KR101392968B1 - Novel autonomously replicating sequence and uses thereof - Google Patents

Abstract

The present invention relates to a novel self-replicating sequence derived from Kluyveromyces marcianus, a plasmid vector containing the self-replicating sequence, a method for producing a transformant using the plasmid, a transformant prepared by the method, A gene library into which a desired gene is introduced into a vector, and a method of screening a gene modified using the gene library. Using the novel self-replicating sequence of the present invention, it is possible to transform K. marxianus with high efficiency, to easily recover the desired gene from the transformed K. marxianus , New proteins, highly active proteins or strong promoters can be screened, making them widely available for industrial production using K. marxianus .

Description

Novel autonomously replicating sequences and uses thereof < RTI ID = 0.0 >

The present invention relates to a novel magnetic replication sequence and its use, and more particularly, to a novel self-replication sequence derived from Cluyveromyces marcianus, a plasmid vector containing the self-replication sequence, A transformant prepared by the above method, a gene library into which the target gene is introduced into the plasmid vector, and a method for screening the gene modified using the gene library.

A thermophilic yeast Cluj Vero My Marcia Seth Augustine (Kluyveromyces marxianus, K. marxianus) is Cluj Vero Mai Seth lactis (Kluyveromyces (Fonseca et al., Appl. Microbiol. Biotechnol. 79, 339, 2008), which is a crabtree-negative yeast similar to lactis . The K. marxianus is an enzyme of industrially valuable inulinase,? -Galactosidase,? -Glucosidase, endopolygalacturonase, protein kinase, carboxypeptidase, aminopeptidase Has been used as a source. In recent years, K. marxianus strains have a variety of carbon source utilization ability and rapid growth ability at high temperature. Therefore, there is an increasing tendency to utilize K. marxianus strains in petroleum alternative biochemistry or bio energy field. The K. marxianus strain is capable of fermenting ethanol at high temperature as compared with the conventional yeast ( S. cerevisiae ), thereby reducing the cooling cost in the fermentation of bioethanol, maintaining the high saccharification efficiency of the substrate, It is very suitable for simultaneous saccharification and fermentation or consolidated bioprocessing because it can prevent contamination of other microorganisms. Up to now, most studies on ethanol production using K. marxianus strains have focused on the development of fermentation processes such as fermentation using various carbon sources or optimization of fermentation methods by strain immobilization. However, recently, by introducing gene recombinant technology, the ethanol production ability of K. marxianus strain Research is underway to improve

Studies on the genetic engineering of Kluyveromyces yeasts have been carried out mainly on the representative strain K. lactis, and the K. marxianus gene manipulation system is in a relatively poor condition. To improve the characteristics of K. marxianus , we first used the pGL2 vector containing the G418 resistance gene and KARS2, an autonomously replicating sequence (ARS) from K. lactis, and the lithium ion method of S. cerevisiae in 1984 Transformation methods have been developed but have not been widely used due to their low efficiency (Das et al., 1984. J Bacteriol 158: 1165). PKD1, the only plasmid found in the genus Kluyveromyces, has properties similar to the 2um plasmid of S. cerevisiae (Falcone et al., 1986. Plasmid 15: 248, Bianchi et al., 1991, Curr. Genet. However, although pKD1 can be used to transform K. marxianus , it has been reported that transformation efficiency and intracellular stability are very low (Chen et al., 1989, Curr. Genet., 95, Bartkeviciute et al., 2000, Enzyme Micro. Technol., 26, 653). An artificial plasmid using the self-replicating sequence derived from K. marxianus chromosome (Ball et al., 1999; Iborra et al., 1994, Zhanag et al., 2003) was developed to remarkably increase the transformation efficiency. However, (Lane et al., 2010, Fungal Biology Reviews, 24, 17). Therefore, in order to maintain the foreign gene in K. marxianus in the long term, it is mainly used as a chromosomal gene introduction method. Especially, in order to increase the copy number of foreign gene, δ sequence, which exists in the yeast chromosome, (Pecota et al., 2007, J. Biotechnol. 127, 408; Yanase et al., 2010, Appl. Microbiol. Biotechnol. 88, 381).

However, in order to utilize the thermophilic yeast K. marxianus as a platform strain to produce a variety of petroleum substitute compounds or bioenergy in the field of biochemistry , the strain should be able to be more efficiently manipulated, It is necessary to construct a gene library and a high speed line system. For this, there is a limit to the chromosome introduction system which has low transformation efficiency and is difficult to recover the gene. Therefore, there is a need for an episomal plasmid vector system that has high transformation efficiency in K. marxianus and is maintained intracellularly and easily gene-recoverable.

Under this background, the present inventors in order to develop a high transfection is independently maintained in the cell has a conversion efficiency also easily epitaxially little plasmid vector system, the gene returnable in K. marxianus, intensive research efforts result, K. marxianus chromosome (ARS) derived from E. coli were selected to obtain a self-replicating sequence excellent in transformation efficiency and intracellular stability. When an episomal plasmid vector containing the same was used, K. marxianus could be transformed with high efficiency , It was confirmed that not only a desired gene can be easily recovered but also a gene library construction and a new protein, a protein showing a high activity or a strong promoter can be screened, thus completing the present invention.

It is an object of the present invention to provide a polynucleotide having novel self-replication activity.

Another object of the present invention is to provide a plasmid vector comprising said polynucleotide.

It is still another object of the present invention to provide a method for producing a transformant using the plasmid vector.

It is still another object of the present invention to provide a transformant produced by the above method.

Yet another object of the present invention is to provide a gene library into which a desired gene is introduced into the plasmid vector.

It is still another object of the present invention to provide a method for screening an improved gene using the gene library.

According to one embodiment of the present invention, there is provided a polynucleotide having a self-replication activity comprising a base sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 4 to 8.

The term "self-replication activity" of the present invention means a property in which a DNA fragment is autonomously replicated in a cell without being transferred to a chromosome. For the purposes of the present invention, the polynucleotide having the self-replication activity may be an autonomously replicating sequence (ARS) derived from K. marxianus , which is preferably a yeast strain, preferably K. marxianus , Saccharomyces S. cerevisiae ), skiing investigation, Schizosaccharomyces pombe ), and the like. However, the present invention is not limited thereto.

According to one embodiment of the present invention, in order to prepare an episome plasmid vector capable of stably expressing a foreign gene introduced into K. marxianus , a self-replicating sequence derived from K. marxianus chromosome was selected. First, the gene for orotidine 5-phosphate decarboxylase ( URA3), which is a selectable marker derived from K. marxianus , was obtained. Next, a genomic DNA library derived from K. marxianus was prepared, and each gene fragment of the library was introduced into Km7155u, a mutant strain of uracil nutrient requirement, to obtain 42 transformants. The plasmid stability was measured by comparing the number of colonies obtained by culturing the transformant in YPD solid medium and minimal medium. As a result, it was confirmed that the six transformants exhibited high plasmid stability of 35% or more.

Thus, plasmids obtained from the above-mentioned six transformants were transformed into Km7155u and Km25571, respectively, and the number of transformants formed per unit plasmid was calculated. As a result, the number of transformants obtained from each of the six transformants All the plasmids showed excellent transformation efficiency. Therefore, it was confirmed that the DNA fragments (KmARS 4, 13, 32, 33, 34 and 39) contained in the six plasmids were self-replicating sequences, As a result of the analysis of the nucleotide sequence of the sequence, it was confirmed that the fragment was a DNA fragment having a size of 633 to 1275 bp as a whole.

The KmARS 4 (SEQ ID NO: 4) was 633 bp and was composed of an AT-rich sequence (SEQ ID NO: 4) generally observed in a eukaryotic cell self-replication sequence. To confirm whether there is a sequence encoding a protein in these sequences, The protein database was searched through NCBI BLASTX search and showed partial homology with proteins from Erwinia pyrifoliae Ep1 / 96 at 124-240 bp.

The KmARS 13 (SEQ ID NO: 5) has an AT-rich sequence of 763 bp (SEQ ID NO: 5), which does not contain a sequence showing homology in the protein database and contains a self-replicating sequence in the entire sequence.

The KmARS 32 (SEQ ID NO: 6) was homologous to the protein from K. lactis NRRL Y-1140 at 3-452 bp and 1085-1270 bp with an AT-rich sequence (SEQ ID NO: 6) of 1275 bp, The replication sequence was determined to be at 453-1084 bp.

The KmARS 33 and 34 (SEQ ID NO: 7) have the same sequence as 719 bp (SEQ ID NO: 7) and showed homology to the protein from K. lactis NRRL Y-1140 at 1-384 bp. it was determined that a self-replicating sequence exists in bp.

The KmARS 39 (SEQ ID NO: 8) is 680 bp (SEQ ID NO: 8), homologous to the protein from K. lactis NRRL Y-1140 at 599-679 bp and the self-replication sequence is present at 1-598 bp (Fig. 3).

According to another embodiment, the present invention provides an episomal plasmid vector comprising said polynucleotide.

The term "plasmid vector " of the present invention means a recombinant vector capable of expressing a desired peptide in a suitable host cell, and is a kind of expression vector that is a genetic construct containing an essential regulatory element operatively linked to the expression of the gene insert. The plasmid vector of the present invention may contain expression regulatory elements such as a promoter, an operator, and an initiation codon as elements contained in a conventional plasmid vector. The initiation codon and the termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide and must be operative when the plasmid vector is introduced into the host cell and must be in a coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

It may also contain a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.

Specific examples of the plasmid vector may be a commercial plasmid vector such as pUC18, pIDTSAMRT-AMP and pBluescript KS +, preferably plasmids derived from Escherichia coli (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis Plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), and more preferably yeast-derived episomal plasmid vectors, but are not particularly limited thereto.

The term "episome" as used herein means an exogenous gene that exists in the cytoplasm and proliferates independently from a chromosome. It may exist in an autonomous state (autonomous state) Or may be introduced independently or may be introduced into a chromosome to maintain its original function.

According to one embodiment of the present invention, the present inventors have confirmed that the above-mentioned six kinds of plasmids are also operative in other yeasts. As a result, it has been confirmed that transformation efficiency is excellent even in other yeasts such as Saccharomyces cerevisiae. It was found that the episomal plasmid vector containing the replication sequence could be used for transformation of K. marxianus as well as other yeast. In addition, the polynucleotide having the self-replication activity of the present invention can be used to improve a gene by screening a protein exhibiting the best activity among various polynucleotides encoding a target protein in K. marxianus .

According to another embodiment, the present invention provides a method for producing a transformant using the plasmid vector and a transformant prepared by the method.

Specifically, a method for producing a transformant capable of expressing a target gene of the present invention comprises the steps of (i) cloning a gene of interest into the plasmid vector of claim 3 to obtain a recombinant plasmid vector; And (ii) introducing the recombinant plasmid vector into a yeast strain. At this time, the target gene is not particularly limited, but can be a gene encoding lipase, and a polynucleotide having a self-replication activity derived from the K. marxianus chromosome, which is a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 4 to 8 .

The term "transfection" of the present invention refers to any action that causes genetically stable genetic events such that the nucleotide fragment migrates into the genome of the host organism to express the desired peptide. Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. Generally, the transformation methods include the CaCl ₂ precipitation method, the Hanahan method which uses a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ method, the electroporation method, the calcium phosphate precipitation method, the protoplasm fusion method, Agrobacterium-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

Used in the transformed host is not particularly limited, Escherichia coli (E. coli) and Escherichia (Escherichia) bacteria belonging to the genus Bacillus subtilis, Bacillus (Bacillus) bacteria belonging to the genus Pseudomonas is footage (such as Pseudomonas (Bacillus subtilis) such putida) may be the same Pseudomonas (Pseudomonas) bacterium belonging to the genus Saccharomyces as MY process three Levy jiae, ski irradiation Caro My process pombe such as yeast, animal cells and insect cells, and preferably may be a yeast strain, more preferably It can be K. marxianus .

According to another embodiment, the present invention provides a gene library into which a polynucleotide is introduced into the above plasmid vector and a method of screening a gene modified using the gene library.

The term "gene library" of the present invention refers to a set obtained by introducing into a vector a fragment obtained by cleaving all the DNA of all chromosomes contained in cells of a desired individual with restriction enzymes and the like. Region, it can be used to screen clones of the gene of interest. For the purpose of the present invention, the gene library may be introduced into the above-described plasmid vector so that the desired gene fragment can be expressed in K. marxianus , which is one of the yeast strains, but is not particularly limited thereto.

Also, a method for screening an improved gene of the present invention includes the steps of: (i) randomly mutating a polynucleotide encoding a desired protein to obtain one or more mutated polynucleotides having various mutations; (Ii) cloning the mutated polynucleotide into an episome plasmid vector comprising a polynucleotide having self-replication activity to obtain a gene library; (Iii) introducing the gene library into a yeast strain to express a target protein; And (iv) comparing the activity of the expressed target protein to select a polynucleotide encoding a mutated protein exhibiting improved activity compared to a control protein expressed from a non-mutated polynucleotide. At this time, the yeast strain may be K. marxianus .

The present invention provides a method for screening a protein having an enhanced activity by introducing a polynucleotide-introduced gene library into a yeast strain, expressing a target protein derived from the polynucleotide, and comparing the activity of the expressed protein . Herein, the polynucleotide is not particularly limited as long as it can code for a protein or a peptide. Alternatively, the polynucleotide may be a fragment obtained by randomly cleaving a genomic DNA of a cell with a restriction enzyme, or a polynucleotide encoding a target protein may be randomly Or mutated polynucleotides having various mutations obtained by mutation in vivo. The gene library into which the polynucleotide is introduced may be obtained by cloning the polynucleotide into a multicloning site of an episomal plasmid vector comprising the polynucleotide having the self-replication activity of the present invention, The polynucleotide may be introduced into the episomal plasmid vector so that the polynucleotide can be introduced into the chromosome by homologous recombination. However, the present invention is not limited thereto, so long as the polynucleotide is constructed so as to be expressed in the host. The yeast strain may be , but not limited to, K. marxianus , Saccharomyces cerevisiae , and ski-investigated caromyces pombe , and the target protein may be a lipase, though not particularly limited thereto .

According to an embodiment of the present invention, a plasmid vector pBTEF-KmArs is prepared (FIG. 5) and an error-prone PCR method is used for screening lipases having higher activity than conventional lipases The mutant library was obtained, and the mutant library was introduced into K. marxianus to obtain about 900 transformants (Fig. 6). The activity of the mutated lipases expressed in the transformants was compared, It was confirmed that lipase showing superior activity to the conventional lipase was expressed in the transformant (Table 5).

Using the novel self-replicating sequence of the present invention, it is possible to transform K. marxianus with high efficiency, to easily recover the desired gene from the transformed K. marxianus , New proteins, highly active proteins or strong promoters can be screened, making them widely available for industrial production using K. marxianus .

1A is a cleavage map of a recombinant plasmid pBlue-KmURA3 containing the URA3 gene derived from Cluyeberomyces marcianus.
Figure 1B is a cleavage map of pBlue-KmURA3-ARS lib, a library of autonomously replicating sequences (ARS) based on the recombinant plasmid pBlue-KmURA3.
1C is a cleavage map of pBlue-KmURA3-ARS, which is a recombinant plasmid into which ARS is introduced into the recombinant plasmid pBlue-KmURA3.
Fig. 2 is a cleavage map of the plasmids pBlue-FR A and pBlue-FR B used as the comparative group.
Fig. 3 is a schematic diagram showing the result of analysis of five kinds of ARS nucleotide sequences.
Fig. 4 is a schematic diagram showing a process for producing an expression vector for expressing the lipase gene in Kluyveromyces marcianus.
5 is a schematic diagram schematically showing the structure of the plasmid vector pBTEF-KmArs.
FIG. 6 is a schematic diagram schematically showing a process for preparing an expression vector for ultra-rapid modification of lipase gene and a process for selecting a highly active lipase.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Cluey Bromyces Marcianus  Origin selection marker Kmura3 Securing

Kluyveromyces marxianus) derived from the URA3 (orotidine 5-phosphate decarboxylase) received pre-sale of K. marxianus KCTC 7155 strain in Daejeon, Korea Research Institute of Bioscience and Biotechnology material for gene cloning gene bank YPD (1% yeast extract, 2% peptone and 2% glucose) medium And genomic DNA was extracted. 1 μl of the extracted sample was subjected to polymerase chain reaction (94 ° C. for 5 minutes; 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute) using the primers of SEQ ID NO: 1 and SEQ ID NO: At 72 ° C for 7 minutes). The amplified gene was recovered by agarose gel electrophoresis through a 1.4 kb fragment. The plasmid pBlue-KmURA3 was prepared by cloning the PCR product of the polymerase chain reaction into pBluescript KS + (FIG. 1A), and the nucleotide sequence of the inserted gene was analyzed. As a result, a 1.4 kb gene containing the URA3 gene from Kluyveromyces marcianus (Bergkamp et al., 1993, Yeast, 9, 677) (SEQ ID NO: 3). 1A is a cleavage map of a recombinant plasmid pBlue-KmURA3 containing the URA3 gene derived from Cluyeberomyces marcianus.

Example  2: Cluey Bromyces Marcianus  Genome DNA  Library Manufacturing

Genomic DNA of Kluyveromyces marcianus was isolated and partially digested with restriction enzyme Sau3AI to obtain a DNA fragment. To this DNA fragment, two nucleotides of G and A were added to Klenow DNA polymerase and then electrophoresed to separate DNA fragments of 500-1,000 bp size. On the other hand, the plasmid pBlue-KmURA3 prepared in Example 1 was digested with restriction enzyme Xho I, and two nucleotides of C and T were added to Clenna DNA polymerase. The DNA fragment of the separated clueiberomyces marcianus was inserted into the digested pBlue-KmURA3 to prepare a clue veromyces marcianus genome DNA library (Fig. 1B). 1B is a cleavage map of pBlue-KmURA3-lib, an ARS library based on the recombinant plasmid pBlue-KmURA3.

After plasmid library was transformed into Escherichia coli DH5α, the transformant was plated on 2YT medium (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride and 2% bactohea) containing 100 μg / ml of ampicillin And cultured overnight at 37 DEG C to obtain a colony of about 5 x 10 < ^{4 &} gt ;. DNA was extracted from these colonies to obtain an amplified plasmid library DNA.

Example  3: Cluey Bromyces Marcianus  Screening of transformed and self-replicating sequences

The library DNA of Cluyveromyces marcianus prepared in Example 2 was transformed into Km7155u, a mutant strain of uracil auxotrophy derived from Kluyveromyces marcianus, according to the method of lithium acetic acid, , 2% glucose and 2% bacto-agar) and cultured at 37 ° C for 2 days to obtain 42 transformants. To confirm whether the plasmid was introduced into C. perfringens marcianus, E. coli DH5? Was transformed with total DNA extracted from 10 randomly selected transformants. E. coli transformants were selected from 2YT medium containing ampicillin, and recombinant plasmid pBlue-KmURA3-ARS was isolated from E. coli transformants. After restriction enzyme analysis, E. coli plasmids containing about 500-1,000 bp DNA (Fig. 1C). 1C is a cleavage map of pBlue-KmURA3-ARS, which is a recombinant plasmid into which ARS is introduced into the recombinant plasmid pBlue-KmURA3.

Example  4: ARS Self-replication activity

In order to confirm the self-replication activity of the DNA fragment obtained in Example 3, plasmid stability and transformation efficiency were confirmed.

Example  4-1: Confirmation of plasmid stability

Each transformant was cultured in a YPD complex medium (1% yeast extract, 2% peptone and 2% glucose) for about 20 generations, and then the same cells were cultured in YPD solid medium (1% yeast extract, 2% Glucose and 2% bacto-agar) and a minimal medium (yeast substrate lacking 0.67% amino acid, nutritional supplement lacking 0.77% uracil, 2% glucose and 2% bactohexa) The number of colonies cultured was compared to determine the stability of selection markers per generation (Table 1). Also, plasmids pBlue-FR A and pBlue-FR B containing a self-replicating sequence (Iborra et al., 1994, Yeast, 10, 1621) derived from Kluyveromyces marcianus showing self-replication activity as a comparative group 2). Fig. 2 is a cleavage map of the plasmids pBlue-FR A and pBlue-FR B used as the comparative group.

The plasmid stability of each transformant Transformant Plasmid stability (%) Transformant Plasmid stability (%) One 32.3 23 25.8 2 15.7 24 25.2 3 32.9 25 33.7 4 42.6 26 27.5 5 33.2 27 32.3 6 27.2 28 19.4 7 31.5 29 21.9 8 26.4 30 17.4 9 16.1 31 31.7 10 16.8 32 37.6 11 11.3 33 70.0 12 15.2 34 67.8 13 38.1 35 9.4 14 28.4 36 19.1 15 19.4 37 19.7 16 9.4 38 24.7 17 24.1 39 51.2 18 11.5 40 1.1 19 30.4 41 27.7 20 12.6 42 17.3 21 23.5 FRA 65.9 22 31.6 FR B 70.9

As shown in Table 1 and FIG. 2, the plasmid stability varied from 1 to 70%. In particular, the transformant 33 showed a stability of 70% similar to that of the comparative group plasmid. Six transformants with plasmid stability of 35% or more were selected and further studies were performed.

Example  4-2: Investigation of transformation efficiency

Plasmids were isolated from the 6 selected transformants and transformed into Km7155u and Km25571 strains, respectively, and the number of transformants per plasmid was determined. Plasmid concentrations were determined by measuring the absorbance at 260 nm using a spectrophotometer. As a control group, pBlue-KmURA3 containing no self-replicating sequence and pBlue-FRA containing ARS were used as a control (Table 2).

The transformation efficiency of each plasmid ARS SEQ ID NO: Number of transformants / 1 DNA DNA KM 7155u KM 25571 S. cerevisiae 4 4 1.2 × 10 ⁵ 7.5 x 10 ⁴ 3.0 x 10 ⁴ 13 5 1.3 x 10 ⁵ 7.6 × 10 ⁴ 3.0 x 10 ⁴ 32 6 1.4 x 10 ⁵ 7.5 x 10 ⁴ 0 33 7 1.1 × 10 ⁵ 7.5 x 10 ⁴ 3.0 x 10 ⁴ 34 7 1.1 × 10 ⁵ 7.5 x 10 ⁴ 8.0 × 10 ³ 39 8 1.2 × 10 ⁵ 7.4 × 10 ⁴ 6.0 x 10 ³ FR-A - 1.3 x 10 ⁵ 7.7 x 10 ⁴ 6.0 x 10 ³ pBlue-KmURA3 - 0 0 0

As shown in Table 2, all the plasmids containing the six self-replicating sequences obtained in the present invention exhibited significantly higher transfection efficiency than the control plasmid pBlue-KmURA3 containing no self-replication sequence, And showed similar transformation efficiencies to the reported Kluyveromyces marcianus in the literature. Therefore, all the plasmids containing the gene obtained from the chromosome of Kluyveromyces marcianus obtained in the present invention can greatly improve the transformation efficiency and can recover the plasmid from the cells after the passage culture, And confirmed that the six DNA fragments thus obtained were self-replicating sequences of Cluyveromyces marcianus. As shown in Table 2, the yeast Saccharomyces cerevisiae was transformed to a similar level and the whole gene of the transformant was isolated The plasmid could be recovered by re-transformation into E. coli. Therefore, it was judged that the self-replicating sequence obtained in the present invention can operate in other yeast.

Example  5: Sequence analysis of the obtained self-replicating sequences

The nucleotide sequences of six plasmids having self-replication ability were analyzed to confirm that DNA fragments of 633 to 1275 bp size were contained. KmARS 4 was composed of AT-rich sequence (SEQ ID NO: 4) which was found to be 633 bp in eukaryotic cell self-replication sequence. First, NCBI BLASTX search was used to determine the protein coding sequence of these sequences. As a result of searching the database, Erwinia and partially homologous with the hypothetical protein derived from pyrifoliae Ep1 / 96. Therefore, it is considered that a self-replicating sequence derived from Kluyveromyces marcianus is present in 1 to 123 bp and 241 to 633 bp except for the region showing homology with the ORF, and the ARS core consensus sequence (5 ' - (A / T) TTTA (C / T) (A / G) TTT (A / T) -3 '). KmARS 13 is an AT-rich sequence of 763 bp (SEQ ID NO: 5), which does not contain homologous sequences in the protein database, and contains a self-replicating sequence in the entire sequence. KmARS 32 is an AT-rich sequence of 1275 bp (SEQ ID NO: 6) and has 3 to 452 bp, Kluyveromyces lactis NRRL Y-1140, and the self-replicating sequence was found to be present at 453 to 1084 bp. KmARS 33 and 34 are the same sequence as 719 bp (SEQ ID NO: 7), with Kluyveromyces lactis NRRL Y-1140, and it was judged that a self-replicating sequence exists in 385 to 719 bp except ORF. KmARS 39 is 680 bp (SEQ ID NO: 8), and Kluyveromyces lactis NRRL Y-1140, and the self-replicating sequence was determined to be present at 1 to 598 bp (Fig. 3). Fig. 3 is a schematic diagram showing the result of analysis of five kinds of ARS nucleotide sequences.

Example  6: Expression of vector-derived foreign proteins including self-replicating sequences

Using the self-replicating sequence of Cluyveromyces marcianus, yeast Candida The plasmid containing the CalB14 gene was introduced with a self-replicating sequence to express CalB14, an antarctic lipase. First, the KmARS 4 gene (SEQ ID NO: 4) was amplified using the primers of SEQ ID NOS: 9 and 10, the KmARS 13 gene (SEQ ID NO: 5) was amplified using the primers of SEQ ID NO: 11 and 12, the KmARS 32 gene 6) was amplified by PCR using the KmARS 33 gene (SEQ ID NO: 7) as the primer of SEQ ID NO: 15 and the KmARS 39 gene (SEQ ID NO: 8) as the primer of SEQ ID NO: The reaction was repeated 25 times at 72 ° C for 1 minute, 72 ° C for 7 minutes once at 94 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 7 minutes, and the fragments were recovered by agarose gel electrophoresis. The polymerase chain reaction product was digested with restriction enzyme SalI and ligated into pBGAP-ST3-CalB14 vector digested with restriction enzyme Xho I to transform E. coli DH5 alpha, and a plasmid in which the KmARS gene was inserted was obtained from the transformant ). Fig. 4 is a schematic diagram showing a process for producing an expression vector for expressing the lipase gene in Kluyveromyces marcianus.

These recombinant plasmids were transformed into Km7155u and Km25571 strains, respectively, and single colonies were selected from the transformants. The lipase activity in the culture medium was analyzed to confirm that each transformant secreted lipase. In the activity analysis, each cell was inoculated into YPD (1% yeast extract, 2% peptone, 2% glucose), cultured at 30 ° C, and then the culture supernatant was compared with each lipase activity by pNPP assay. 20 μl of the enzyme solution was added to 1 ml of the reaction solution (100 μl of 50 mM pNPP, 950 μl of ethyl alcohol and 50 mM Tris buffer, pH 7.5), reacted for 5 minutes, and absorbance was measured at 410 nm (Table 3 and Table 4) .

Analysis of lipase activity of Km7155u transformants ARS number Lipase activity (U / L) ARS numer Lipase activity (U / L) ARS4-1 42 ARS33-1 19 ARS4-2 41 ARS33-2 20 ARS4-3 42 ARS33-3 20 ARS4-4 42 ARS33-4 22 ARS4-5 43 ARS33-5 21 ARS13-1 27 ARS39-1 20 ARS13-2 27 ARS39-2 21 ARS13-3 26 ARS39-3 19 ARS13-4 27 ARS39-4 20 ARS13-5 27 ARS39-5 20 ARS32-1 20 FRA-1 62 ARS32-2 20 FRA-2 63 ARS32-3 20 FRA-3 63 ARS32-4 19 FRA-4 63 ARS32-5 19 FRA-5 62

Analysis of lipase activity of Km25571 transformants ARS number Lipase activity (U / L) ARS numer Lipase activity (U / L) ARS4-1 40 ARS33-1 19 ARS4-2 40 ARS33-2 20 ARS4-3 41 ARS33-3 20 ARS4-4 41 ARS33-4 21 ARS4-5 40 ARS33-5 20 ARS13-1 68 ARS39-1 19 ARS13-2 66 ARS39-2 19 ARS13-3 67 ARS39-3 19 ARS13-4 66 ARS39-4 20 ARS13-5 67 ARS39-5 20 ARS32-1 20 FRA-1 21 ARS32-2 20 FRA-2 22 ARS32-3 19 FRA-3 21 ARS32-4 19 FRA-4 20 ARS32-5 18 FRA-5 21

As shown in Tables 3 and 4, most of the transformants showed lipase activity, indicating that the vector containing the self-replicating sequence was maintained in the cells to express and secrete the exogenous lipase. However, the activity of lipase was different depending on the strains used and self - replicating sequences. As shown in Table 3, ARS4 and FR-A showed excellent lipase activity in Km7155u, and ARS4 and 13 showed excellent lipase activity in Km25571 as shown in Table 4. FR-A, which was used as a control, showed very low activity in strain Km25571, indicating a large difference according to strains.

Example  7: Episome  Using a plasmid Lipase  Gene super fast improvement

PCR for the preparation of the vector from which the ST3-CalB14 gene had been removed by using a primer having the nucleotide sequence of SEQ ID NOS: 19 and 20 having the Eco RI recognition sequence as the template and pBTEF-ST3-CalB14-KmARS as the template for the high activity lipase screening To obtain a KmARS gene of 1.2 kb. The KmARS gene thus obtained was digested with restriction enzyme Eco RI and inserted into a vector from which ST3-wt CalB-KmARS obtained by digesting with the same restriction enzyme was removed, to prepare plasmid vector pBTEF-KmArs (FIG. 5 is a schematic diagram schematically showing the structure of the plasmid vector pBTEF-KmArs.

In order to obtain the mutant library using the error-prone PCR method, the mutagenesis enzyme using the primers having the nucleotide sequences of SEQ ID NOs: 21 and 22 was used as the template for pBTEF-ST3-wt CalB-KmARS PCR random mutagenesis kit (Clontech) was performed. Mutagenesis induces two mutations per kb in accordance with the instructions included in the product, once at 30 ° C at 94 ° C; Reaction at 94 占 폚 for 30 seconds, 68 占 폚 for 1 minute and 30 seconds 25 times; 1 < / RTI > at 68 < RTI ID = 0.0 > The recovered agarose gel via electrophoresis fragment with 1% tri beauty Lin (tributyrin) the UD in the medium treated with Pst and Bam Ⅰ HⅠ added pBTEF-KmARS vector in vivo recombinant method to introduce about 900 transformants into the Km7155u strain (Fig. 6). FIG. 6 is a schematic diagram schematically showing a process for preparing an expression vector for ultra-rapid modification of lipase gene and a process for selecting a highly active lipase.

As shown in FIG. 6, when the expression vector was contained, it was confirmed that active rings appeared around most strains.

Ten strains with relatively large active rings were selected and their lipase activities were compared by UD medium supplemented with 1% tributyrin and pNPP (FIG. 7). Fig. 7 is a photograph and a chart showing the results of comparing lipase activities exhibited in different transformants. As shown in FIG. 7, it was confirmed that the activity of the strain No. 2 was increased about twice that of wt, and that of strain No. 7 was increased about 4.6 times that of wt.

Nucleotide sequences of strains No. 2 and No. 7 with increased activity were analyzed to determine whether mutation occurred on the signal peptide portion (ST3) and amino acid sequence (wt CalB) of lipase (Table 5).

The signal peptide region and the mutation in the amino acid sequence of the selected mutant Strain number ST3 wt CalB 2 Q2L G41V 7 - L278R

As shown in Table 5, it was confirmed that each of the strains produced lipases in which a part of the amino acid sequence was mutated.

Therefore, the genetic library can be constructed directly from Cluyveromyces marcyanus using the self-replicating sequence obtained in the present invention, so that a new protein can be excavated or a protein showing high activity can be screened directly or a strong promoter can be screened Applications will be possible.

Attach an electronic file to a sequence list

Claims (13)

A polynucleotide having a self-replication activity consisting of any one of the nucleotide sequences of SEQ ID NOS: 4 to 8.
The method according to claim 1,
Kluyveromyces marxianus ) chromosome.
An episome plasmid vector comprising the polynucleotide of claim 1.
The method of claim 3,
Is an episomal plasmid vector represented by a cleaved map in Fig. 1C.
(I) cloning a gene of interest into the plasmid vector of claim 3 to obtain a recombinant plasmid vector; And
(Ii) introducing the recombinant plasmid vector into a yeast strain to transform the target gene.
6. The method of claim 5,
Wherein the target gene is a gene encoding lipase.
6. The method of claim 5,
Wherein the plasmid vector comprises a polynucleotide having self-replication activity consisting of any one of the polynucleotide sequences selected from the group consisting of SEQ ID NOS: 4-8.
6. The method of claim 5,
Wherein the yeast strain is a strain of Kluyveromyces marcianus.
9. A transformant which is produced by the method according to any one of claims 5 to 8 and capable of expressing a gene of interest.
A gene library in which one or more polynucleotides are introduced into the plasmid vector of claim 3.
11. The method of claim 10,
Wherein the polynucleotide is obtained by randomly mutating a polynucleotide encoding a target protein as a template.
(I) randomly mutating a polynucleotide encoding a desired protein as a template to obtain at least one mutated polynucleotide having various mutations;
(Ii) cloning the one or more mutated polynucleotides into the episomal plasmid vector of claim 3 comprising a polynucleotide having self-replication activity to obtain a gene library;
(Iii) introducing the gene library into a yeast strain to express a target protein; And
(Iv) comparing the activity of the expressed target protein to select a polynucleotide encoding a mutated protein exhibiting improved activity compared to a control protein expressed from a non-mutated polynucleotide. ≪ / RTI >
13. The method of claim 12,
Wherein the yeast strain is a strain of Kluyveromyces marcianus.

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