KR101230638B1 - Kluyveromyces marxianus having superior heat resistance and ethanol productivity - Google Patents

Abstract

The present invention relates to a Kluyveromyces Marchanus mutant strain having excellent heat resistance and ethanol productivity, and a method for producing ethanol using the same. Cluiberomyces Marchanus mutant strain of the present invention is very excellent in heat resistance and ethanol productivity, and can be easily used for ethanol production because it can be simultaneously glycated and fermented during ethanol production using biomass, preferably fibrous biomass. have.

Description

Kluyveromyces marxianus having superior heat resistance and ethanol productivity

The present invention relates to a Kluyveromyces Marchanus strain having excellent heat resistance and ethanol productivity, and a method for producing ethanol using the same.

Recently, interest in bioethanol has increased due to environmental concerns and concerns about depletion of fossil fuels. Among them, interest and research on fibrous bioethanol, which can avoid collisions with food resources and bring about a significant reduction in raw material costs, are increasing.

The fibrous bioethanol is obtained through ethanol fermentation using microorganisms such as bacteria or yeast, based on fibrous biomass such as barley straw, silver grass, and waste wood. Raw materials are much cheaper than saccharide and starch biomass because they mainly use fibrous biomass such as agricultural and forestry waste resources, but relatively expensive in pretreatment and saccharification process to obtain sugar from fibrous biomass. There is a difficulty in industrialization.

In general, fibrous biomass is difficult to directly saccharify, so that tissues are degraded by pretreatment such as acid, base treatment, ammonia treatment, hydrothermal treatment, and steam depletion treatment, followed by cellulose, hemi-cellulose, and beta-glucosidase. The action of complex glycosylation enzymes such as to obtain mainly glucose and sugar in the form of sugar. After the sugar is obtained, it is not much different from the conventional ethanol fermentation and distillation using microorganisms, but the cost of pretreatment and saccharification is more than half of the total fibrous bioethanol production cost, so that the development of glycoenzyme production technology and efficient process technology and Development of good ethanol fermentation microorganisms is required.

In particular, the glycosylation of fibrous biomass is optimal glycosylation at a high temperature of 45-50 ℃ due to the characteristics of the fibrous glycosylase, ethanol fermentation microorganisms used in the ethanol fermentation industry, mainly yeast at 30-35 ℃ below 40 ℃ Optimal fermentation is carried out, and even at high temperatures above 40 ° C., most growth is difficult. Therefore, at present, since the saccharification process and the fermentation process must be completely separated, there is a problem that causes inconvenience in the process and takes a lot of operating costs. Therefore, at the same time, it is necessary to develop a method for producing ethanol by saccharification and fermentation, and for this purpose, the development of a yeast strain capable of ethanol fermentation even at a high temperature of 40 ° C. or more is urgently needed.

The present inventors have developed a novel strain having excellent heat resistance and ethanol productivity, and completed the present invention by effectively ethanol production by simultaneously glycosylating and fermenting the strain.

Therefore, the present invention is Saccharomyces Saccharomyces accession number KCTC11250BP cerevisiae ) and Kluyveromyces with excellent heat resistance and ethanol productivity produced by protoplast fusion of Kluyveromyces Marchanus under accession number KCTC17631. marxianus ) aims to provide variant strains.

It is another object of the present invention to provide a probiotic composition for ethanol production containing the Kluyveromyces Marchanus mutant strain or a culture thereof.

In another aspect, the present invention is to provide a method for producing ethanol comprising the step of glycosylation and fermentation by treating the Cluyberomyces Marchanus strain and glycosylase on a biomass substrate.

In order to solve the above problems, the present invention is the Saccharomyces cerevisiae with accession number KCTC11250BP ( Saccharomyces cerevisiae ) and Kluyveromyces with excellent heat resistance and ethanol productivity produced by protoplast fusion of Kluyveromyces Marchanus under accession number KCTC17631. marxianus ) provides a variant strain.

The present invention also provides a probiotic composition for ethanol production containing the Kluyveromyces Marchanus mutant strain or a culture thereof.

In another aspect, the present invention provides a method for producing ethanol comprising the step of glycosylation and fermentation by treating the Kluyveromyces Marchanus mutant strain and glycosylase on a biomass substrate.

Hereinafter, the present invention will be described in more detail.

Slucharomyces cerevisiae of the Kluyveromyces Marchanus mutant strain of the present invention accession number KCTC11250BP ( Saccharomyces cerevisiae ) and Accession No. KCTC17631 is a strain having excellent heat resistance and ethanol productivity produced by protoplast fusion of Kluyveromyces Marchanus.

The Kluyveromyces Marchanus mutant strain is produced by protoplast fusion of Saccharomyces cerevisiae with accession number KCTC11250BP with excellent ethanol productivity and Kluyberomyces Marshanus with accession number KCTC17631 with excellent heat resistance while having excellent ethanol productivity. It is also characterized by heat resistance.

The Kluyveromyces Marchanus mutant strain may be prepared by protoplasty fusion with Saccharomyces cerevisiae with accession number KCTC11250BP or its mutant and Kluyveromyces Marshanus with accession number KCTC17631 or a mutant thereof. Mutant refers to a strain into which Saccharomyces cerevisiae, Accession No. KCTC11250BP, or a selection marker capable of identifying Kluiberomyces Marchanus, Accession No. KCTC17631 is introduced.

Specifically, the mutant of Saccharomyces cerevisiae may be a respiratory deficient mutant of Saccharomyces cerevisiae prepared by treating the strain with a substance causing mutation, preferably Acriflavine.

In addition, the mutant of Kluyveromyces Marchanus is a leucine produced by treating the strain with a substance causing mutation, preferably EMS (ethylmethansulphate) and NTG (N-methyl-N'-nitro-N-nitrosoguanidine). The biosynthetic gene associated with Isoleucine and Valine of Veromyses Marchanus may be a trophyotropic mutant.

Plasma fusions used to prepare the Kluyveromyces Marchanus mutant strains can be performed according to methods known in the art.

Protoplast fusion is a technique of fusion of two strains with different traits to create a new high-quality hybrid strain having good traits of both cells. In some protozoa, the protoplasts of two haploid cells are combined without nuclear fusion. This is also the case. The plasma fusion is not limited thereto, but the two strains are cultured in a logarithmic state, and then treated with a cell wall degrading enzyme such as Lyticase under appropriate buffer conditions to remove the cell wall, and then two cells in the plasma state are removed. Mixing in fusion buffer conditions to effect fusion.

The Kluyveromyces Marchanus mutant strain of the present invention is not limited thereto but preferably has a growth temperature of 28 to 48 ° C, more preferably 35 to 43 ° C, and an ethanol fermentation titration temperature of 32 to 45 ° C, more preferably. Preferably it is 35-41 degreeC. Therefore, it is possible to grow even above 40 ℃ and ethanol fermentation is also possible features. In addition, the Kluyveromyces Marchanus mutant strain of the present invention can be grown at pH 3 to pH 10, preferably pH 4 to pH 6.

In addition, in one embodiment of the present invention, the 26S rDNA D1 / D2 and ITS sequences of the Kluyveromyces marchanus mutant strain may be represented by the nucleotide sequence of SEQ ID NO: 5, and are registered in the NCBI database based on the sequence information. As a result of sequencing and homology analysis of 26S rDNA D1 / D2 and ITS region of other yeast strains, it can be seen that the Kluyveromyces Marchanus mutant strain of the present invention is a novel strain.

Kluyveromyces Marchanus mutant strain according to one embodiment of the present invention (C. 16: CHY1612) may be a strain deposited on September 3, 2010 with the accession number KCTC11757BP to the Korea Biotechnology Research Institute Gene Bank.

Cluiveromyces Marchanus mutant strains of the present invention can be cultured according to methods known to those skilled in the art in a conventional strain culture medium, YPD (Yeast extract Peptone Dextrose, 10 g / L Yeast Extract (Difco) for ethanol production ), 20 g / L Peptone (Difco), 50 g / L Glocose (Junsei) medium.

Cluyveromyces Marchanus mutant strains of the present invention can maintain the growth and ethanol production ability at the biomass, preferably fibrous biomass saccharification temperature of 45 ~ 50 ℃ level, ethanol using biomass Simultaneous saccharification and fermentation are possible at the time of manufacture, so it can be easily used for ethanol production.

Meanwhile, the probiotic composition for ethanol production of the present invention is characterized by containing the Kluyveromyces Marchanus mutant strain or a culture thereof.

The composition is a probiotic composition that can be used to produce ethanol from biomass, wherein the composition contains the Kluyveromyces Marchanus mutant strain, or a culture thereof, and the culture typically comprises the Kluyveromyces Marchanus mutant strain. Refers to those cultured according to the strain culture method of. For the cultivation of the mutant strain, but not limited thereto, may be preferably cultured using YPD (10 g / L Yeast Extract (Difco), 20 g / L Peptone (Difco), 50 g / L Glocose (Junsei)) medium. Can be.

The probiotic composition for ethanol production is not limited to this, but preferably, the Kluyveromyces marchanus mutant strain may be included in an amount of 80% to 90% for powders and 0.5% to 1.5% for cultures based on the total weight of the composition. .

The biomass may be, but is not limited to, fibrous biomass or starch biomass, more preferably fibrous biomass, as specifically described below.

On the other hand, the ethanol production method of the present invention is characterized in that it comprises the step of glycosylation and fermentation by treating the Cluiberomyces Marchanus mutant strain and glycosylase on a biomass (Biomass) substrate.

The saccharification means a process of producing a sugar by hydrolyzing the biomass by an enzyme, and there is no limitation on the kind of the saccharification enzyme, but preferably carbohydrase, cellulase, amylase, hemicellulase and glucosidase. It may be at least one selected from the group consisting of. The saccharification may be performed for 5 to 72 hours by adding glycosylase to 0.06 to 25% by weight of the substrate, but not limited thereto.

The biomass may be, but is not limited to, fibrous biomass or starch biomass, and more preferably fibrous biomass.

The fibrous biomass may also be referred to as cellulose biomass or lignocellulosic biomass, but is not limited thereto, and may be at least one selected from the group consisting of wood, surplus agricultural products, and herbaceous crops. It may be at least one selected from the group consisting of corn stalk, rapeseed and palm fruit bunch (EFB).

The biomass used as the substrate of the present invention may be pretreated by one or more processes selected from the group consisting of high temperature, high pressure ammonia pretreatment, acid, base treatment, and hydrothermal treatment to assist in lignin removal and fiber decomposition. Pretreatment of the biomass may be additionally included in the ethanol production method of the present invention. The pretreatment method can be easily selected and applied by those skilled in the art according to methods known in the art.

In the ethanol production method of the present invention, the Kluyveromyces Marchanus mutant strain is not limited thereto, but 0.5 to 5 g yeast / L (2 x 10 ⁸ to 8 x 10 ⁹ viable cells / g yeast) or mutation to the substrate culture. The strain culture may be treated with 3 to 10% (v / v) with respect to the substrate culture and the treatment time is not limited thereto, but may preferably be 24 to 84 hours. If the treatment is less than the numerical range may not be effective fermentation by the Kluyveromyces Marchanus mutant strain, if the value exceeds the numerical range may be ineffective compared to the throughput or treatment time and may not be economical.

Ethanol production method of the present invention is an important feature that can be performed at the same time saccharification and fermentation process. This is because the Kluyveromyces Marchanus mutant strains of the present invention can be grown at high temperatures and have excellent ethanol production capability at high temperatures.

By simultaneously performing the saccharification and fermentation process, the sugar produced by saccharification is simultaneously consumed to decrease the substrate inhibition of saccharification enzyme, thereby increasing the saccharification rate. The reduction can lead to an increase in ethanol productivity.

Therefore, the ethanol manufacturing process of the present invention is not limited thereto, but preferably, the glycoberomyces marchanus mutant strain and glycosylase are administered together with a biomass substrate at 35 ° C. to 45 ° C. to simultaneously perform glycosylation and fermentation. It can be a process that can be done.

In particular, the ethanol production method of the present invention is not limited thereto, but preferably, when the simultaneous glycation and fermentation process is carried out as described above, by treating the Kluyveromyces Marchanus mutant strain and glycosylase on a biomass substrate, Firstly saccharified and fermented at a temperature higher than or equal to 50 ° C or lower, and then saccharified and fermented secondly at a temperature higher than or equal to 30 ° C and lower than 40 ° C, more preferably, firstly at 40 ° C to 45 ° C, then 32 It can be saccharified and fermented at a second temperature from ℃ to 38 ℃. The primary saccharification and fermentation is not limited thereto, but may be preferably performed for 5 to 24 hours, and the secondary saccharification and fermentation may be performed for 48 to 67 hours but not limited thereto.

The secondary saccharification fermentation may be performed through natural cooling after primary saccharification and fermentation, and specifically, after saccharification and fermentation, secondary saccharification and fermentation may be performed by gradually lowering the temperature by natural cooling without using additional cooling means. Therefore, the fermentation efficiency can be increased and the cooling cost can be effectively reduced.

The numerical range is considered in consideration of the saccharification efficiency and ethanol production efficiency, including the temperature conversion process as described above to perform the saccharification and fermentation process in two stages to increase the saccharification efficiency in the first stage and the ethanol fermentation efficiency in the second stage to the final saccharification Efficiency and ethanol production efficiency can be maximized .

In particular, co-glycosylation and fermentation using the temperature conversion process can perform the saccharification process with a saccharification rate similar to the saccharification rate of the saccharification optimum temperature, and at the same time the fermentation process at a similar efficiency to that of the optimum temperature of ethanol fermentation Can be done. This completes the saccharification and ethanol fermentation within the time required only for the saccharification of the conventional fibrous ethanol production, it is possible to produce ethanol very effectively.

 Ethanol production method of the present invention is not limited to this, but preferably may be carried out by a process selected from the group consisting of a batch process, a semi-continuous process and a continuous process.

Cluiberomyces Marchanus mutant strain of the present invention is very excellent in heat resistance and ethanol productivity, and can be easily used for ethanol production because it can be simultaneously glycated and fermented during ethanol production using biomass, preferably fibrous biomass. have.

1 is a graph comparing ethanol fermentation capacity with CHY1012 and CHY1703 used for protoplast fusion with Cluyveromyces Marchanus CHY1612 of the present invention. (A: 35 ° C, B: 41 ° C)
Figure 2 is a diagram showing the morphological observation results of the Cluy Veromyces Marchanus CHY1612 of the present invention.
Figure 3 is a diagram showing the position of the primers used in the sequencing of the Cluyveromyces Marchanus CHY1612 of the present invention.
4 is a diagram showing 26S rDNA D1 / D2 and ITS region of Cluyveromyces Marchanus CHY1612 of the present invention. (Black section: ITS1, 5.8SrDNA and ITS2, blue section: 26S rDNA D1 / D2)
5 is a graph measuring the amount of ethanol produced and the residual amount of glucose after the simultaneous saccharification and fermentation at 45 ° C. with the Cluyveromyces Marchanus CHY1612 of the present invention.
6 is a graph comparing the results obtained by applying the temperature conversion process (45-> 35 ° C) in the simultaneous glycosylation and fermentation using the Cluyveromyces Marchanus CHY1612 of the present invention and ethanol production and glucose residues otherwise. .

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Reference Example  1>

Selection of strains with high ethanol productivity and strains with high heat resistance

In order to effectively perform plasma fusion to obtain excellent traits of two strains, a screening marker is needed to select only excellent fusion strains after fusion of two strains. Therefore, the present inventors first gave the Saccharomyces cerevisia CHY1011, which has excellent ethanol productivity, and gave a triphenyltetrazolium chloride (TTC) respiratory deficiency marker with a different color reaction after the medium culture of Triphenyltetrazolium chloride (TTC). Seth Marchanus KCTC17631 was determined to be a nutritional marker capable of growing in minimal medium only by adding missing amino acids. Such a selection marker can be used as an index for selecting only fusion strains after fusion.

<1-1> Respiratory Deficiency Mutations Saccharomyces Cerevisiae CHY1012 Screening

Saccharomyces cerevisiae CHY1011 is an industrial strain with high ethanol productivity or does not have any selection markers, making it difficult to improve and select strains. Therefore, we tried to induce a respiratory deficiency mutation for CHY1011 and use it as a screening marker. The respiratory deprivation mutations do not decompose TTC during the intermediate culture of Triphenyltetrazolium chloride (TTC) on the yeast colonies of solid medium, so that the color reaction does not occur, thereby maintaining white color. Normal cells can be used as a selection marker after fusion because the color reaction occurs in red color in TTC medium culture.

Specifically, CHY1011 was incubated with logarithmic water to prepare a respiratory deprivation mutant, and then diluted to an appropriate concentration to treat the mutagen, Acriflavine 100 uM for 30 minutes. Treated cultures were plated in YPD solid medium (10 g / L Yeast Extract (Difco), 20 g / L Peptone (Difco), 20 g / L Glocose (Junsei), 20 g / L Agar (junsei)) After 48-72 hours of incubation, TTC solution containing low concentration agar was incubated in layers to separate only white colonies without color reaction.

Among the strains obtained by the above method, ethanol productivity was confirmed, and finally, strains similar to CHY1011 (KCTC11250BP) and ethanol productivity were selected for Saccharomyces cerevisiae and designated as CHY1012. Used.

<1-2> Nutritional requirements  Mutation Cluey Bromyces Marchanas CHY1703 Screening

Kluyveromyces marxianus ) KCTC17631 (Bio Resources Center, Biotechnology Research Institute Accession No .: KCTC17631) is a strain capable of growing and fermenting ethanol even at a high temperature of 40 ℃ or higher, and ethanol productivity does not reach industrial strains and does not have a selection marker. This is difficult. Therefore, in order to use the nutritional marker for KCTC17631, KCTC17631 was incubated with logarithmic water, diluted to an appropriate concentration, 1 mg / ml of mutant EMS (ethylmethansulphate) and NTG (N-methyl-N'-nitro-N-nitrosoguanidine). ) 5mg / ml were treated for 1 hour and 3 hours, respectively, and then plated in a solid medium and incubated at 35 ° C for 48-72 hours.

The resulting colonies were YPD solid medium (10 g / L Yeast Extract (Difco), 20 g / L Peptone (Difco), 20 g / L Glocose (Junsei), 20 g / L Agar (junsei)) and minimum medium (MM) , 6.7 g yeast nitrogen base w / o aa (Difco), 20 g / L glucose (Junsei), 20 g / L Agar (junsei), respectively, were cultured in nutrient requirement strains that did not grow in minimal medium. Strain obtained by the above method is ^{Iso - (Isoleucine), Val -} (Valine) mutant and that, Cluj Vero My process mareusya bonus KCTC17631 the ethanol productivity was significantly different. The strain was named CHY1703 and was used as a parent strain with the trophic marker of the present fusion.

< Example  1>

Preparation of Strains of the Invention by Protoplast Fusion

Saccharomyces cerevisiae of <Reference Example 1-1> CHY1012 is excellent in ethanol productivity, but difficult to grow at a temperature of 41 ℃ or more, there is a problem that ethanol productivity is significantly reduced at high temperatures as described above. On the other hand, Cluiberomyces Marchanus CHY1703 of <Reference Example 1-2> is capable of growing and fermenting ethanol even at a temperature of 40 ° C. or higher, but ethanol productivity is lower than that of the strain at a temperature of 40 ° C. or lower.

Therefore, in order to prepare a protoplast fusion strain having excellent characteristics of both strains, the two parent strains were cultured in logarithmic state at 32 ° C, and then reaction buffers (0.06M phosphate buffer, 0.2% β-mercaptoethanol, 0.6M KCl, 0.8, respectively). M sorbitol) was enzymatically treated at 33 ° C. for 30 minutes using Lyticase (Sigma).

The cell walls were removed by the respective enzyme treatments, and the two strains prepared in a protoplast state were mixed 1: 1, and a fusion buffer (30% PEG 6000, 50 mM CaCl ₂ , 10 mM Tris-HCl buffer, pH 7.5) was added. 60 minutes or 120 minutes at 35 ° C. was used to induce fusion.

The mixed solution prepared to induce the fusion was a minimal solid medium (MM, 6.7 g yeast nitrogen base w / o aa (Difco), 20 g / L glucose (Junsei), 20 to prevent the growth of unfused nutrient-forming mutant CHY1703 g / L Agar (junsei)), and similarly non-heat-resistant breathing mutant CHY1012 was incubated for 48-72 hours at 41 ℃ high temperature to prevent growth.

After obtaining the strain forming colonies by the cultivation, and subcultured in a minimal solid medium to confirm the growth, and to determine whether the lack of respiration by culturing TTC medium, only heat-resistant fusion strain recovered the function by plasma fusion The primary markers were selected using the selection markers for selection.

In order to ensure genetic stability, the selected strains were passaged several times, and afterwards, only normal strains capable of growing at 41 ° C. in a minimum solid medium and turning red as a result of TTC middle layer culture were secondarily selected.

  The secondary screened strains were cultured in YPD medium (3 g / L Yeast Extract (Difco), 5 g / L Peptone (Difco), 110 g / L Glocose (Junsei)) to perform an ethanol fermentation experiment, each strain By comparing the fermentation ability of these strains at high temperature conditions, 41 ℃ excellent ethanol productivity was selected as the final excellent strain was named as CHY1612.

The selected CHY1612 strain was deposited on September 3, 2010 with the accession number KCTC 11757BP to the Korea Biotechnology Research Institute Gene Bank.

< Example  2>

Heat resistance and ethanol productivity verification of the strain of the present invention

Ethanol productivity of CHY1612, the strain of the present invention prepared in <Example 1> is industrial YPD medium (2.5 g / L Yeast Extract (Difco), 2.5 g / L Peptone (Difco), 0.25 g / L MgSO ₄ ㆍ 7H General fermentation conditions (A, 35 ° C. in FIG. 1) and good strain selection conditions (FIG. 1 of FIG. 1) using ₂ 0 (Junsei), 1 g / LK ₂ HPO ₄ (Junsei), 140 g / L Glocose (Junsei) B, 41 ° C.) and the results are shown in FIG. 1.

As shown in FIG. 1, the strain of the present invention was expected to be useful as an ethanol fermentation strain showing not only the best fermentation ability at high temperature but also a final level of ethanol productivity similar to CHY1012 at normal fermentation temperature. In particular, it was expected to be suitable as a strain of co-glycosylated fermentation using the characteristics of high temperature fermentation.

< Example  3>

The CHY1612  Identification of the strain

The CHY1612 strain of the present invention, which can be grown at the high temperature and has an excellent ethanol fermentation yield, was identified based on morphological observation results, biochemical properties, and DNA sequencing of 26S rDNA D1 / D2 and ITS. It was.

<3-1> Morphological Characteristics

First, each strain was incubated for 18 hours in YPD medium (3 g / L Yeast Extract (Difco), 5 g / L Peptone (Difco), 20 g / L Glocose (Junsei)) for morphological observation. Observation at a magnification of 600 times.

As a result of the morphological observation, the strain of the present invention was elliptical or rod-shaped as shown in FIG. 2, shorter than CHY1012 of <Reference Example 1>, but similar to CHY1703 of <Reference Example 2>, but slightly larger in size than CHY1703. It was also confirmed that breeding was possible by the germination method, the anaerobic anaerobic, the growth temperature was 28 to 48 ° C, and the ethanol fermentation titration temperature was 32 to 45 ° C.

<3-2> Biochemical Characteristics

Biochemical characteristics of the strain of the present invention was used API20 C AUX, a yeast magnetization confirmation kit of the French company Biomerieux, and the results of the magnetization was expressed as-, +, ++, +++ . The biochemical properties of the strain are shown in Table 1 compared with CHY1012 of <Reference Example 1> and CHY1703 of <Reference Example 2>.

CHY1012 CHY1703 CHY1612 Glucose +++ +++ +++ Glycerol - +++ +++ 2-Keto-D-Gluconate - - - L-Arabinose - - - D-Xylose - - - Adonitol - + ++ Xylitol - ++ +++ D-Galactose +++ +++ +++ Inositol - - - D-Sorbitol - +++ +++ a-Methyl-D-Glucoside - - - N-Acetyl-D-Glucosamine - - - D-Cellobiose - - - D-Lactose - - - D-Maltose +++ - ++ D-Saccharose (Sucrose) +++ +++ +++ D-Trehalose - - - D-Melezitose - - - D-raffinose - + +++

-API 20C AUX + Iso (Isoleucine), Val (Valine) added

<3-3> sequencing

The analysis of the nucleotide sequence for identifying the yeast strain used molecular biological classification through 26S rDNA D1 / D2 sequencing and molecular biological classification through ITS (internal transcribed spacer) sequencing. DNA sequencing was performed in the following manner.

Specifically, the CHY1612 strain of the present invention was cultured, and the cells were recovered by centrifugation of the culture solution, followed by genomic DNA extraction from the cells. For the extracted genomic DNA, polymerase chain reaction (PCR) was performed using an ITS5 primer (5`-GAAGTAAAAGTCGTAACAAGG-3`) of SEQ ID NO: 1 and an NL4 primer (5`-GGTCCGTGTTTCAAGACGG-3`) of SEQ ID NO: 3). It was performed according to Table 2 and Table 3 below.

Usage (ul) Mold (10ng / ul) One Primer (F, R) 10 pmole/ul 2 each 10X Taq pol buffer (Takara, Japan) 5 dNTP (Takara, Japan) 4 Taq polymerase (Takara, Japan) 0.3 D.W. 35.7 Sum 20

Temperature Cycle number Predenaturation 95 ℃ / 5 min Amplification 95 ℃ / 30 sec 30 54 ℃ / 30 sec 72 ℃ / 90 sec Final Extension 72 ℃ / 7 min

The polymerase chain reaction amplified DNA of about 1300bp including 26S rDNA D1 / D2 and the ITS region.

The PCR product was cloned using T-vector (New England biolabs) and sequenced through SolGent Co. (Korea), a sequencing sponsor, to obtain 26S rDNA D1 / D2 and ITS sequences. For more accurate sequencing, sequencing includes NL1 primer of SEQ ID NO: 4 (5`-GCATATCAATAAGCGGAGGAAAAG-3`) and two ITS4 primers of SEQ ID NO: 2 (5`-TCCTCCGCTTATTGATATGC-3`) of about 1300bp of PCR product. ) Was further provided for analysis.

Figure 3 shows the sequence and its position of the primer for PCR and sequencing.

As a result of the analysis, the nucleotide sequences shown in SEQ ID NO: 5 and FIG. In particular, in FIG. 4, 26S rDNA D1 / D2 and ITS regions are separately indicated.

Homology and homology analysis of 26S rDNA D1 / D2 and ITS regions of other yeast strains registered in the NCBI database using the nucleotide sequences, respectively, showed that species and genus were cloned into Cluiberomyosis marshanas. Since it has a unique 26S rDNA D1 / D2 and ITS sequence which is different from the previously reported nucleotide sequence of other Kluyveromyces Marchanus, it was designated as Kluiberomyces Marchanus CHY 1612.

Table 4 shows the results of nucleotide search of the 26S rDNA D1 / D2 and ITS region of the Kluyveromyces Marchanus CHY 1612 using the NCBI database.

Strain 26s rDNA D1 / D2, ITS region NCBI registration number Kluyveromyces marxianus Identities = 923/926 (99%), Gaps = 2/926 (0%) AF543841 Kluyveromyces marxianus Identities = 907/910 (99%), Gaps = 1/910 (0%) AB480229 Kluyveromyces marxianus isolate VA 116042-03 Identities = 719/721 (99%), Gaps = 0/721 (0%) AY939806 Kluyveromyces marxianus isolate AS2.1549 Identities = 717/721 (99%), Gaps = 0/721 (0%) EU019227 Kluyveromyces marxianus isolate F4-3 Identities = 713/724 (98%), Gaps = 3/724 (0%) EU019224 Kluyveromyces marxianu s strain ATCC 4135 Identities = 672/676 (99%), Gaps = 0/676 (0%) DQ249191 Kluyveromyces lactis isolate UOA / HCPF BUL74 Identities = 716/747 (95%), Gaps = 6/747 (0%) GQ376079 Kluyveromyces dobzhanskii Identities = 612/635 (96%), Gaps = 5/635 (0%) AJ401722

< Example  4>

The CHY1612 Using Fibrous system  ethanol Simultaneous saccharification  Fermentation

The CHY1612 yeast of the present invention is useful for fibrous ethanol co-glycosylation fermentation because it can grow and ethanol fermentation even at high temperatures. The yeast can be grown and fermented even under high temperature fibrous glycosylation conditions of 45 ° C., so that the simultaneous saccharification fermentation process can be applied. By increasing the saccharification rate, and ethanol fermentation proceeds simultaneously with the saccharification can increase the ethanol productivity by reducing the total saccharification, fermentation time.

<4-1> Biomass  Pretreatment

Fibrous biomass is used barley straw, but the present invention is not limited to the barley straw to present a preferred embodiment.

Barley straw provided by RDA consists of 39.0% cellulose, 19.8% hemicelluloses, 23.0% lignin and 1.7% ash. 10% (w / v) of barley straw pulverized to 1 mm or less using a crusher and a grinder was pretreated at 150 ° C. for 90 minutes using a 15% (v / v) ammonia solution. The composition of dried barley straw after pretreatment is shown in Table 5 below.

Configuration (%) After pretreatment Cellulose (glucose) 57.1 Hemicellulose (Xylose) 19.2 Lignin 14.7 moisture 5.5 Ash 2.3

<4-2> pretreatment Biomass  By temperature Glycation  compare

In general, fibrous glycosylase has an optimum activity at a high temperature of 45-50 ℃ and low activity at 32-35 ℃ the general ethanol fermentation temperature. The barley straw pretreated in <Example 4-1> was subjected to enzyme saccharification experiment according to each temperature to confirm the difference.

80 g of the pretreated barley straw was mixed with 200 mL of 0.1 M sodium citrate buffer (pH 4.8) and 211 mL of distilled water, followed by complex cellulose (Novozyme, NS50013) 30 FPU (filter paper unit) / g and β-glucosidase for saccharification. (Novozyme, NS50010) 30 CBU (cellobiase unit) / g was added and glycosylated at 150 rpm for 72 hours at each temperature condition in the table below in shake incubator. The saccharification solution of which saccharification was completed was analyzed for sugar using HPLC, and the saccharification rate was calculated as follows.

Temperature condition Per conversion (g / L) Glycation rate (%) Relative Glucose Rate (%) 50 ℃ 79.8 90 100 45 ° C 78.4 88.4 98.2 45-> 35 ℃ 72.9 82.2 91.3 35 ℃ 58.8 66.2 73.6

As shown in Table 6, the lower the temperature was, the lower the glycosylation activity was, the lower the glycosylation rate was confirmed. Thus, in order to obtain the same saccharification rate even at low fermentation temperature in the saccharification of fibrous biomass, it is necessary to add more expensive saccharification enzymes. Yeast is essential.

<4-3> of the present invention CHY1612 Simultaneous by Glycation  And fermentation

For the reason of <Example 4-2>, the use of a heat-resistant fusion yeast, Kluyveromyces Marchanus CHY1612 in the fibrous co-glycosylation fermentation enables co-glycosylation and fermentation at the 43-45 ° C level. Simultaneous glycosylation and fermentation do not require a separate saccharification process, as well as a reduction in the initial equipment cost, and by directly consuming the sugars produced during saccharification, thereby reducing the substrate inhibition of glycase and increasing the saccharification efficiency, and total ethanol fermentation time. Reduction results in an increase in ethanol productivity.

In order to confirm the effect as described above, co-glycosylation and fermentation of the fibrous biomass using the Cluiberomyces Marchanus CHY1612 of the present invention at 45 ℃, specifically, as shown in the <Example 4-2> 80 g of the pretreated barley straw of Example 4-1 was sterilized by mixing 200 mL of 0.1 M sodium citrate buffer (pH 4.8) and 211 mL of distilled water, and 30 FPU / g of complex cellulose for glycosylation (Novozyme, NS50013). 30 CBU / g of β-glucosidase (Novozyme, NS50010) was added and 35 using YPD medium (3 g / L Yeast Extract (Difco), 5 g / L Peptone (Difco), 50 g / L Glocose (Junsei)). 5% (v / v) of the CHY1612 culture pre-incubated for 18-24 hours at ℃ was incubated at 45 ℃ co-glycosylation to measure the production of ethanol and glucose and the results are shown in FIG.

As shown in FIG. 5, it can be seen that simultaneous glycosylation and fermentation are possible based on the fibrous biomass when using Cluiberomyces Marchanus CHY1612 even at a high temperature of 45 ° C.

However, while the glycosylation rate of glycosylation enzyme was optimal (90% level), ethanol productivity according to Equation 2 below did not reach an optimal level (efficiency-90% level).

<4-4> of the present invention CHY1612 Simultaneous by Glycation  And fermentation process improvement

In the fibrous ethanol co-glycosylation fermentation, the glycosylation rate is excellent at the optimum saccharification temperature (43-50 ℃), but the ethanol fermentation efficiency is low, and the ethanol fermentation efficiency is 32-38 ℃ at the optimum fermentation temperature of general yeast. In order to find the optimal conditions for simultaneous saccharification and fermentation, in the present invention, the saccharification rate is excellent, but in order to find the optimal conditions for the simultaneous saccharification and fermentation, the saccharification-oriented simultaneous saccharification and fermentation are performed at the optimum temperature of saccharification and then naturally cooled to fermentation-simultaneous saccharification at the optimum fermentation temperature. And fermentation to improve both glycation and fermentation efficiency to an optimal level.

Specifically, simultaneous glycosylation and fermentation at 35 ° C, simultaneous glycosylation and fermentation at 45 ° C, and naturally cooling after 24 hours at 45 ° C, which is the temperature conversion process of the present invention, simultaneously glycosylation and fermentation at 35 ° C were performed. 3> and the results thereof are compared and described in FIG.

As shown in FIG. 6, it can be seen that the temperature conversion process combining the optimal saccharification and the optimum fermentation conditions in the simultaneous saccharification and fermentation using the Cluiberomyces Marchanus CHY1612 of the present invention is the best.

In addition, the glycosylation rate and ethanol efficiency were calculated using the sugar analysis and ethanol analysis results and Equations 1 and 2 according to the respective conditions, and are shown in Table 7 below.

Coclinization
Fermentation time (h) Glucose (g / L) Xylose (g / L) Ethanol (g / L) Per conversion
(g / L) Glycation rate
(%) Efficiency
(%) 45 ° C 71 18 21.3 22.2 75.3 90.8 57.7 45-35 ℃ 71 0 19.9 34.3 74.0 89.3 90.6 35 ℃ 71 0 18.6 31.0 67.3 81.2 90

As shown in Table 7, the simultaneous saccharification fermentation using the temperature conversion process (45-> 35 ℃) showed a saccharification rate similar to the saccharification rate (90%) of the optimum temperature of saccharification, ethanol efficiency and fermentation efficiency of the optimum temperature The most ethanol was produced, which was similar to 90.6%. In the present invention, when the input amount of biomass is increased, the ethanol production may also be increased, and the 90.6% efficiency of using the invention strain is an industrially useful result. In addition, it can be seen that the ethanol productivity is also very good because the ethanol fermentation is completed within 72 hours for the saccharification of fibrous ethanol production.

Korea Biotechnology Research Institute KCTC11757 20100903

<110> CHANGHAE ETHANOL CO., LTD. <120> Kluyveromyces marxianus having superior heat resistance and          ethanol productivity <130> DPP20104444KR <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ITS5 primer <400> 1 gaagtaaaag tcgtaacaag g 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> 223 ITS4 primer <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> 223 NL4 primer <400> 3 ggtccgtgtt tcaagacgg 19 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NL1 primer <400> 4 gcatatcaat aagcggagga aaag 24 <210> 5 <211> 1313 <212> DNA <213> Artificial Sequence <220> <223> 26S rDNA D1 / D2 and ITS of CHY1612 <400> 5 ggaagtaaaa gtcgtaacaa ggtttccgta ggtgaacctg cggaaggatc attaaagatt 60 atgaatgaat agattactgg gggaatcgtc tgaacaaggc ctgcgcttaa ttgcgcggcc 120 agttcttgat tctctgctat cagttttcta tttctcatcc taaacacaat ggagtttttt 180 ctctatgaac tacttccctg gagagctcgt ctctccagtg gacataaaca caaacaatat 240 tttgtattat gaaaaactat tatactataa aatttaatat tcaaaacttt caacaacgga 300 tctcttggtt ctcgcatcga tgaagaacgc agcgaattgc gatatgtatt gtgaattgca 360 gattttcgtg aatcatcaaa tctttgaacg cacattgcgc cctctggtat tccagggggc 420 atgcctgttt gagcgtcatt tctctctcaa acctttgggt ttggtagtga gtgatactcg 480 tctcgggtta acttgaaagt ggctagccgt tgccatctgc gtgagcaggg ctgcgtgtca 540 agtctatgga ctcgactctt gcacatctac gtcttaggtt tgcgccaatt cgtggtaagc 600 ttgggtcata gagactcata ggtgttataa agactcgctg gtgtttgtct ccttgaggca 660 tacggcttta accaaaactc tcaaagtttg acctcaaatc aggtaggagt acccgctgaa 720 cttaagcata tcaataagcg gaggaaaaga aaccaaccgg gattgcctta gtaacggcga 780 gtgaagcggc aaaagctcaa atttgaaatc tggcgtcttc gacgtccgag ttgtaatttg 840 aagaaggcga ctttgtagct ggtccttgtc tatgttcctt ggaacaggac gtcatagagg 900 gtgagaatcc cgtgtggcga ggatcccagt tatttgtaaa gtgctttcga cgagtcgagt 960 tgtttgggaa tgcagctcta agtgggtggt aaattccatc taaagctaaa tattggcgag 1020 agaccgatag cgaacaagta cagtgatgga aagatgaaaa gaactttgaa aagagagtga 1080 aaaagtacgt gaaattgttg aaagggaagg gcatttgatc agacatggcg tttgcttcgg 1140 ctttcgctgg gccagcatca gttttagcgg ttggataaat cctcgggaat gtggctctgc 1200 ttcggtagag tgttatagcc cgtgggaata cagccagctg ggactgagga ttgcgacttt 1260 tgtcaaggat gctggcgtaa tggttaaatg ccgcccgtct tgaaacacgg acc 1313

Claims (10)

Prepared by protoplast fusion of Saccharomyces cerevisiae , Accession No. KCTC11250BP, and Kluiberomyces Marchanus, Accession No. KCTC17631,
Having heat resistance and ethanol productivity,
Kluyveromyces marxianus variant strain (Accession Number: KCTC11757BP). According to claim 1, wherein the Kluyveromyces Marchanus mutant strain has a growth temperature of 28 to 48 ℃, the appropriate temperature of ethanol fermentation of the Kluyveromyces Marchanus mutant strain. The method of claim 1, wherein the 26S rDNA D1 / D2 and ITS sequence of the Kluyveromyces Marchanus mutant strain is represented by the nucleotide sequence of SEQ ID NO: 5 Kluyveromyces Marchanus mutant strain. delete A method for producing ethanol comprising the step of glycosylation and fermentation by treating the Kluyveromyces marchanus mutant strain and glycosylase of any one of claims 1 to 3 on a biomass substrate. The method of claim 5, wherein the biomass is a fibrous biomass or a starch biomass. The method of claim 5, wherein the saccharification and fermentation is carried out simultaneously. The method of claim 5, wherein the Kluyveromyces Marchanus mutant strain and glycosylation enzyme is treated with a biomass substrate to be first glycosylated and fermented at 40 ° C. or more and 50 ° C. or less, and then, at 30 ° C. or more and less than 40 ° C. Ethanol manufacturing method that is saccharified and fermented with tea. The method of claim 8, wherein the primary saccharification and fermentation is performed for 5 to 24 hours, and the secondary saccharification and fermentation is performed for 48 to 67 hours. The method of claim 5, wherein the ethanol production method is an ethanol production method is carried out by a process selected from the group consisting of a batch process, a semi-continuous process and a continuous process.

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