KR20130025701A - Improved microorganism variants having ehtanol producing ability using glycerol or crude glycerol and method for producing ethanol using the same - Google Patents

Abstract

PURPOSE: A mutant microorganism with high ethanol productivity and a method for producing the same are provided to produce ethanol from glycerol or waste glycerol. CONSTITUTION: A mutant microorganism with high ethanol productivity from glycerol or waste glycerol is prepared by deleting a gene encoding lactate dehydrogenase(LdhA). The microorganism is selected from bacteria, yeast, and fungi. The bacteria are selected from a group consisting of genus Klebsiella, genus Lactobacillus, genus Clostridium, and genus Enterobacter. [Reference numerals] (AA) Glycerol consumption(g/L); (BB) EtOH yield; (CC) Conversion ratio(mol/mol)

Description

Improved Microorganism Variants Having Ehtanol Producing Ability Using Glycerol or Crude Glycerol and Method for Producing Ethanol Using the Same} from Glycerol or Waste Glycerol

The present invention relates to a mutant microorganism having increased ethanol production ability using glycerol or waste glycerol as a carbon source and a method for producing ethanol using the same. More specifically, the enzyme activity of alcohol dehydrogenase (Adh II) is increased. , Lactate dehydrogenase (Lactate) in mutant microorganisms with high ethanol production from glycerol or waste glycerol with reduced enzymatic activity of 1,3-propanediol oxidoreductase (DhaT) Deletion of genes encoding dehydrogenase (LdhA) or overexpressing genes encoding pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldehdyde dehydrogenase (AdhII)) Pertaining to Mutant Microorganisms and a Method for Preparing Ethanol Using the Same Will.

Currently, as a major by-product of the biodiesel manufacturing process, waste glycerol is generated in an amount equivalent to about 10% (w / w) of total production (Johnson and Taconi, 2007). This waste glycerol not only affects the price of the traditional glycerol market, but also emerges as an important environmental problem such as treatment cost since it cannot be directly released to the environment (da Silva et al . 2009). Therefore, there is an active development of a method for converting low-cost waste glycerol into industrially valuable materials, including fuels and bioactive substances.

Glycerol is used as a fermentative nutrient by several bacteria, including the genus Lactobacillus , genus Clostridium , genus Enterobacter , and genus Klebsiella , to propanediol. And various compounds including ethanol. Recently, the Gonzales research group produced ethanol from glycerol by anaerobic fermentation of E. coli, which was traditionally known as a glycerol nonfermentable microorganism (Dharmadi et al . 2006; Gonzalez et al . 2008; Murarka et al . 2008). Compared to the price of producing ethanol from corn starch-derived glucose, the price from glycerol is about 40% lower (Yazdani and Gonzalez, 2007). Therefore, the study of metabolic engineering of microbial strains for the effective fermentation conversion of ethanol and by-products from glycerol (Yazdani and Gonzalez 2008; Durnin et al . 2009).

The highest yield of ethanol fermentation production from glycerol reported to date is 19.5 g / L at 35 hours incubation time in fed batch culture process of Klebsiella oxytoca and the productivity at that time is 0.56 g / L Level (Yang G et al. 2007). In fermentation culture with recombinant E. coli, the maximum fermentation yield of ethanol from glycerol was 20.7 g / L in 96-hour incubation, but the hourly productivity was 0.22 g / L, much lower than that of Krebsiaa oxytoca (Durnin et al . 2009). .

Therefore, the present inventors have made intensive efforts to develop microorganisms that produce industrially useful ethanol with high efficiency using glycerol or waste glycerol as a carbon source, and as a result, the lactate dehydrogeze in mutant microorganisms having high ethanol performance by gamma irradiation Deleting the naize gene, and overexpressing the pyruvate dehydrogenase gene and the aldehyde dehydrogenase gene, it was confirmed that the ethanol production capacity is improved and completed the present invention.

An object of the present invention is to delete the lactate dehydrogenase gene in mutant microorganisms having high ethanol production by gamma irradiation, overexpressing the pyruvate dehydrogenase gene and aldehyde dehydrogenase gene to improve the ethanol production ability The present invention provides a mutant microorganism and a method of manufacturing the same.

Another object of the present invention 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 the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) The present invention provides a mutant microorganism having a high ethanol production ability from glycerol or waste glycerol with reduced activity.

In the present invention, the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is reduced. In mutant microorganisms with high ethanol production from glycerol or waste glycerol, genes encoding pyruvate dehydrogenase (Pdc) and genes encoding aldehyde dehydrogenase (alddhdyde dehydrogenase (AdhII)) are introduced. By providing a new mutant microorganism further improved ethanol production ability.

In the present invention, the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is reduced. In mutant microorganisms with high ethanol production from glycerol or waste glycerol, the gene encoding Lactate dehydrogenase (LdhA) is deleted and the gene encoding pyruvate dehydrogenase (Pdc) And overexpressing and introducing a gene encoding an aldehyde dehydrogenase (aldehdyde dehydrogenase (AdhII)) to provide a new mutant microorganism further improved ethanol production ability.

In addition, the present invention (a) by irradiating gamma rays to the microorganism increases the enzyme activity of alcohol dehydrogenase (AdhII) and 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT Mutating to decrease enzymatic activity; (b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. Obtaining a microorganism; And (c) deleting the gene encoding the lactate hydrogenase from the obtained microorganism. The method provides a method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol, which comprises.

In addition, the present invention (a) by irradiating gamma rays to the microorganism increases the enzyme activity of alcohol dehydrogenase (AdhII) and 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT Mutating to decrease enzymatic activity; (b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. Obtaining a microorganism; And (c) overexpressing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (aldhdyde dehydrogenase, AdhII) in the obtained microorganism, or Provided is a method for producing a mutant microorganism having high ethanol production from waste glycerol.

In addition, the present invention (a) by irradiating gamma rays to the microorganism increases the enzyme activity of alcohol dehydrogenase (AdhII) and 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT Mutating to decrease enzymatic activity; (b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. 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 (AdhII)). It provides a method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol comprising the step of overexpressing the gene encoding the.

The present invention also provides a method for producing ethanol, wherein the mutant microorganism is cultured in a medium containing glycerol or waste glycerol as a carbon source to produce ethanol, and then ethanol is recovered from the culture solution.

The present invention has the effect of providing a mutant microorganism that produces ethanol with high efficiency by using glycerol or waste glycerol as a carbon source by irradiating gamma rays to a microorganism and deleting or overexpressing a specific gene. 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 ethanol, a high value-added product, using waste glycerol which is a by-product of the biodiesel industry with high efficiency.

Figure 1 is a schematic diagram showing the metabolic pathway of glycerol fermentation of Krebsiella pneumoniae.
Figure 2 shows the ethanol production of the mutant microorganisms used in the control and the present invention ((a) K. pneumoniae Cu strain, (b) K. pneumoniae GEM167 strain).
Figure 3 shows the ethanol production according to the pH of K. pneumoniae GEM167 mutant strains.
Figure 4 shows the ethanol production according to the air injection amount of K. pneumoniae GEM167 mutant strains.
5 shows ethanol production by fed-batch culture using pure glycerol of K. pneumoniae GEM167 mutant strain.
6 shows ethanol production by fed-batch culture using waste glycerol of K. pneumoniae GEM167 mutant strain.
Figure 7 shows the production method (A) of the K. pneumoniae GEM167 ΔldhA variant strain and Southern hybridization results (B) confirming the ldhA mutation.
Figure 8 shows the result of pure glycerol fermentation and ethanol production by fed- batch culture of K. pneumoniae GEM167 ΔldhA mutant strains.
9 shows the results of biodiesel waste glycerol fermentation and ethanol production by fed- batch culture of K. pneumoniae GEM167 ΔldhA mutant strains.
Figure 10 shows an improved ethanol production route of K. pneumoniae GEM167 strain.
Figure 11 shows the glycerol (a) or waste glycerol (b) fed-batch culture and ethanol production results of the K. pneumoniae GEM167 / pBR-pdc-adhII strain.
Figure 12 shows the glycerol (a) or waste glycerol (b) fed-batch culture and ethanol production of K. pneumoniae GEM167 ΔldhA / pBR-pdc-adhII strain.

Unless otherwise defined, 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, the enzyme activity of alcohol dehydrogenase (AdhII) is increased by irradiating gamma rays to the Klebsiella pneumoniae strain and 1,3-propanediol oxidoreductase (1,3) A microorganism having a mutant having reduced enzymatic activity of -propanol oxidoreductase (DhaT) was produced, and it was confirmed that the mutant microorganism can produce ethanol with high efficiency using glycerol or waste glycerol as a single carbon source.

In addition, in the present invention, a mutation in which the enzyme activity of the alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) is decreased. It was confirmed that the ethanol production capacity can be improved by deleting the lactate hydrogenase gene additionally in the microorganisms generated.

In the present invention, a mutation occurs in which the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is decreased. It was confirmed that ethanol production could be improved by overexpressing a gene encoding pyruvate dehydrogenase (Pdc) and an aldehyde dehydrogenase (aldehdyde dehydrogenase (AdhII)) in microorganisms.

In one embodiment of the present invention, gamma-rays were irradiated with 1 kGy 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. As a result of comparing the amount of metabolite production of mutant microorganisms, the amount of metabolite of glycerol oxidative metabolite was significantly increased, and the amount of metabolite of glycerol oxidative metabolite was significantly increased compared to the control, especially ethanol production was higher than that of the control. It was confirmed that the increase of about 8 times.

The above-mentioned mutant microorganisms were 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 the glycerol fermentable microorganisms, an alcohol dehydrogenase (AdhII) was encoded. Mutations in the genes or genes encoding 1,3-propanediol oxidoreductase (DhaT) result in changes in the enzyme activity encoded by the genes or biosynthetic pathways involving enzymes It is estimated that some or a substantial portion of the change induces the conversion of glycerol to high efficiency ethanol.

In another embodiment of the present invention, the enzyme activity of the mutant microorganisms irradiated with gamma-ray was measured. As a result, the enzyme activity of alcohol dehydrogenase (AdhII) was greatly increased when cultured in a medium containing glucose as a single carbon source. On the other hand, when cultured in a medium containing glycerol as a single carbon source, the enzymatic activity of alcohol dehydrogenase (Adh II) was increased by three times compared to the control and 1,3-propanediol oxidder. Enzyme activity of reductase (1,3-propanol oxidoreductase, DhaT) was found to be lower than that of the control. From this result, it can be seen that the change in the enzyme activity of the mutant microorganisms causes a change in the glycerol fermentation metabolic pathways to increase the product production of glycerol oxidative metabolic pathways and in particular to increase the ethanol production.

In the present invention, the enzyme activity of alcohol dehydrogenase (Adh II) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is reduced. In mutant microorganisms with high ethanol production from glycerol or waste glycerol, the mutant microorganism further improved the ability to produce ethanol from glycerol or waste glycerol by further deleting the gene encoding Lactate dehydrogenase (LdhA). It is about.

In the present invention, a mutation occurs in which the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is reduced. In the case of ethanol production of glycerol as a carbon source using an existing microorganism, an excess amount of lactate is produced, and in order to suppress the production of lactate, the activity of the lactate dehydrogenase enzyme in the mutant microorganism is suppressed. Reduced.

Specifically, in one embodiment of the present invention, the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) Lactate dehydrogenase gene was deleted by homologous recombination in the microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP) in which this decreased mutation occurred. As a result, ethanol production was 28.9 g / L at 21.5 g / L. It was confirmed that the increase.

Accordingly, an aspect of the present invention is a mutation in which a gene encoding lactate dehydrogenase (LdhA) is deleted in a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP) having high ethanol production from glycerol or waste glycerol. It relates to a microorganism.

In another aspect, the present invention, the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is increased In mutant microorganisms with high ethanol production from reduced glycerol or waste glycerol, genes encoding pyruvate dehydrogenase (Pdc) and genes encoding aldehyde dehydrogenase (aldhdyde dehydrogenase (AdhII)) The present invention relates to a mutant microorganism which further improves the ability to produce ethanol from glycerol or waste glycerol by introducing and overexpressing.

In the present invention, a mutation occurs in which the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is reduced. Enzymatic activity of pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldhdyde dehydrogenase (AdhII)) to enhance the pathway to produce ethanol via aldehydes via aldehydes as carbon sources Increased.

Specifically, in one embodiment of the present invention, the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) Genes encoding pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldhdyde dehydrogenase (AdhII)) in the microorganism Klebsiella pneumoniae GEM167 (Accession Number KCTC11742BP), in which mutations with reduced activity have occurred. The gene was transformed using a recombinant vector, and as a result, the ethanol production was confirmed to increase from 21.5g / L to 25g / L.

Thus, another aspect of the present invention is a gene and aldehyde dehydrogenase encoding pyruvate dehydrogenase (Pdc) in a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP) having high ethanol production from glycerol or waste glycerol. The present invention relates to a mutant microorganism overexpressed by introducing a gene encoding genease (aldehdyde dehydrogenase (AdhII)).

In another aspect of the present invention, the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is increased. In mutant microorganisms with high ethanol production from reduced glycerol or waste glycerol, the gene encoding Lactate dehydrogenase (LdhA) is deleted and pyruvate dehydrogenase (Pdc) is removed. The present invention relates to a novel mutant microorganism which further improves the ability to produce ethanol from glycerol or waste glycerol by introducing and overexpressing the gene encoding and the gene encoding aldehyde dehydrogenase (aldhdyde dehydrogenase (AdhII)).

In the present invention, a mutation occurs in which the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) is reduced. To inhibit the production of lactate, to reduce the activity of lactate dehydrogenase enzymes in the mutant microorganisms, and to enhance the pathway of producing ethanol via aldehydes as a carbon source, Enzyme activity of pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldehdyde dehydrogenase (AdhII)) was increased.

Specifically, in one embodiment of the present invention, the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) In the microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP), in which mutations with reduced activity have occurred, the lactate dehydrogenase gene is deleted by homologous recombination and encodes pyruvate dehydrogenase (Pdc). Genes and genes encoding aldehyde dehydrogenase (aldehdyde dehydrogenase (AdhII)) were transformed using a recombinant vector, and as a result, the ethanol production was confirmed to increase from 21.5g / L to 31.0g / L.

Thus, another aspect of the present invention, in a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP) having high ethanol production from glycerol or waste glycerol, deletes the gene encoding the lactate dehydrogenase (LdhA). The present invention relates to a mutant microorganism overexpressed by introducing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (AdhII).

In the present invention, the microorganism may be bacteria, yeast, and the like, preferably bacteria. The bacterium may be, for example, genus Klebsiella , genus Lactobacillus , genus Clostridium , or genus Enterobacter , more preferably Krebssi. Klebsiella pneumoniae , or a microorganism capable of using glycerol or waste glycerol as a carbon source is not limited thereto.

The present invention also relates to (a) irradiation of gamma rays to microorganisms to increase the enzymatic activity of alcohol dehydrogenase (AdhII) and to 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT Mutating to decrease enzymatic activity; (b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. Obtaining a microorganism; And (c) relates to a method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol comprising the step of deleting the gene encoding the lactate hydrogenase in the obtained microorganism.

In another aspect, the present invention (a) by irradiating gamma rays to the microorganism increases the enzyme activity of alcohol dehydrogenase (Adh II) and 1,3-propanediol oxidase deductase (1,3-propanol oxidoreductase (DhaT), the mutation to reduce the enzymatic activity; (b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. Obtaining a microorganism; And (c) overexpressing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (aldhdyde dehydrogenase, AdhII) in the obtained microorganism, or It relates to a method for producing mutant microorganisms having high ethanol production from waste glycerol.

In another aspect, the present invention (a) by irradiating gamma rays to the microorganism increases the enzyme activity of alcohol dehydrogenase (Adh II) and 1,3-propanediol oxidase deductase (1,3-propanol oxidoreductase (DhaT), the mutation to reduce the enzymatic activity; (b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. 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 (AdhII)). It relates to a method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol comprising the step of overexpressing the gene encoding the.

In the present invention, the gamma ray may be irradiated at an appropriate dose depending on the type of mutant microorganism, for example, may be irradiated at a dose of 0.1 kGy to 10 kGy. Preferably from 0.5 kGy to 5 kGy, more preferably from 0.5 kGy to 3 kGy, most preferably from 1 kGy dose.

In another aspect, the present invention relates to a method for producing ethanol, comprising 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 ethanol from the culture. will be.

In one embodiment of the present invention K. pneumoniae GEM167 and mutant strains of the present invention The ethanol production yield was compared with the previously reported results. As shown in Table 10, the maximum ethanol production yield of 31 g / L was obtained in the K. pneumoniae GEM167 ΔldhA strain overexpressing the Pdc-AdhII enzyme gene. This is much better than the existing maximum production of 20.7 g / L (recombinant Escherichia coli).

In the industrial process of producing ethanol from glycerol, it is expected to be economical when the production yield is about 40-50 g / L. Considering the production yield of the mutant strains of the present invention, it is judged that commercial value is very high if the cultivation process is optimized.

In another embodiment of the present invention, in order to determine the effect of pH on the ethanol production of mutant microorganisms, culturing the mutant microorganisms while varying the pH of the culture medium to compare the ethanol production, neutrality of pH 6 and pH 7 It was confirmed that high ethanol productivity was shown under the conditions, and the results of analysis of ethanol production of mutant microorganisms according to air injection amount showed that ethanol production was high when aereation was performed at 0.1vvm and 0.5vvm.

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

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

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

50 ml flasks of Klebsiella pneumoniae Cu strains were cultured and recovered when the OD _600nm was 0.4-0.6. The cells were recovered by PBS buffer (8g NaCl, 0.2g KCl, 1.44g Na ₂ HPO ₄ , 0.24g KH ₂ Suspended in PO ₄ in 1 L of distilled H ₂ O, pH 7.4) and irradiated with gamma rays at 1 kGy, and when the killing rate was about 99%, the suspended cells were appropriately diluted with sterile water and plated in LB medium. After obtaining a total of 26,000 colonies, a medium containing glycerol as the only carbon source [20 g / L glycerol was added to 1 L of 0.1 M potassium phosphate buffer (pH 7.0), followed by 2 g / L (NH ₄ ) ₂ SO ₄ , 0.2 g / L MgSO ₄ , 0.002 g / L CaCl ₂ 2H ₂ O, 1 g / L yeast extract, 1 ml iron solution (5 g / L FeSO ₄ 7H ₂ O), 4 ml HCl (37%, w / v), 1 ml trace element solution (70 mg / L ZnCl ₂ , 100 mg / L MnCl ₂ 4H ₂ O, 60 mg / LH ₃ BO ₃ , 200 mg / L CoCl ₂ 4H ₂ O, 20 mg / L CuCl ₂ H ₂ O, 25 mg / L NiCl ₂ 6H ₂ O, 35 mg / L Na2MoO ₄ 2H2O, 4 ml HCl (37%, w / v)]] and cultured at 120 rpm at 37 ° C. At the same time, mutant strains with improved ethanol production were isolated by analyzing the residual amount of glycerol in the culture supernatant and the production of metabolites including ethanol by liquid chromatography. 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 vvm using 5 L fermenter, and compared with the control strain, Cu strain.

As a result, in the mutant strains, 1,3-propanediol and 3-hydroxypropionic acid, which are metabolites of the glycerol reduction metabolic pathway, were hardly produced, and the amount of metabolites of the glycerol oxidative metabolic pathway was significantly higher than that of the control. It appeared to increase. In particular, the production of ethanol showed an extremely high ethanol production (high performance) to 8.6 g / L, an increase of about 8 times from 1.1 g / L (Fig. 2, Table 1).

Comparison of Metabolite Production (g / L) of K. pneumoniae Cu Strain and GEM 167 Mutant Strain Carbon source Strain 1,3-Propanediol 3-Hydroxypropionic acid Ethanol Lactic acid Succinic acid 2,3-
Butandiol Acetic acid Glycerol K. pneumoniae Cu 7.93 1.0 1.1 0.5 0.5 0 3.5 K.
pneumoniae GEM 167 0.2 0 8.6 1.4 0.7 0.35 1.0 Glucose K.
pneumoniae Cu 0.15 0 1.9 0.5 0.4 0.25 2.9 K. pneumoniae GEM 167 0.07 0 1.7 0.6 0.4 0.25 2.5

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

Example 2: K. pneumoniae Enzyme Activity Verification of GEM167 Mutant Strain

In order to measure the enzyme activity of the mutant strain K. pneumoniae GEM167 prepared in Example 1, after recovering the cells cultured in the same medium containing glycerol or glucose as the only carbon source, 50 mM potassium phosphate buffer (pH 7.5 ), And then dissolved again and ground by sonication.

Enzyme Activity Assay for Measuring Enzyme Activity of Alcohol Dehydrogenase (AdhII) A mixed solution was prepared by adding glycine-KOH buffer (pH 9.0) (50 mM) and NAD + (1 mM) to the culture strain pulverized solution. The reaction was started by adding 100 mM ethanol to the prepared mixture. The reaction was carried out for 30 minutes at 37 ℃ was measured for absorbance at 340 nm (Postma E. et al , Appl Environ Microbiol. , 55: 468-477, 1989).

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 culture strain pulverized solution so that the final concentration was 20 mM, and the final concentration was 10 mM. Add vitamin B ₁₂ to react at 37 ℃ for 5 minutes. Then, 1M citrate buffer (pH 3.6) was added and MBTH 0.1% was added, followed by reaction at 100 ° C. for 10 minutes, and the absorbance at 315 nm was measured.

Enzyme Activity Assay for Measuring Enzyme Activity of Glycerol Dehydrogenase (DhaD) A mixed solution was prepared by adding glycine-KOH buffer (pH 9.0) (50 mM) and NAD + (1 mM) to the culture strain pulverized solution. The reaction was started by adding 100 mM glycerol to the prepared mixture. The reaction was carried out for 30 minutes at 37 ℃ and the absorbance was measured at 340 nm.

Enzyme activity assay mixtures for measuring enzymatic activity of 1,3-propanediol oxidoreductase (DhaT) were glycine-KOH buffer (pH 9.0) (50 mM) and Prepared by adding NAD + (1 mM), the reaction was started by adding 100 mM 1,3-propanediol to the prepared mixture. The reaction was carried out for 30 minutes at 37 ℃ and the absorbance was measured at 340 nm.

As a result, as shown in Table 2, the alcohol dehydrogenase (AdhII) enzyme activity of the GEM 167 mutant strain when cultured in glycerol medium increased more than three times compared to 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 (AdhII) enzyme activity was not significant. A marked increase in alcohol dehydrogenase (AdhII) enzyme activity in glycerol medium is a result consistent with the metabolite production assay above. Meanwhile, 1,3-propanediol oxidoreductase (DhaT) enzyme activity was decreased in the GEM 167 mutant strain, which is consistent with the decrease in 1,3-propanediol production of the mutant strain. Is the result.

Enzyme Activity of K. pneumoniae Cu and GEM 167 Mutant Strains Carbon source Strain DhaB DhaT DhaD AdhII Glycerol K. pneumoniae Cu 0.15 0.31 2.5 0.6 K. pneumoniae GEM 167 0.25 0.21 2.9 1.9 Glucose K. pneumoniae Cu 0.26 0.22 1.08 0.41 K. pneumoniae GEM 167 0.27 0.19 1.22 0.74

Enzyme units are expressed as unit / g protein.

Example 3: Analysis of Ethanol Productivity of Mutant Strains According to pH

In order to determine the effect of pH on ethanol production of the mutant strain K. pneumoniae GEM167, when the culture was terminated by changing the pH of the culture medium to 5, 6, 7 and 8 using the same method as in Example 1 above. The ethanol production of the mutant strains was measured while maintaining up to. 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 shown that the cell growth and ethanol productivity is very poor.

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

In order to examine the effect of aeration on the ethanol production of the mutant strain K. pneumoniae GEM167, using the same method as in Example 1, areation conditions were 0.0 vvm, 0.1 vvm, 0.5 vvm, 1.0 vvm, 2.0 vvm and 3.0, respectively. Incubated differently with vvm. As a result, as shown in Figure 4, it can be seen that the ethanol production is higher when the aeration to 0.1vvm, 0.5vvm than in the case of 0.0 vvm without aeration at all, that is, to make the aeration properly It was shown to be advantageous for production.

Example 5: Ethanol Productivity Analysis from Glycerol of Mutant Strains

In the same manner as in Example 1, GEM 167 was fermented and cultured by a fed-batch method while maintaining areation at 0.5 vvm and pH at 6.8 to 7.2. As a result, as shown in FIG. 5 and Table 3, the maximum yield of ethanol at 23 hours culture was 21.5 g / L, and the hourly yield of 0.93 g / L showed the highest level of ethanol production at the highest level known to date.

Fermentation of K. pneumoniae GEM 167 Mutant Strain by Fed-Batch Method division 23 hours of incubation 1,3-Propanediol 0.45 g / L 3-Hydroxypropionic acid 0 g / L Acetic acid 0.4 g / L Lactic acid 11.5 g / L Succinic acid 1.3 g / L 2,3-Butanediol 2.1 g / L Ethanol 21.5 g / L Productivity (Ethanol) 0.93 g / Lh Molar yield (Ethanol / Glycerol) 0.62 mol / mol

Example 6: Mutation Strains Ethanol Productivity Analysis from Waste Glycerol

In the same manner as in Example 1, GEM 167 was fermented and cultured using waste glycerol, a biodiesel by-product as the only carbon source, while maintaining areation at 0.5 vvm and pH at 6.8 to 7.2. As a result, as shown in Figure 6 and Table 4, the maximum production of ethanol at 23 hours incubation was 19.9 g / L, the hourly production was 0.87 g / L showed a similar ethanol production of pure glycerol.

Fermentation Culture of K. pneumoniae GEM 167 Mutant Strain Using Waste Glycerol division 23 hours of incubation 1,3-Propanediol 0.6 g / L 3-Hydroxypropionic acid 0 g / L Acetic acid 0.2 g / L Lactic acid 10.6 g / L Succinic acid 0.7 g / L 2,3-Butanediol 1.8 g / L Ethanol 19.9 g / L Productivity (Ethanol) 0.87 g / Lh Molar yield (Ethanol / Glycerol) 0.70 mol / mol

Example 7: K. pneumoniae  Improvement of GEM167 Mutant Strain: Preparation of Lactate-deficient derivative

(1) Preparation of K. pneumoniae GEM167 ΔldhA mutant strain

As shown in Table 3 and Table 4, ethanol produced 21.5 g / L of ethanol and 11.5 g / L of lactate in the glycerol fermentation culture of the K. pneumoniae GEM167 mutant strain, leading to a major competitive metabolite of ethanol. It can be seen that is lactate.

Therefore, if the production of lactate in the GEM167 strain inhibits the production of ethanol can be expected to increase, in order to confirm this, the lactate-deleted mutant strain was prepared from the GEM167 mutant strain and analyzed the characteristics of glycerol fermentation and ethanol production.

The manufacturing process of the lactate deficient GEM167 mutant strain (GEM167 ΔldhA ) is briefly described as follows. In order to remove the lactate dehydrogenase gene ( ldhA ) that catalyzes the lactate production on the chromosome of the GEM167 strain, 0.9 kb of the up and down sequences of the ldhA gene were identified as P1, P2 primer (upstream) or P3, respectively. After amplification using P4 primers (downstream), the antibiotics apramycin-resistant genes were inserted between the homologous recombination DNA cassettes. The prepared DNA cassette ( ldhA gene upstream sequence-apramycin resistance gene- ldhA gene downstream sequence) was introduced into K. pneumoniae GEM167 strain by electroshock method, and then the DNA cassette was homologous recombination. A transformant inserted into chromosomal DNA and showing apramycin resistance was isolated.

Primer sequence used to prepare GEM167 ΔldhA strain division Base sequence P1 (upstream of ldhA ) 5-CAGCCAGACGGGAATAGCTT-3 (SEQ ID NO: 1) P2 (upstream ldhA ) 5-CGCCAATTTTCTGGTGCTTCA GATATC GCCTCAAGGTCGACGTTGTTAA-3 (SEQ ID NO: 2) P3 (downstream ldhA ) 5-TTAACAACGTCGACCTTGAGGC GATATC TGAAGCACCAGAAAATTGGCG-3 (SEQ ID NO: 3) P4 (downstream ldhA ) 5-AGCTCGATGGTTCGGCGATT-3 (SEQ ID NO: 4)

Southern hybridization was performed on the isolated transformants to confirm that the DNA cassette was correctly inserted into the ldhA gene. The chromosome DNA of the parent strain GEM167 and the resulting transformants were treated with restriction enzyme Mlu I, and hybridization was performed using the downstream sequence of ldhA gene as a probe. As expected, 3.2 kb of GEM167 strain was obtained. The DNA fragment was found to bind 2.3 kb of DNA fragment in the transformants (FIG. 7). On the other hand, Apra azithromycin transformants a resistance gene obtained show that it does not combine DNA fragments present in the as contrast GEM167 strain the binding of the same DNA fragment in the transformant when used as probes in the ldhA gene on the chromosome O It was confirmed that it was correctly substituted by the pramycin resistance gene.

(2) Lactate dehydrogenase enzyme activity verification

Lactate dehydrogenase enzyme protein activity was analyzed by culturing the GEM167 ldhA strain in the medium used in Example 1 containing glycerol or glucose. The recovered cultured cells were washed with 50 mM potassium phosphate buffer (pH 7.5), resuspended, pulverized by the sonication method, and used as an enzyme source. Enzyme activity assay mixture for measuring LdhA enzyme activity was prepared by adding glycine-KOH buffer (pH 9.0) (50mM) and NAD + (1mM) to the culture strain pulverized solution, and added 100 mM lactate to the prepared mixture. The reaction started. The reaction was carried out for 30 minutes at 37 ℃ was measured for absorbance at 340nm. As a result, as shown in Table 6, it was confirmed that the lactate dehydrogenase enzyme activity in the GEM167 ΔldhA strain significantly decreased.

Lactate Dehydrogenase Activity of K. pneumoniae GEM167 and GEM167 ΔldhA Mutants Strain LdhA activity (U / mg) GEM167 0.99 ± 0.02 GEM167 Δ ldhA 0.08 ± 0.01

Example 8: K. pneumoniae  GEM167 ΔldhA  Ethanol Production by Feeding Cultivation of Mutant Strains

Fermentation of the K. pneumoniae GEM167 ΔldhA mutant strain by a fed- batch method using 2 L of the medium used in Example 1 in a 5 L fermenter while maintaining a culture temperature of 37 ° C., agitation speed of 200 rpm, an air injection speed of 0.5vvm, and a pH of 6.8 to 7.2 The culture was analyzed for glycerol fermentation and ethanol production.

When pure glycerol was used as a nutrient source, the production of lactate in GEM167 Δ ldhA compared to the GEM167 strain as shown in Fig. 8 and Table 7 (pure glycerol fermentation culture with K. pneumoniae GEM167 Δ ldhA mutant strain) was 11.5 g / It was confirmed that the L was significantly reduced from 1.4 g / L. In proportion to the decrease in lactate production, the maximum production of ethanol was 28.9 g / L and the hourly production was 1.2 g / L.

On the other hand, in the fed-batch fermentation of GEM 167 Δ ldhA using biodiesel industrial by-product waste glycerol as a nutrient source, as shown in FIG.

Example 9: K. pneumoniae  Improvement of GEM167 Mutant Strain: Overexpression of Pdc-adhII Gene

(1) Construction of a Pdc-adhII Gene Overexpression Vector of Zymomonas mobilils

As confirmed in Example 8, it was confirmed that the production of ethanol was greatly increased by the removal of the LdhA gene, which catalyzes the production of lactate, a major competitive metabolite of ethanol, in the K. pneumonae GEM167 strain. As a second method to increase the production of ethanol in the GEM167 strain, overexpressing the pyruvate dehydrogenase (Pdc) and aldehyde dedygenase (aldehdyde dehydrogenase (AdhII)) genes of ethanol producing enzymes derived from Z. mobilis In the ethanol production pathway, pyruvate to acetaldehyde conversion and acetaldehyde to ethanol conversion were accelerated (FIG. 10).

A brief description will be given of the construction of the Pdc-AdhII enzyme gene overexpression vector. 1.8kb of Pdc gene with 1.15kb of the gene, respectively AdhII Ppdc-F of (5'- TCTAGA atgagttatactgtcggtacctatttagc-3 '(SEQ ID NO: 5); italic - Xba I sites), Ppdc-R (5'- CTCGAG CTGCAG ctagaggagcttgttaacaggcttac-3 '(SEQ ID NO: 6); Italic- Xho I site, underline-indicates Pst I site) and PaldB-F (5'- AGATCT atggcttcttcaactttttatattcc-3' (SEQ ID NO: 7); Italic- Bgl II site), PaldB-R (5'- CTCGAG TCTAGA ttagaaagcgctcaggaagagtt-3 '(SEQ ID NO: 8); italic- Xho I site, underline-represents Xba I site) primers were used to amplify Z. mobilis chromosomal DNA as a template. The lacZ promoter sequence (P _lacZ -aldB ), a potent gene expression promoter, is also expressed in PlacZ-aldB-F (5'- GAATTC agcgggcagtgagcgcaa-3 '(SEQ ID NO: 9); Italic- Eco RI site) and P _lacZ -aldB-R ( 5'- CTCAGA AGATCT agctgtttcctgtgtgaaattg-3 (SEQ ID NO: 10), italic- Xho I site, underline- Bgl II site) amplified using a primer. The amplified DNA fragments were cloned into pGEM TEasy vector (Promega, USA) and sequenced to confirm that gene amplification was correct.

PGEM-P _lacZ -aldB , pGEM-P _lacZ -aldB-pdc . Were constructed by inserting AdhII, Pdc genes downstream of the LacZ promoter, and then reinserting the P _lacZ -aldB-pdc cassette into the pBR322 vector to obtain pBR-aldB. -pdc was prepared and introduced into K. pneumoniae Cu, GEM167, and GEM167 ldhA strains, respectively.

(2) Pdc-adhII enzyme activity verification

The pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (AdhII) enzyme activities of the recombinant cells were 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. Enzyme activity assay mixture for measuring Pdc enzyme activity was added to imidazole hydrochloride buffer (pH 6.5) (40mM), MgCl ₂ (5mM), thiamine pyrophosphate (0.2mM) and NADH (0.15mM) to the culture strain grinding solution, Prepared by addition of alcohol dehydrogenase (Boehringer) to 88U. Then, 50 mM pyruvate was added to start the reaction. The reaction was carried out for 30 minutes at 30 ℃ and then measured the absorbance at 340nm. Enzyme activity assay mixture for measuring AdhII enzyme activity was prepared by adding glycine-KOH buffer (pH 9.0) (50mM) and NAD + (1mM) to the culture strain pulverized solution, and reaction was performed by adding 100mM ethanol to the prepared mixture. Started. The reaction was carried out for 30 minutes at 30 ℃ and then measured the absorbance at 340nm.

As a result, as shown in Table 8, it was confirmed that the Pdc and AdhII enzyme activity was increased in the recombinant cells into which the pBR-aldB-pdc plasmid was introduced.

Pdc and Adh Enzyme Activities in K. pneumoniae Strains Pdc Adh Cu 0.78 ± 0.01 0.61 ± 0.02 GEM167 1.45 ± 0.03 1.92 ± 0.02 GEM167 Δ ldhA 1.39 ± 0.01 1.79 ± 0.01 GEM167 / pBR- adhB-pdc 2.12 ± 0.02 2.67 ± 0.03 GEM167 Δ ldhA / pBR- adhB-pdc 2.25 ± 0.01 2.88 ± 0.01

Enzyme activity (U / mg protein)

Example 10 Pdc-AdhII Overexpression K. pneumoniae  Ethanol Production by Feeding Cultivation of Strains

K. pneumoniae mutant strain overexpressing the Pdc-AdhII enzyme gene while maintaining a culture temperature of 37 ° C., agitation speed of 200 rpm, an air injection rate of 0.5vvm, and a pH of 6.8 to 7.2 using a 2L medium described in Example 1 in a 5 L fermentor. The fermentation cultures were fed in a fed-batch method to analyze glycerol fermentation and ethanol production, and the results are shown in FIGS. 11 and 12. As shown in Table 9 (Pure glycerol fermentation culture with Pdc-AdhII overexpressing K. pneumoniae recombinant strain), when the Pdc-AdhII gene was overexpressed, the ethanol production was 21.5 g / L in the GEM167 strain and the GEM167 Δ ldhA strain, respectively. Was increased from 25 g / L to 28.9 g / L to 31 g / L. Investigation of the stability of the pBR-pdc-adhII plasmid during fermentation showed that about 93% of cells retained the plasmid after 150 generations.

Example 11: K. pneumoniae  Comparison of Ethanol Production Capacity of Mutant Strains

K. pneumoniae GEM167 and its variants identified in the above examples The yield of ethanol was compared with the results of previously reported known strains.

As shown in Table 10, the K. pneumoniae GEM167 ΔldhA strain overexpressing the Pdc-AdhII enzyme gene showed a maximum ethanol production yield of 31 g / L, compared to the existing maximum yield of 20.7 g / L (recombinant Escherichia coli). Excellent results. In the industrial process of producing ethanol 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, it is judged that commercial value is very high if the cultivation process is optimized.

Comparison of Ethanol Production Capacity from Glycerol of Known Strains Strain name Culture method Ethanol production literature density
g / l Productivity
g / l / h Enterobacter aerogenes Hu-101 Batch 10 0.83 Ito et al., J Biosci Bioeng, 100, 260, 2005 Escherichia coli EH05 Batch 20.7 0.22 Durnin et al., Biotechnol Bioeng, 103,148, 2009 Klebsiella oxytoca M5al Fed-batch 19.5 0.56 Yang et al., Appl Microbiol Biotechnol, 73, 1017, 2007 Klebsiella pneumoniae M5a1 Fed-batch 18 0.28 Cheng et al., Process Biochem, 42, 740, 2007 K. pneumoniae GEM167 Fed-batch 21.5 0.93 Invention K. pneumoniae GEM167 / pBR- pdc - adh Fed-batch 25.0 0.78 Invention K. pneumoniae GEM167 Δ l d hA Fed-batch 28.9 0.81 Invention K. pneumoniae GEM167 Δ l d hA / pBR- pdc - adh Fed-batch 31.0 0.89 Invention

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Institute of Bioscience and Biiotechnology <120> Improved Microorganism Variants Having Ehtanol Producing Ability          Using Glycerol or Crude Glycerol and Method for Producing Ethanol          Using the same <130> P11-B152 <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cagccagacg ggaatagctt 20 <210> 2 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cgccaatttt ctggtgcttc agatatcgcc tcaaggtcga cgttgttaa 49 <210> 3 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttaacaacgt cgaccttgag gcgatatctg aagcaccaga aaattggcg 49 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agctcgatgg ttcggcgatt 20 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tctagaatga gttatactgt cggtacctat ttag 34 <210> 6 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctcgagctgc agctagagga gcttgttaac aggcttac 38 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 agatctatgg cttcttcaac tttttatatt cc 32 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ctcgagtcta gattagaaag cgctcaggaa gagtt 35 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gaattcagcg ggcagtgagc gcaa 24 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctcagaagat ctagctgttt cctgtgtgaa attg 34

Claims (25)

From glycerol or waste glycerol with increased enzymatic activity of alcohol dehydrogenase (AdhII) and reduced enzymatic activity of 1,3-propanediol oxidoreductase (DhaT) In a mutant microorganism having high ethanol production, a mutant microorganism having high ethanol performance from glycerol or waste glycerol which has deleted a gene encoding Lactate dehydrogenase (LdhA).
The method of claim 1, wherein the microorganism is a mutant microorganism having high ethanol performance from glycerol or waste glycerol, characterized in that selected from the group consisting of bacteria, yeast and mold
According to claim 2, wherein the bacteria is selected from the group consisting of genus Klebsiella , genus Lactobacillus , genus Clostridium and genus Enterobacter . A mutant microorganism having high ethanol production performance from glycerol or waste glycerol.
The mutant microorganism which deleted the gene which codes lactate dehydrogenase (LdhA) in the mutant microorganism Klebsiella pneumoniae GEM167 (Accession Number KCTC11742BP) which has high ethanol performance from glycerol or waste glycerol.
From glycerol or waste glycerol with increased enzymatic activity of alcohol dehydrogenase (AdhII) and reduced enzymatic activity of 1,3-propanediol oxidoreductase (DhaT) In mutant microorganisms with high ethanol production, glycerol or lung glycerol overexpressed by introducing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (AdhII) Mutant microorganism having high performance from ethanol.
The method of claim 5, wherein the microorganism is a mutant microorganism having high ethanol performance from glycerol or waste glycerol, characterized in that selected from the group consisting of bacteria, yeast and fungi
The method of claim 5, wherein the bacteria is selected from the group consisting of genus Klebsiella , genus Lactobacillus , genus Clostridium , and genus Enterobacter . A mutant microorganism having high ethanol production performance from glycerol or waste glycerol.
In a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP) with high ethanol production from glycerol or waste glycerol, a gene encoding pyruvate dehydrogenase (Pdc) and an aldehyde dehydrogenase (alddhdyde dehydrogenase (AdhII)) A mutant microorganism overexpressed by introducing a gene encoding a.
From glycerol or waste glycerol with increased enzymatic activity of alcohol dehydrogenase (AdhII) and reduced enzymatic activity of 1,3-propanediol oxidoreductase (DhaT) In mutant microorganisms with high ethanol production, genes encoding Lactate dehydrogenase (LdhA) are deleted, genes encoding pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase A mutant microorganism having high ethanol-producing ability from glycerol or waste glycerol overexpressed by introducing a gene encoding azede (aldehdyde dehydrogenase, AdhII).
10. The method of claim 9, wherein the microorganism is a mutant microorganism having high ethanol performance from glycerol or waste glycerol, characterized in that selected from the group consisting of bacteria, yeast and fungi
The method of claim 10, wherein the bacteria is selected from the group consisting of genus Klebsiella , genus Lactobacillus , genus Clostridium , and genus Enterobacter . A mutant microorganism having high ethanol production performance from glycerol or waste glycerol.
In a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP) with high ethanol-producing ability from glycerol or waste glycerol, the gene encoding Lactate dehydrogenase (LdhA) is deleted and pyruvate dehydrogenase (pyruvate) A mutant microorganism overexpressed by introducing a gene encoding dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (AdhII).
Method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol comprising the following steps:
(a) Irradiation of gamma rays to microorganisms increased the enzyme activity of alcohol dehydrogenase (AdhII) and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT). Mutating to decrease;
(b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. Obtaining a microorganism; And
(c) deleting the gene encoding the lactate hydrogenase in the obtained microorganism.
The method of claim 13, wherein the microorganism is selected from the group consisting of bacteria, yeast and fungi.
The genus Klebsiella , genus Lactobacillus , genus Clostridium and genus Enterobacter are selected from the group consisting of Genus Enterobacter . A method for producing a mutant microorganism having high ethanol performance from glycerol or waste glycerol.
The method of claim 13, wherein the gamma rays are irradiated at a dose of 1 kGy.
Method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol comprising the following steps:
(a) Irradiation of gamma rays to microorganisms increased the enzyme activity of alcohol dehydrogenase (AdhII) and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT). Mutating to decrease;
(b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. Obtaining a microorganism; And
(c) overexpressing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding aldehyde dehydrogenase (aldhdyde dehydrogenase, AdhII) in the obtained microorganism.
18. The method of claim 17, wherein the microorganism is selected from the group consisting of bacteria, yeast and mold.
The method of claim 18, wherein the bacteria are selected from the group consisting of genus Klebsiella , genus Lactobacillus , genus Clostridium , and genus Enterobacter . A method for producing a mutant microorganism having high ethanol performance from glycerol or waste glycerol.
18. The method according to claim 17, wherein the gamma rays are irradiated at a dose of 0.1 kGy to 10 kGy.
Method for producing a mutant microorganism having high ethanol production from glycerol or waste glycerol comprising the following steps:
(a) Irradiation of gamma rays to microorganisms increased the enzyme activity of alcohol dehydrogenase (AdhII) and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT). Mutating to decrease;
(b) the enzyme activity of alcohol dehydrogenase (AdhII) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is reduced. Obtaining a microorganism; And
(c) deleting the gene encoding the lactate hydrogenase from the obtained microorganism, the gene encoding pyruvate dehydrogenase (Pdc) and the aldehyde dehydrogenase (aldehdyde dehydrogenase (AdhII)) Overexpressing the coding gene.
22. The method of claim 21, wherein the microorganism is selected from the group consisting of bacteria, yeasts and molds.
The method of claim 22, wherein the bacteria are selected from the group consisting of genus Klebsiella , genus Lactobacillus , genus Clostridium , and genus Enterobacter . A method for producing a mutant microorganism having high ethanol performance from glycerol or waste glycerol.
22. The method of claim 21, wherein the gamma rays are irradiated at a dose of 0.1 kGy to 10 kGy.
The method for producing ethanol, wherein the mutant microorganism of any one of claims 1 to 12 is cultured in a medium containing glycerol or waste glycerol as a carbon source to produce ethanol, and then ethanol is recovered from the culture solution.

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