KR20210039115A - Transformed Saccharomyces cerevisiae having improved productivity of bio-ethanol and use thereof - Google Patents

Abstract

The present invention provides a recombinant vector for building a single enzyme saccharification and fermentation system, wherein the recombination vector includes an AGAH71 gene sequence represented by SEQ ID NO: 1, an AGAG1 gene sequence represented by SEQ ID NO: 2, and an NABH558 gene represented by SEQ ID NO: 3; a transformed Saccharomyces cerevisiae strain having an improved bioethanol production ability and including the recombinant vector; and a method for producing bioethanol through a single enzyme saccharification and fermentation system using the transformed Saccharomyces cerevisiae strain. When ethanol is produced from agarose through a recombinant Saccharomyces cerevisiae strain of the present invention, biomasses (substrates) do not have to be preprocessed. Also, by unifying a saccharification and fermentation process, the unity and simplification of steps are ensured. Accordingly, the recombinant Saccharomyces cerevisiae strain can be applied to various fields of producing ethanol from biomasses.

Description

Transformed Saccharomyces cerevisiae having improved productivity of bio-ethanol and use thereof

The present invention relates to a transformed Saccharomyces cerevisiae strain with improved bioethanol production capability by enabling simultaneous saccharification and fermentation of agar and a method for producing agar bioethanol using the same.

Fossil fuels, which currently account for about 80% of the world's energy demand, have limited reserves of fossil fuels, which pose a problem of energy security, and climate such as global warming due to emissions of greenhouse gases such as carbon dioxide generated when fossil fuels are burned. By causing various problems including environmental problems such as the threat of change, discussions are being actively conducted around the world to reduce the use of fossil fuels, and the demand for alternative energy is increasing.

Accordingly, there is an urgent need to develop an alternative fuel to be used in place of fossil fuels, and bio-energy is referred to as such an alternative fuel.

The bioenergy refers to an energy source made from biomass such as animals, plants, and microorganisms, and is also referred to as biomass energy. In the method of using the biomass as an energy source, in the past, a method such as direct combustion was mainly used, but today, a biochemical conversion process using enzymes or microorganisms to convert organic matter or organic waste; Photobiological transformation process using photosynthetic microorganisms; And a method of producing a liquid fuel such as ethanol, methanol, biodiesel, Fischer-Tropsch diesel, and/or gaseous fuel such as hydrogen and methane through a thermochemical conversion process using thermal energy and a chemical catalyst. Research is in progress. In recent years, the energy source obtained from biomass, that is, the biomass energy source, can be stored and regenerated, and the necessity of the research is increasing as a new energy source in that it does not cause environmental problems.

In accordance with the necessity of such research, studies to replace fossil fuels have recently been conducted worldwide, and various methods have been studied for efficient hydrolysis of the biomass, which is a biopolymer material, and conversion to a fuel material.

Existing research on biomass energy sources mainly focused on the use of plant oils or the conversion of woody biomass to bioenergy, but this has the disadvantage of being difficult to apply on a commercial scale, as well as the problem of world food shortages. In connection with the like, a problem was pointed out that it is difficult to use plants as an alternative fuel.

Accordingly, there is an urgent need to develop alternative fuels that can replace fossil fuels other than using land plants or woody biomass related to food problems. Interest in research for producing bio-energy such as hydrogen or methane or bio-energy such as bioethanol from marine plants using a bio-energy source using existing marine plants, and specifically microorganisms is increasing.

In this regard, one of the fields of interest in recent years is research on the production of bioethanol using ethanol fermentation, and bioethanol is not only used for fuels and beverages, but also for many uses such as pharmaceuticals, cosmetics, and industrial raw materials. It is used, and its production is increasing year by year. For this reason, there is a need to develop a technology for producing ethanol that is cheaper and more productive. In order to manufacture inexpensive and highly productive ethanol, many studies are being conducted in the direction of making the fermentation conditions as good as possible, and studies to reduce cost and accelerate the process are being attempted (Kadar et al., 2004; Won et al. ., 2012).

Meanwhile, programs to produce biofuels from grain have recently been criticized for being less environmentally friendly in terms of rising food prices and energy and land use invested in the grain manufacturing process.

Algae are known to have several advantages over other biomass sources such as corn, which are more environmentally friendly. Algae can produce more fuel per area of land cultivated compared to corn or the like, or can grow on land that is not suitable for conventional agriculture due to salinity. According to Michael Briggs of the University of New Hampshire, the North American desert could provide more than enough land for algae ponds to meet all the demands for US transportation fuel. Recently, interest in alternative energy is starting to increase again due to high oil prices and increasing energy demands in emerging countries such as China and India.

In addition, since Korea is made up of sea on three sides, seaweed can be easily obtained. Among seaweeds, especially red algae, there is a large amount of agar (agar), a natural polysaccharide, which can be widely used in food, pharmaceutical, cosmetics, and other industries. The annual output of seaweed is 914,715 M/T (Agar: 3,716 M/T) for 10 years, which can be obtained relatively easily, but the utilization rate is low. (fs.fips.go.kr, 2010, Agricultural and Fishery Production Statistics Division, Social Statistics Bureau, National Statistical Office)

Algae grows rapidly, and green algae in the upper layer, brown algae in the middle layer, and red algae in the lower layer mainly grow according to the depth of the sea, so through complex culture, it has a lower land use rate and higher productivity than terrestrial biomass. In addition, seaweeds do not require a de-lignin (delignification) process and are easy to decompose because there is no hardly degradable lignin in lignocellulosic biomass.

However, conventionally, in order to produce bioethanol from agar, biomass pretreatment has to be carried out, and for this purpose, the yeast strain is subjected to saccharification with fermented sugar so that biomass (polysaccharide) can be fermented using acid hydrolysis and enzymes. It had to go through the fermentation process used.

Accordingly, the present inventors provide a genetically recombined yeast strain capable of simultaneous saccharification and fermentation in order to improve agar bioethanol production capacity, and provide a bioethanol production method using the same.

An object of the present invention provides a recombinant vector comprising a NABH558 gene represented by AGAG1 gene sequence and SEQ ID NO: 3 shown in AGAH71 gene sequence, SEQ ID NO: 2 as shown in SEQ ID NO: 1, for building a single enzyme saccharification and fermentation systems do.

In addition, another object of the present invention is to provide a transformed Saccharomyces cerevisiae strain with improved bioethanol production capability comprising the recombinant vector.

In addition, another object of the present invention is to provide a method for producing bioethanol through a single enzyme saccharification and fermentation system using the Saccharomyces cerevisiae strain of the present invention.

The present invention for achieving the above object includes a NABH558 gene represented by AGAG1 gene sequence and SEQ ID NO: 3 shown in AGAH71 gene sequence, SEQ ID NO: 2 as shown in SEQ ID NO: 1, for building a single enzyme saccharification and fermentation systems It provides a recombinant vector.

In addition, the present invention provides a transformed Saccharomyces cerevisiae strain with improved bioethanol production capability including the recombinant vector.

In addition, the present invention provides a method for producing bioethanol through a single enzyme saccharification and fermentation system using the Saccharomyces cerevisiae strain of the present invention.

The present invention provides a recombinant vector comprising a NABH558 gene represented by AGAG1 gene sequence and SEQ ID NO: 3 shown in AGAH71 gene sequence, SEQ ID NO: 2 as shown in SEQ ID NO: 1, for building a single enzyme saccharification and fermentation system. In addition, the present invention provides a transformed Saccharomyces cerevisiae strain with improved bioethanol production capability including the recombinant vector, and when producing ethanol from agarose through the strain , Biomass (substrate) pretreatment is not required, and by unifying the saccharification and fermentation process, it can be applied to various fields of producing ethanol from biomass by presenting the possibility of uniformity and simplification of the process.

1A is a diagram illustrating a process in which agarose is decomposed by agarase.
1B is a diagram showing the structure of gene expression cassettes (EC) of a recombinant plasmid for expressing an agarase gene.
2 is a diagram comparing agarase activity of host strains including pGMFα-AgaH71, pGMFα-AgaG1 and pGMFα-NABH plasmids.
3 is a diagram showing the results of analyzing the degree of transcription of the agarase gene through RT-PCR.
4 is a diagram showing a hydrolysis product decomposed from agarose through a TLC chromatogram.
lane 1: galactose
lane 2: AHG (3,6-anhydro-L-galactose), NA2 (neoagarobiose), NA4 (neoagarotetraose)
lane 3: Agarose + AgaH71
lane 4: Agarose + AgaG1
lane 5: Agarose + HGN (AgaH71-AgaG1-NABH)
5 is a diagram showing the result of comparing the ethanol production ability from agarose using the 2805/pGMFα-HGN strain. (Close circles and open circles represent culture solutions (CS) and culture cells (CC) of the 2805/pGMFα-HGN strain, respectively)

The present invention provides a recombinant vector comprising a NABH558 gene represented by AGAG1 gene sequence and SEQ ID NO: 3 shown in AGAH71 gene sequence, SEQ ID NO: 2 as shown in SEQ ID NO: 1, for building a single enzyme saccharification and fermentation system.

As an embodiment of the present invention, the present invention is a recombinant vector pGMFα-AgaH71 comprising the AGAH71 gene derived from Pseudoalteromonas hodoensis H7 represented by SEQ ID NO: 1, Alteromomas sp. Recombinant vector pGMFα-AgaG1 comprising AGAG1 gene derived from GNUM-1, recombinant vector pGMFα-NABH comprising NABH558 gene derived from Gayadomonas joobiniege G7 represented by SEQ ID NO: 3 and genes represented by SEQ ID NOs 1 to 3 It provides a recombinant vector pGMFα-HGN containing all of.

In the present invention, the term “vector” refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host, in particular recombinant yeast. The vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are currently the most commonly used form of vectors, in the specification of the present invention “plasmid” and “vector” are sometimes used interchangeably. However, the present invention includes other forms of vectors that have functions equivalent to those known or become known in the art.

In the present invention, the "recombinant vector" includes any cloning or expression vector containing the cloned gene(s) of interest.

In the present invention, "expression" refers to a process by which a polypeptide is produced from a structural gene. The process includes transcription of the gene into mRNA, and translation of such mRNA into the polypeptide(s).

In addition, the present invention provides a transformant transformed with the expression vector of the present invention.

The present invention provides a transformed Saccharomyces cerevisiae strain with improved agar bioethanol production capacity including AGAH71, AGAG1 and NABH558 genes.

The microorganisms of the present invention are microscopic organisms with a size of 0.1 mm or less that cannot be seen with the naked eye, and may include microorganisms capable of producing ethanol from biomass without limitation, but may be preferably yeast ( Saccharomyces cerevisiae ). Yeast has the advantage of being easy to separate cells and ensuring human stability compared to other microorganisms.

In the present invention, the "transformed Saccharomyces cerevisiae" strain is two kinds of beta-agarose and one kind of neo-agarobiose hydrolase (NABH) to produce ethanol from agarose. ), and the enzyme includes, without limitation, enzymes known in the art, preferably two kinds of beta- agalases are AGAH71 derived from Pseudoalteromonas hodoensis H7 and Alteromomas sp. It is derived from AGAG1 GNUM-1, Bios hydrolase agar in neo one species is derived from NABH558 Gayadomonas joobiniege G7.

In the present invention, the yeast may include, without limitation, yeast strains known to those skilled in the art, preferably, Saccharomyces cerevisiae 2805 strain.

In the present invention, "transformation" means that DNA is introduced into a host so that DNA can be replicated as an extrachromosomal factor or by completion of chromosomal integration.

In one embodiment, the present invention provides two types of beta-Niagara gene encoding a dehydratase AGAH71 and AGAG1, said vector prepared by introducing the gene NABH558 to encode the BIOS hydrolase as neo agar one kind of To provide a transformed yeast strain. The enzyme gene includes, without limitation, enzymes known in the art, preferably two kinds of beta- agalases are AGAH71 derived from Pseudoalteromonas hodoensis H7 and Alteromomas sp. It is derived from AGAG1 GNUM-1, Bios hydrolase agar in neo one species is derived from NABH558 Gayadomonas joobiniege G7.

Recombinant yeast having the recombinant vector may use a variety of gene delivery methods including, but not limited to, transformation, transduction, conjugal mating, or electroporation, and these methods are well suited in the field of molecular biology. It is known.

In addition, the present invention provides a method for producing bioethanol through a single enzyme saccharification and fermentation system using the Saccharomyces cerevisiae strain of the present invention.

More specifically, the method for producing bioethanol according to the present invention is

(a) constructing a pGMFα-HGN plasmid gene expression cassette containing the AGAH71, AGAG1 and NABH558 genes;

(b) transforming the plasmid containing the gene expression cassette constructed in step (a) into the Saccharomyces cerevisiae strain using a high-efficiency transformation method; And

(c) culturing the Saccharomyces cerevisiae strain transformed in step (b) in a medium containing agarose; It may include.

The present invention also provides a method for producing ethanol comprising the step of culturing the transformed Saccharomyces cerevisiae strain described above.

The medium and culture conditions can be appropriately selected and used according to the host cell. When culturing for ethanol production, conditions such as temperature, pH of the medium, and culture time can be appropriately adjusted to suit the growth of cells and mass production of proteins. Culture conditions are preferably the cells are cultured at 30°C for 48 hours to express each gene first, and then transferred to a medium containing agarose and cultured at 40°C for 48 hours to add agarose to fermentable sugars. Induce decomposition, and incubate for 72 hours at 30° C. to induce ethanol fermentation.

Recombinant yeast according to the present invention does not require pretreatment of biomass (substrate), and by unifying the saccharification and fermentation process to present the possibility of uniformity and simplification of the process, it can be applied to various fields of producing ethanol from biomass.

Terms that are not otherwise defined in the present specification have the meanings commonly used in the technical field to which the present invention pertains.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1. Construction of expression plasmid

As starting strains for the expression and production of agarase gene, Saccharomyces cerevisiae BY4742, 2805 and FY834 strains were sold in Yeast Genetic Resource Center (YGRC) and used for experiments, and for the production of agarase, AGAH71, AGAG1, NABH558 Genes were used, and the genes were Pseudoalteromonas hodoensis H7, Alteromomas sp. GNUM-1, derived from Gayadomonas joobiniege G7. As shown in Fig. 1A, agarose is neo-agarohexose, neo-agarotetraose, neo-agarobiose, di-galactose and 3,6-anhydrous-L-galactose by each agarase. It is decomposed sequentially into (3,6-anhydro-L-galactose). Therefore, in order to effectively express the AGAH71, AGAG1, and NABH558 genes in the Saccharomyces cerevisiae strain, pGMFα-AgaH71, pGMFα-AgaG1 and pGMFα-NABH plasmids were constructed, respectively. The agarase gene is provided by the pGEX-AgaH71, pGEX-AgaG1 and pET-NABH558 plasmids, respectively, and pGMFα-XYLP plasmid was used as a vector for subcloning each agarase gene. pGMFα-XYLP comprises a galactose induction promoter GAL10 promoter, and a mating factor signal sequence (mating factor α signal sequence, MFαs.s) for efficient secretion production of the agarase, a URA3 gene as a selection marker. The strains used and the plasmids (vectors) used for the construction of the recombinant plasmid are shown in Table 1 below, and Escherichia coli DH5α strain was used to amplify each plasmid and sub-cloning the agarase gene.

[Table 1]

Each gene was linked under the control of the inducible yeast GAL10 promoter and fused to the Saccharomyces cerevisiae mating factor signal sequence (MFαs.s). To construct each plasmid, it was cloned using In-Fusion HD cloning kit (Clontech Laboratories, Inc. Takara).

First, in order to construct the pGMFα-AgaH71 plasmid, pGMFα-AgaH71 plasmid containing a component that regulates the inducible expression of the AGAH71 gene by inserting a PCR product containing the 824 bp AGAH71 gene into the SalI-XbaI site of the pGMFα-XYLP plasmid Was constructed (Fig. 1B). The PCR product containing the AGAH71 gene with the SalI-XbaI restriction site uses the pGEX-AgaH71 plasmid as a template and AgaH71-F and AgaH71-R as a forward primer and a reverse primer, respectively. Produced. The oligonucleotides used are shown in Table 2 below.

[Table 2]

The PCR products containing the AGAG1 gene (906 bp) and the NABH558 gene (1080 bp), respectively, and having a SalI-XbaI restriction site were pGEX-AgaG1 and pET-NABH558 plasmids as templates, and AgaH71-F/AgaH71-R and NABH- F/NABH-R was each amplified using a primer set. The resulting AGAG1 and NABH558 gene products were ligated to the XbaI-SalI site of the pGMFα-XYLP plasmid, respectively, to prepare pGMFα-AgaG1 and pGMFα-NABH plasmids.

In addition , in order to construct a pGMFα-HGN plasmid for simultaneous expression of AGAH71, AGAG1 and NABH558 genes, the pGMFα-NABH plasmid was used as a starting plasmid.

AGAG1 gene expression cassette ( GAL10p-MFαs.s-AGAG1-GAL7t ) was amplified using the pGMFα-AgaG1 plasmid as a template, In-Gal10p-F/In-Gal7t-R primer set, and the pGMFα-NABH plasmid By cloning into the SacI restriction enzyme site, pGMFα-GN plasmid (9.3 kb) was constructed. In addition, the AGAH71 gene expression cassette ( GAL10p-MFαs.s-AGAH71-GAL7t ) was amplified using the same primer set as a template for the pGMFα-AgaH71 plasmid, and the amplified product was cloned into the pGMFα-GN plasmid. A pGMFα-HGN plasmid (11.2 kb) containing all of the AGAH71, AGAG1 and NABH558 gene expression cassettes (EC) was constructed in the same manner as described above (Fig. 1B).

The sequence of genes in the pGMFα-HGN plasmid was determined by the agar digestion sequence of each enzyme shown in Fig. 1A.

The expression plasmid produced by the above process has an MFα signal sequence (MFα = Saccharomyces cerevisiae α-Mating factor) for product secretion by the recombinant agarase gene, and the signal sequence is bound to the agarase gene. It was made to be (Fig. 1B). The predicted intersection of each signal sequence and the agarase gene was confirmed using DNA sequencing of the pGMFα-AgaH71, pGMFα-AgaG1 and pGMFα-NABH plasmids.

The original target sequence for MFα propeptide removal is KREAEAEA, and yeast KEX2 endopeptidase recognizes the dibasic peptide K-R and cleaves the peptide following KR. Therefore, even in the constructed pGMFα-AgaH71, pGMFα-AgaG1 and pGMFα-NABH expression plasmids, the peptide behind the dibasic residue Lys-Arg by KEX2 endopeptidase is cleaved to secrete mature agarase.

Example 2. Saccharomyces cerevisiae with improved agar bioethanol production ability ( Saccharomyces cerevisiae ) Strategy for constructing 2805/pGMFα-HGN strain

Using agar biomass, Saccharomyces cerevisiae 2805/pGMFα-HGN strain, a yeast strain in which genes were recombined, was constructed so that saccharification and fermentation processes can be simultaneously performed without saccharification of biomass.

First, Saccharomyces cerevisiae BY4742, 2805 and FY834 strains, which are uracil nutrient requirements and heat-resistant, were used to select host strains for expression of agarase genes.

AGAH71, AGAG1, and NABH558 genes were used for the agarase production, and the agarase genes are provided by the pGEX-AgaH71, pGEX-AgaG1 and pET-NABH558 plasmids, respectively, as a vector for subcloning each agarase gene. The pGMFα-XYLP plasmid was used. pGMFα-XYLP comprises an agar mating factor alpha signal sequence (MFα ss ) Efficient secretion of galactose induction promoter GAL10 promoter and the secretion signal sequence for the production of a raise, and a URA3 gene as a selection marker.

In order to select an appropriate host strain for agarase expression, pGMFα-AgaH71, pGMFα-AgaG1 and pGMFα-NABH plasmids were transformed from galactose into Saccharomyces cerevisiae BY4742, 2805 and FY834 strains, respectively, having excellent ethanol production ability. . Thereafter, 10 clones were randomly selected from each strain by culturing in SC-Ura medium, and the agarase activity of these clones was analyzed by direct staining using an iodine solution. Among the above clones, clones with high agar resolution were selected and cultured in 10 ml of YPDG medium for 48 hours, and enzymatic reactions of beta-agarase and NABH were performed at 45° C. and 40° C., respectively. After that, the agarase activity was compared through DNA assay.

As a result, as shown in FIG. 2, when the Saccharomyces cerevisiae 2805 strain was used as the host strain, the agarase activity was 1.2 to 2.8 times superior to other host strains, and the beta-agarase activity was NABH activity. Appeared higher than. Accordingly, Saccharomyces cerevisiae 2805 strain was selected as a host strain for expression of the agarase gene.

The strain transformed with the pGMFα-HGN plasmid into the selected Saccharomyces cerevisiae 2805 strain was named Saccharomyces cerevisiae 2805/pGMFα-HGN, and the strain was 3 agar-degrading enzymes (2 types It secretes and produces beta-agarase and one kind of neoagarobiose hydrolyase (NABH) extracellularly.

Example 3. Culture medium and culture conditions of transformed yeast

The plasmid prepared in Example 1 was transformed into Saccharomyces cerevisiae BY4742, 2805 and FY834 using a high-efficiency transformation method, and then selected in SC (synthetic complete) medium. In order to induce expression of the agarase gene, the yeast transformant is 10 ml (in vitro tube culture) or 50 ml (flask culture) YPDG [yeast extract 10 g, peptone 20 g, dextrose 10 g and galactose 10 g / liter] galactose inducing gene Incubated at 30° C. for 48 hours in a medium for expression.

In order to obtain ethanol from agarose, yeast transformants were cultured at 30° C. in 10 ml YPG [yeast extract 10 g, peptone 20 g and galactose 20 g/liter] medium. Then, the yeast transformant cultured for 48 hours was cultured (CS, cell + medium) or cultured cells (CC, cell pellet) in 50 ml of YPA [yeast extract 10 g, peptone 20 g and agarose 10 g/ L] was inoculated in the medium.

In order to proceed with the saccharification process, it was cultured at 40°C for 48 hours, and then converted to 30°C to proceed with the fermentation process and cultured for 72 hours.

The optical density (OD) of the yeast transformant was periodically measured using a spectrophotometer at 600 nm, and the dry cell weight (DCW) was converted by the conversion factor, 0.31 DCW (g/l) / OD.

Example 4. Measurement of agarase activity

The decomposition activity of agarase against agarose was measured using the DNS reducing sugar quantification method (3,5-dinitrosalicylic acid (DNS) method).

The enzyme solution was reacted for 10 minutes at 40°C or 45°C in 20 mM Tris-HCl (pH 7.0) buffer containing 0.2% agarose. After DNS was added, the enzyme mixture was cooled for 10 minutes, and after performing spectrophotometric activity measurement at 540 nm, the amount of free reducing sugar was measured based on galactose. In this assay condition, the activity of 1 unit of enzyme was defined as the amount of enzyme that produced 1 μmol of reducing sugar in 1 minute. In addition, after direct staining with an iodine solution (0.12 M of KI and 0.05 M of I ₂ included), agarase-expressing cells were measured to measure agarase-decomposing activity.

The signal sequence (MFαs.s) was bound to the expression plasmids of AGAH71, AGAG1 and NABH558 for secretory production of two beta-agarases and NABH, and Saccharomyces cerevisiae 2805/pGMFα-AgaH71, 2805/pGMFα-AgaG1 and The activity and secretion efficiency of the recombinant beta-agarase and NABH of the 2805/pGMFα-NABH transformant were analyzed.

In order to compare the agarase activity analysis, the clones having the highest expression level in the in vitro culture were selected, and then the expression rates (activity) and secretion efficiency of beta-agarase and NABH of each transformant are shown in Table 3 below.

As a result, as shown in Table 3, most of the beta-agarase and NABH activities were found in the extracellular medium (supernatant), and the secretion efficiency was 87% to 96%. NABH activity was lower than that of beta-agarase activity, but it was found to be efficiently secreted from the extracellular fraction (96%).

In the enzymatic glycosylation process for direct production of ethanol from agarose, the cooperation of three genes was required.A pGMFα-HGN plasmid having all of the AGAH71, AGAG1 and NABH558 gene expression cassettes was prepared, and the plasmid was used as saccharomyces. Levisiae 2805 strain was transformed. Saccharomyces cerevisiae 2805/pGMFα-HGN transformants were incubated in YPDG medium for 48 hours to induce beta-agarase and NABH.

As a result, as shown in Table 3 below, the total activity of agarase including beta-agarase and NABH was 1.47 unit/ml, and it was confirmed that 90% of the total agarase was successfully secreted from the supernatant. .

[Table 3]

Example 5. Reverse Transcription PCR (RT-PCR) for measuring the degree of gene transcription of the transformed Saccharomyces cerevisiae strain

RT-PCR was performed to measure the degree of transcription of AGAH71, AGAG1 and NABH558 genes of the transformed Saccharomyces cerevisiae strain.

Total RNA of each transformant was obtained from Saccharomyces cerevisiae 2805/pGMFα-AgaH71, 2805/pGMFα-AgaG1, 2805/pGMFα-NABH and 2805/pGMFα-HGN strains, and total RNA of each strain was obtained from Chomczynski and Sacchi. After extraction by the method of, oligo dT was used as a primer, and 1 μg of total RNA was synthesized in a 20 μl reaction system using HyperScript First-Strand Synthesis Kit (GeneAll). The cDNA synthesis was performed under the conditions of 30°C for 10 minutes, 42°C for 60 minutes and 72°C for 15 minutes. The synthesized cDNA was used as a template for PCR, and AgaH71-F/R, AgaG1-F/R, NABH-F/R and ACT1-F/R primer sets were used to amplify the AGAH71, AGAG1, NABH558 and ACT1 genes. Each was used, and the ACT1 gene was used as a control.

As a result, as shown in FIG. 3, was able to verify that the AGAH71 and AGAG1 gene was successfully amplified, the level of transcription AGAH71 gene was confirmed that there are no differences between the AGAG1 gene. However, it was confirmed that the transcription level of the NABH558 gene in the 2805/pGMFα-NABH and 2805/pGMFα-HGN strains was lower than that of other genes. Therefore, the low NABH activity in the 2805/pGMFα-NABH and 2805/pGMFα-HGN strains is thought to be caused by the decrease in the transcription level of the NABH558 gene, and the reason or mechanism for this is not yet clearly identified, but optimization of the promoter, It will be overcome by codon changes and gene rearrangements.

Example 6. TLC analysis for measurement of hydrolysis products

In order to measure the resolution of agar by the recombinant strain, the hydrolysis product of agarose by recombinant agarase was detected and measured by thin layer chromatography (TLC) using a silica gel 60 plate.

A reaction mixture was prepared using 20 mM Tris-HCl (pH 7.0) buffer containing 0.3% agarose substrate and an agarase enzyme, and the reaction was carried out at 40 to 45°C for 24 hours. Thereafter, the reaction mixture was dispensed on a silica gel 60 plate by 6 μl, and developed twice using n-butanol/ethanol/water (5:3:2, v/v/v) as a solvent. The separated product was confirmed by spraying sulfuric acid (dissolved in ethanol, 10% w/v) and heating the plate to 110°C.

As a result, as shown in lane 3 of FIG. 4, it was confirmed that beta-agarose produced by the 2805/pGMFα-AgaH71 strain can decompose agarose into neoagarotetraose and neoagarohexose. .

In addition, as shown in lane 4 of FIG. 4, it was confirmed that the beta-agarase produced in the 2805/pGMFα-AgaG1 strain has excellent resolution to decompose agarose into neoagarotetraose and neoagarobiose. However, since the neo-agarobiose hydrolase produced in the 2805/pGMFα-NABH strain cannot directly degrade agarose because it uses neo-agarobiose as a substrate, the beta-agarase produced in the 2805/pGMFα-HGN strain. And the simultaneous decomposition of agarose by NABH was attempted.

As a result, as shown in lane 5 of FIG. 4, agarose was decomposed into galactose, 3,6-anhydro-L-galactose (3,6-anhydro-L-galactose, AHG), and neo-agarobiose. It was confirmed that some of them were decomposed into neo-agarohexose.

Accordingly, it was confirmed that galactose was directly produced from agarose through enzymatic saccharification using the 2805/pGMFα-HGN strain.

Example 7. Measurement of ethanol production ability

The ethanol-producing ability of the genetically recombined Saccharomyces cerevisiae 2805/pGMFα-HGN strain was measured as follows.

First, for induction of agarase gene expression, Saccharomyces cerevisiae 2805/pGMFα-HGN strain was pre-cultured in YPG [YP + 2% galactose] medium for 48 hours, and the culture solution (CS) and cultured cells (CC) were For saccharification and ethanol fermentation, preheated YPA [YP + 1% agarose] medium was inoculated. Agarose is easily solidified at low temperatures, and the cells were cultured at 40°C for 48 hours to induce hydrolysis of agarose into fermentable sugars, and then for ethanol fermentation at 30°C for 72 hours. Cultured. Agarase was allowed to hydrolyze agarose into fermentable sugars for 48 hours.

The concentration of ethanol was measured by HPLC (Agilent 1100 series, Agilent. Inc., USA) using a RID (refractive index detector), and an Aminex HPX-87H column (Biorad) was used at 65°C, and the moving flow rate was (5 mM sulfuric acid). It proceeded under the condition of 0.6ml/min.

As a result, it was confirmed that the cell density gradually increased for 96 hours as the Saccharomyces cerevisiae 2805/pGMFα-HGN strain hydrolyzed agarose using galactose. In addition, as the fermentation proceeded, the concentration of ethanol increased, and as a result of performing the fermentation for 96 hours after C.S and C.C inoculation, respectively, the concentration of ethanol increased to 1.97 g/l and 1.89 g/l. At the end of the fermentation process, the galactose concentration was about 1.2 g/l, and it was confirmed that the concentration of other hydrolysis products (AHG and NA2) was maintained.

In the present invention, the saccharification and ethanol fermentation process of agarose was unified using a single recombinant yeast strain, and as a result of unified enzymatic saccharification and fermentation (USF) for 10 g/l agarose, ethanol concentration Was measured at a maximum of 1.97 g/l.

Therefore, the single enzyme saccharification and fermentation system (USF) of the present invention does not require pretreatment of biomass (substrate) in order to produce ethanol from agarose, which is the unity of the process in the process of producing ethanol from agarose. By presenting the possibility of oversimplification, it can be applied to various fields of producing ethanol from biomass.

<110> Dong-eui University Industry-Academic Cooperation Foundation <120> Transformed Saccharomyces cerevisiae having improved productivity of bio-ethanol and use thereof <130> PN1905-244 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> AGAH71 gene derived from Pseudoalteromonas hodoensis H7 <400> 1 gcagctgatt ggagcccttt tagtattcca gcacaagcag gcgcaggtaa aagctggcag 60 ttacaaagtg tttcagatga gtttaactac attgcacaac ctaataataa accagctgct 120 tttaataatc gttggaacgc atcttatata aatgcctggc taggtcctgg cgatacagaa 180 tttagcgctg gccactcata tacaactggc ggggcgcttg gtctgcaagc aaccgaaaaa 240 gcaggtacga acaaagttct ctcaggtatt atttcttcta aagcaacctt tacttacccc 300 ctatacctag aggcgatggt aaaaccaaca aacaacacta tggcaaatgc tgtatggatg 360 cttagtgctg attctaccca agaaatcgat gctatggagt catatggtag cgaccgaatt 420 ggccaagagt ggtttgacca acgtatgcat gttagtcatc acgtatttat tcgagaccca 480 tttcaagatt accagccaaa ggacgctggc tcttgggttt acaacaacgg agaaacatac 540 cgcaataaat ttcgtagata tggagtacat tggaaggatg cgtggaattt agattactat 600 atagatggtg ttttggttcg aagtgtctct ggcccaaata ttattgatcc tgaaaactat 660 actaacggaa caggcttaaa caagcctatg cacataatac tggatatgga acatcagcca 720 tggcgagacg ttaagcctaa tgcatctgag cttgcagatc ccaataaaag tatattttgg 780 gtagattgga tacgagttta taaagcccag taa 813 <210> 2 <211> 849 <212> DNA <213> Artificial Sequence <220> <223> AGAG1 gene derived from Alteromomas sp. GNUM-1 <400> 2 gcgattcctt tcatcagcaa ggccactgaa aaaagttttg atgttgatga agcaagccca 60 ttaatgccat cgtttgtcgc attcgaagat acaacgcccg ccggtatgac atggcaaaaa 120 gttgaagctc tttccgatga gtttaatcag tcgtgggacg cgtctaaatg gaaaagatca 180 aattggaatt attctgacac tcctgtgaat atggttgata caaactcagg ggtggagaat 240 ggctaccttt ggattagtgc tacgctggat gattcaacag aagaaagctg gtttaaaact 300 tcacgggtac attctaaagc gaaaattagc tttcctatgt acacagaaac acgtttgaaa 360 gttgcacaca tagcggctta taacacattt tggttgaata atggtgatgc agataatcga 420 gatgaaattg atattattga gattaattct gatccaactt gtggggagaa tgatgaatat 480 ccttggcaga tgaattctca gtattttatt gttaaaaatg gagaaacaga gcgtaataaa 540 gggccttgga gcacgaaaaa attatcggat gccaacaccc gtaaaggcgt gacgtggaac 600 caggactatc atgtatttgg cgcatggtgg aaagatgaac acaatgtaca gttttacctg 660 aatggggaac cggcgggtca tgtagtgagc agtcagcctt tcacgttaca gcaggagctg 720 atttgggatc tctggacgca agacagttct tgggtttgcg gcctgccaga aaaagaagac 780 ttactagacc ataagcgcaa tacaatgaag gttgactggg ttaggacatg gaagttagtg 840 tccaaataa 849 <210> 3 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> NABH558 gene derived from Gayadomonas joobiniege G7 <400> 3 gcagctgatt ggagcccttt tagtattcca gcacaagcag gcgcaggtaa aagctggcag 60 ttacaaagtg tttcagatga gtttaactac attgcacaac ctaataataa accagctgct 120 tttaataatc gttggaacgc atcttatata aatgcctggc taggtcctgg cgatacagaa 180 tttagcgctg gccactcata tacaactggc ggggcgcttg gtctgcaagc aaccgaaaaa 240 gcaggtacga acaaagttct ctcaggtatt atttcttcta aagcaacctt tacttacccc 300 ctatacctag aggcgatggt aaaaccaaca aacaacacta tggcaaatgc tgtatggatg 360 cttagtgctg attctaccca agaaatcgat gctatggagt catatggtag cgaccgaatt 420 ggccaagagt ggtttgacca acgtatgcat gttagtcatc acgtatttat tcgagaccca 480 tttcaagatt accagccaaa ggacgctggc tcttgggttt acaacaacgg agaaacatac 540 cgcaataaat ttcgtagata tggagtacat tggaaggatg cgtggaattt agattactat 600 atagatggtg ttttggttcg aagtgtctct ggcccaaata ttattgatcc tgaaaactat 660 actaacggaa caggcttaaa caagcctatg cacataatac tggatatgga acatcagcca 720 tggcgagacg ttaagcctaa tgcatctgag cttgcagatc ccaataaaag tatattttgg 780 gtagattgga tacgagttta taaagcccag taa 813

Claims (6)

Recombinant containing AGAH71 gene sequence, gene NABH558 represented by AGAG1 gene sequence and SEQ ID NO: 3 as shown in SEQ ID NO: 2 as shown in SEQ ID NO: 1 vector The method of claim 1,
The AgaH71 gene is a recombinant vector, characterized in that derived from Pseudoalteromonas hodoensis H7 The method of claim 1,
The AgaG1 gene is Alteromomas sp. Recombinant vector characterized by being derived from GNUM-1 The method of claim 1,
The NABH558 gene is a recombinant vector, characterized in that derived from Gayadomonas joobiniege G7 A single enzyme glycosylation and fermentation system comprising the recombinant vector of claim 1, including two types of beta-agarase AgaH71 and AgaG1 and one type of neoagarobiose hydrolyase (NABH). Saccharomyces cerevisiae strain, characterized in that it produces ethanol from agarose through a single process (a) constructing a pGMFα-HGN plasmid gene expression cassette containing the AGAH71, AGAG1 and NABH558 genes;
(b) transforming the plasmid containing the gene expression cassette constructed in step (a) into the Saccharomyces cerevisiae strain using a high-efficiency transformation method; And
(c) culturing the Saccharomyces cerevisiae strain transformed in step (b) in a medium containing agarose;
Method for producing bioethanol through a single enzyme saccharification and fermentation system comprising

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