae GEM167 AldhA mutant strains were maintained using a culture medium of 37 ^° C., agitation speed of 200 rpm, air injection rate of 0.5wm, and pH of 6.8 to 7.2 using 2L of medium used in Example 1 in a 5L fermenter. Fermentation and cultivation by fed-batch method analyzed glycerol fermentation and ethanol production.

When pure glycerol was used as a nutrient source, it was confirmed that the production of lactate was significantly reduced from 11.5 g / L to 1.4 g / L in GEM167 l / ¾4, as shown in FIG. 8 and Table 7. Could. In proportion to the decrease in lactate production, the maximum production of ethanol increased significantly to 28.9 g / L and 1.2 g / L per hour.

<124>

<125> [Table 7]

Isolation of Pure Glycerol by I pneumniae Qm MdhA Mutant

On the other hand, GEM 167 Δ / fed-batch fermentation culture using biodiesel industrial by-product waste glycerol as a nutrient source, as shown in Figure 9 was observed to be similar to pure glycerol. <128>

<i29> Example 9: ^. Improvement of the wew? C / a ^ EM167 Mutant Strain: Overexpression of the Pdc-adhll Gene

<i30> (1) Construction of Zymomonas mobilils Pdc-adhll Gene Overexpression Vector

<i3i> As shown in Example 8, the production of ethane was significantly increased by the removal of the LdhA gene, which catalyzes the production of lactate, a major competitive metabolite of ethane 1 :, in the. ^ / «70 3 ^ ΕΜ167 strain. It was confirmed that. As a second method to increase the production of ethanol in the GEM167 strain, overexpressing the pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldhdyde dehydrogenase, Adhll) genes derived from Z. mobilis The conversion of pyruvate to acetaldehyde and the conversion of acetaldehyde to ethane in the production route were facilitated (FIG. 10).

<i32> Pdc-Adhll enzyme gene overexpression of the production of the vector briefly described as follows. Pbdc-F (5'-7n fi4atgagttatactgtcggtacctatttagc-3 '(SEQ ID NO: 5); Italic-balsite) and Ppdc-R (5'-Cra 6CTGCAGctagaggagcttgttaacaggcttac-3' (1.8kb Pdc gene and 1.15kb Adhll gene, respectively) SEQ ID NO: 6) italic site, underlined-/ ¾l site) and PaldB-F (5'-¾tggcttcttcaactttttatattcc-3 '(SEQ ID NO: 7); italic- _< gJII site), PaldB-R (5'- Z. mobilis chromosomal DNA was amplified using a C7C 6TCTAGAttagaaagcgctcaggaagagtt-3 '(SEQ ID NO: 8); italic site, underline-¾al site) primer. In addition, the strong gene expression promoter / ac promoter sequence (Ρ-aldB) was expressed as PlacZ-aldB-F (5'-477 agcgggcagtgagcgcaa-3 '(SEQ ID NO: 9); italic- ^ oRI site) and P _lacZ -aldB-R ( 5 '-CTCAGAkQMCl agctgt t tcctgtgtgaaat tg-3 (SEQ ID NO:

10), Italic-¾osite, underline-¾7IIsite) and amplified using a primer. The amplified DNA fragments were cloned into pGEM TEasy vector (Proraega, USA) and sequenced to confirm that gene amplification was correct.

<i33> pGEM-P _{/ ac}厂 a 1 clB, pG -P _lacZ -a 1 cIB-pdc. were constructed by inserting the Adhll and Pdc genes downstream of the LacZ promoter, followed by P _/ ^ -a / ^-/ c: The cassette was reinserted into a _P BR322 vector to prepare pBR-akiB-jxk and introduced into K. pneumoniae Cu, GEM16 and GEM167 / dM strains, respectively, by the electrolayered method.

(2) Validation of Pdc-adhll Enzyme Activity

Pyruvate decarboxylase (Pdc) and aldehyde dihydrate of recombinant cells Logenase (Adhll) enzyme activity was analyzed. The cultured cells were washed with 50 mM potassium phosphate buffer (pH 7.5), resuspended, crushed by the sonication method, and used as an enzyme source. The enzyme activity assay mixture for measuring Pdc enzyme activity was imidazole hydrochloride buffer (pH-6.5) (40 mM) in culture strain grinding solution.

MgCl ₂ (5 mM), thiaminepyrophosphate (0.2 mM) and NADH (0.15 mM) were added, and alcohol dihydrogenase (Boehringer) was added to make 88U. Then reaction was started by adding 50 mM pyruvate. The reaction was reacted for 30 minutes at 30 ^° C. and then the absorbance was measured at 340 nm. Enzyme activity assay mixture to measure Adhll enzyme activity was prepared by adding glycine-KOH buffer (pH 9.0) (50mM) and NAD + (ImM) to culture strain pulverized liquid, and added 100 mM ethane to the prepared mixture. The reaction began. The reaction was reacted for 30 minutes at 30 ^° C. and then the absorbance was measured at 340 nm.

As a result, as shown in Table 8, it was confirmed that the Pdc and Adhll enzyme activity was increased in the recombinant cells to which the pBR-a / d »-/ ¾ / c plasmid was introduced.

<137>

<138> [Table 8]

Pdc and Adh Enzyme Activities in K. pneumoniae Strains

Enzyme activity (U / mg protein) Example 10: Ethanol production by fed-batch culture of Pdc-Adhll overexpressing K pneumoniae strains

K overexpressed the Pdc-Adhll enzyme gene while maintaining a culture temperature of 37 ^° C., agitation speed of 200 rpm, air injection speed of 0.5wm, and pH of 6 · 8 to 7.2 using 2L of the medium described in Example 1 in a 5L fermenter. Pneumoniae mutant strains were fermented and cultured in a fed-batch method to analyze the fermentation and ethanol production of glyce, and the results are shown in FIGS. 11 and 12. As shown in Table 9, when the Pdc-Adhll gene was overexpressed, ethanol production increased from 21.5 g / L to 25 g / L and 28.9 g / L to 31 g / L in the GEM167 and GEM167 l / ¾4 strains, respectively. Appeared to be. Investigation of the stability of the pBR-pdc-adhl plasmid during fermentation showed that approximately 93% of cells It was confirmed to have a plasmid.

<143>

<Table> Table 9

sheep

Net ^. τ ^λ

Example 11 Production Capacity of Ethane of ^ .p / iTO7 / ae Mutant Strains

Ethanol production yield of K. pneumoniae GEM167 and its mutant strains identified in the above example was compared with the results of previously reported known strains.

As shown in Table 10, the K. pneumoniae GEM167 Δ IdhA strain overexpressing the Pdc-Adhll enzyme gene showed a yield of 31 g / L of ethane, which was 20.7 g / L (recombinant Escherichia coli). This is much better than the previous one. In the industrial process of producing ethane from glycerol, it is expected to be economical when the production yield reaches 40-50 g / L. Considering the production yield of the mutant strains of the present invention, if the cultivation process is optimized, the commercial value is judged to be very high.

<150> [Table 10]

Comparison of Ethanol Production Capacity from Glacese of Known Strains

<151>

<! 52> The specific parts of the present invention have been described in detail above, and for those skilled in the art, these specific descriptions are only preferred embodiments, and thus the scope of the present invention is limited. It will be clear. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<153>

<154> accession number

<155> Depositary Name ： Korea Research Institute of Bioscience and Biotechnology

<156> accession number ： KCTC11742BP

<157> Date of Trust ： 20100813

<158> [Industrial availability]

The present invention has the effect of providing a mutant microorganism that produces ethane with high efficiency by irradiating gamma rays to the microorganisms as a carbon source of glycerol or waste glycerol. The mutant microorganism according to the present invention has little production of metabolites in the glycerol reduction metabolic pathway, and is industrially useful by producing high value-added product ethane using waste glycerol which is a by-product of the biodiesel industry. .

<160>

[SEQ ID NO Free Text]

<161> An electronic file is attached.

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT

issued pursuant to Rule 7.1

TO ： KIM, Chul Ho

Jeonbuk Branch, Korea Research Institute of Bioscience and Biotechnology

1404 Sinjeong-dong, Jeongeup-si, Jeonbuk 580-185

Republic of Korea

Fomi BP / 4 (KCTC Form 17) sole page

Claims

[Range of request]

[Claim 1]

Ethanol from glycerol or waste glycerol with increased enzyme activity of alcohol dehydrogenase (Adhll) and reduced enzyme activity of 1,3-propanediol oxidoreductase (DhaT) Mutant microorganism with high performance.

[Claim 2]

The method of claim 1, wherein the microorganism is a mutant microorganism having a high performance of ethane from glycerol or waste glycerol, characterized in that selected from the group consisting of bacteria, yeast and fungi.

[Claim 3]

The genus Clostridium V genus Enterobacter of claim 2, wherein the bacterium is Genus / ebs / e // a, Genus Lactobacinus, Genus ClostridiumV genus Enterobacter. A mutant microorganism having high performance in ethane from glycerol or waste glycerol, characterized in that selected from the group consisting of one.

[Claim 4] ^"

Mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC I1742BP) having high performance from glycerol or waste glycerol.

[Claim 5]

Glycer or waste glycerol with increased enzymatic activity of alcohol dehydrogenase (Adhll) and reduced enzymatic activity of 1,3-propaneol oxidoreductase (1,3—propanol oxidoreductase (DhaT)) A mutant microorganism having ethanol high performance from glycerol or waste glycerol which has deleted a gene encoding Lactate dehydrogenase (LdhA) in a mutant microorganism having high ethanol production from.

[Claim 6]

The method of claim 5, wherein the microorganism is in the group consisting of bacteria, yeast and fungi A mutant microorganism having high ethanol production from glycerol or waste glycerol, characterized in that it is selected.

[Claim 7]

The method of claim 6, wherein the bacterium is genus Crabciella (genus / ebs / e // a), genus Lactobacillus (genus ac oZac /// i / s), genus Clostridium V A mutant microorganism having high performance of ethane from glyce or waste glycerol, characterized in that it is selected from the group consisting of genus Enterobacter.

[Claim 8]

The mutant microorganism which deleted the gene which codes lactate dehydrogenase (LdhA) in the mutant microorganism Klebsiella GEM167 (Accession Number KCTC11742BP) which has high ethanol performance from glycerol or waste glycerol.

[Claim 9]

Glycer or waste glycerol with increased enzymatic activity of alcohol dehydrogenase (Adhll) and reduced enzymatic activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)) In mutant microorganisms with high production of ethane, genes encoding pyruvate dehydrogenase (Pdc) and genes encoding aldehyde dehydrogenase (aldhdyde dehydrogenase, Adhll) were overexpressed. A mutant microorganism having high ethanol production from glycerol or waste glycerol.

[Claim 10]

The method of claim 9, wherein the microorganism is a mutant microorganism having high ethanol production from glycerol or waste glycerol, characterized in that selected from the group consisting of bacteria, yeast and bear.

[Claim 11]

The genus Clostridium ^ Enterobac of claim 10, wherein the bacterium is Genus / e /? S / e // a, Genus iactobaci! Jus, Genus Clostridium. Genus Enterobacter ^, characterized in that selected from the group consisting of Mutant microorganisms having high ethanol production from serul or waste glycerol.

[Claim 12]

Genes encoding pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldehdyde dehydrogenase) in mutant microorganisms Klebsiella pneumoniae GEM167 Accession No. KCTC11742BP, which have high performance in glycerol or waste glycerol. , Mutant microorganisms overexpressed by introducing a gene encoding Adhll).

[Claim 13]

Glycer or waste glycerol with increased enzyme activity of alcohol dehydrogenase (Adhll) and reduced enzyme activity of 1,3-propanediol oxidoreductase (1,3—propanol oxidoreductase (DhaT)) Genes encoding lactate dehydrogenase (LdhA), genes encoding pyruvate dehydrogenase (Pdc), in mutant microorganisms having high ethanol production from A mutant microorganism having high ethanol production from glyce or waste glycerol overexpressed by introducing a gene encoding aldehyde dehydrogenase (aldhdyde dehydrogenase, Adhll).

[Claim 14]

14. The mutant microorganism having high ethanol production from glycerol or waste glycerol according to claim 13, wherein the microorganism is selected from the group consisting of bacteria, yeast and mold.

[Claim 15]

15. The genus Clostridium V enterobacter genus according to claim 14, wherein the bacterium is genus Crabciella (genus / ebs / e // a), genus Lactobacillus (genus 3c obac // s), genus Clostridium V. (genus Enterobacter) A mutant microorganism having high ethanol production from glyce or waste glycerol, characterized in that selected from the group consisting of.

[Claim 16]

Mutant microorganisms with high ethanol production from glycerol or waste glycerol In Klebsiella e ™ o; 2 / ae GEM167 (Accession No. KCTC11742BP), the gene encoding Lactate dehydrogenase (LdhA) was deleted and pyruvate dehydrogenase (Pdc) A mutant microorganism overexpressed by introducing a gene encoding the gene and a gene encoding the aldehyde dehydrogenase (aldhdyde dehydrogenase, Adhll). .

[Claim 17]

A method for producing a mutant microorganism having high performance in glycerol or waste glycerol comprising the following steps:

(a) Irradiation of microorganisms with gamma rays increases the enzyme activity of alcohol dehydrogenase (Adhll) and the enzyme activity of 1, 3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)). Mutating to decrease;

(b) the enzyme activity of the alcohol dehydrogenase (Adhll) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is changed to decrease Obtaining microorganisms present.

[Claim 18]

18. The method of claim 17, wherein the microorganism is selected from the group consisting of bacteria, yeast, and mold.

[Claim 19]

19. The genus Enterobacter of claim 18, wherein the bacterium is composed of the genus Crabciella (genus, genus Lactobacillus (genus ^ ac obac / // s), genus ClostridhimV genus Enterobacter. Method for producing a mutant microorganism having high ethanol production from glyce or waste glycerol, characterized in that selected from the group.

[Claim 20]

18. The method of claim 17, wherein the gamma rays are irradiated at a dose of 0.1 kGy to 10 kGy, mutant microorganisms having high ethanol performance from glycerol or waste glycerol How to make water

[Claim 21]

A method for producing a mutant microorganism having high performance in ethane from glycerol or waste glycerol comprising the following steps:

(a) Gamma-irradiation of microorganisms increases the enzyme activity of alcohol dehydrogenase (Adhll) and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreduct se, DhaT) Mutating such that it is reduced;

(b) the enzyme activity of the alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is mutated to reduce the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase (DhaT)). Obtaining a microorganism present; And

(c) deleting the gene encoding the lactate hydrogenase from the obtained microorganism.

[Claim 22]

22. The method according to claim 21, wherein the microorganism is selected from the group consisting of bacteria, yeast and gourds.

[Claim 23]

According to claim 22 7 The bacteria is genus Clostridium V genus Enterobacter genus Lactobaci J Jus A method for producing a mutant microorganism having high performance in ethane from glycerol or waste glycerol, characterized in that selected from the group consisting of.

[Claim 24]

22. The method of claim 21, wherein the gamma rays are irradiated at a dose of 0.1 kGy to 10 kGy to produce mutant microorganisms having high ethanol performance from glycerol or waste glycerol.

[Claim 25] A method for preparing a mutant microorganism having high performance in ethane from glycerol or waste glycerol comprising the following steps:

(a) Irradiation of gamma rays to microorganisms increased the enzyme activity of alcohol dehydrogenase (Adhn) and the enzyme activity of 1,3-propanedi oxidoreductase (1,3-propanol oxidoreductase (DhaT) Mutating to decrease;

(b) a variation in which the enzyme activity of the alcohol dehydrogenase (Adhn) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is reduced Obtaining a prepared microorganism; And

(c) overexpressing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (Adhll) in the obtained microorganism.

[Claim 26]

26. The method of claim 25, wherein the microorganism is selected from the group consisting of bacteria, yeast and gourds.

[Claim 27]

27. The method of claim 26, wherein the bacterium is Genus ebs / e / a, Genus ^ aciobac / J // s, Genus Clostridium ^ Enterobacter. A method for producing a mutant microorganism having high ethanol production from glasers or waste glycerol, characterized in that it is selected from the group consisting of genus Entero cter.

[Claim 28]

26. The method of claim 25, wherein the gamma rays are irradiated at a dose of 0.1 kGy to 10 kGy to produce mutant microorganisms having high ethanol performance from glycerol or waste glycerol.

[Claim 29]

Method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol comprising the following steps: (a) Irradiation of microorganisms with gamma rays increases the enzyme activity of alcohol dehydrogenase (Adhll) and the enzyme activity of 1, 3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)). Mutating to decrease;

(b) mutated to increase the enzyme activity of the alcohol dehydrogenase (Adhll) and to reduce the enzyme activity of 1,3-propanediol oxidoreductase (DhaT). Obtaining a microorganism present; And

(c) deleting the gene encoding the lactate hydrogenase from the obtained microorganism, and the gene encoding pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldehdyde dehydrogenase (Adhll)); Overexpressing the coding gene.

[Claim 30]

30. The method of claim 29, wherein the microorganism is selected from the group consisting of bacteria, yeast and mold.

[Claim 31]

The genus Clostridhim genus Clostridhim genus Clostridhim according to claim 30, wherein the bacterium is genus Crabciella (genus / ebs / e //), genus Lactobacillus (genus ac obac // i / s), genus Clostridhim genus (genus Enterobacter)-a method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol, characterized in that selected from the group consisting of.

[Claim 32]

30. The method of claim 29, wherein the gamma rays are irradiated at a dose of 0.1 kGy to 10 kGy to produce mutant microorganisms having high ethanol performance from glycerol or waste glycerol.

[Claim 33]

The method for producing ethanol, wherein the mutant microorganism of any one of claims 1 to 16 is cultured in a medium containing glycerol or waste glycerol as a carbon source to produce ethane, and then ethanol is recovered from the culture solution.