JP7145140B2 - Genome rearrangement method and its use - Google Patents

Description

The present specification relates to a genome rearrangement method, its use, and the like.

Attempts have been made to efficiently obtain useful organisms by inducing genetic recombination in genomic DNA in various organisms such as cells and plants to promote genome rearrangement.

As one method for inducing genetic recombination, a DNA double-strand cleavage enzyme such as TaqI is expressed in cells such as yeast, and the DNA double-strand cleavage enzyme is heated at a high temperature of 40°C or higher for about 60 minutes or less. is activated by a short-time treatment to induce DNA cleavage and recombination artificially at high temperature and in a short time (Patent Documents 1 to 3).

JP 2011-160798 A JP 2006-141322 A JP 2012-44883 A

However, with the method of activating the DNA double-strand cleavage enzyme for a short period of time by such high-temperature and short-time treatment, it is difficult to control the frequency of DNA double-strand breaks under the treatment conditions. That is, depending on the temperature or treatment time, cutting was insufficient and sufficient genetic recombination was not performed, while cells were weakened due to excessive cutting. As a result, in some cases, it is difficult to rearrange the genome as intended, or it is difficult to obtain sufficient genome rearrangement efficiency.

On the other hand, the action of DNA double-strand breaking enzymes is thought to remarkably suppress the survival and proliferation of cells. Therefore, it was not realistic to operate the DNA double-strand cleavage enzyme beyond the cell cycle, for example, the range of 2 hours to several hours in the case of yeast.

The present specification provides a more practical genome rearrangement method, that is, a genome rearrangement method capable of controlling the level of genome rearrangement and obtaining excellent genome rearrangement efficiency, and its use.

The present inventors performed a DNA double-strand break treatment, unlike the conventional method (high temperature and short time), under environmental conditions where cells can grow sufficiently (environmental conditions for cell growth). Surprisingly, we obtained the knowledge that the genome rearrangement level can be controlled at will and the genome rearrangement efficiency can be dramatically increased by the action of . Based on such knowledge, this specification provides the following means.

(1) A method for genome rearrangement in a eukaryotic organism, comprising:
1 or 2 or more steps of genome rearrangement in which a protein having DNA double-strand breaking activity is allowed to temporarily act in a cell of a eukaryote under conditions of a growth environment of the cell to perform genetic recombination. ,Method.
(2) The method according to (1), wherein the protein is a thermostable DNA double-strand cleavage enzyme derived from a thermophile.
(3) The method according to (1) or (2), wherein the protein is one or more selected from BclI, Sse9I, BstUI, TfiI, TseI, Tsp45I, TaqI and PhoI.
(4) The method according to any one of (1) to (3), wherein the protein is one or more selected from Sse9I, BstUI, TfiI, TseI, Tsp45I and TaqI.
(5) The method according to any one of (1) to (4), wherein the protein is TaqI.
(6) The method according to any one of (1) to (5), wherein the genome rearrangement step is a step of inducibly expressing the protein.
(7) The method according to any one of (1) to (6), wherein the genome rearrangement step is a step of allowing the protein to act for 1 hour or more and 72 hours or less.
(8) The method according to any one of (1) to (7), wherein the cell growth environmental conditions include a temperature at which the cells exhibit sufficient growth potential.
(9) The method according to any one of (1) to (8), wherein the eukaryotic organism is a plant.
(10) The induction method according to any one of (1) to (9), wherein the eukaryotic organism is a microorganism.
(11) The method according to any one of (1) to (10), wherein the eukaryotic organism is yeast.
(12) The method according to any one of (1) to (11), wherein the eukaryotic organism is a homothalmic yeast.
(13) After performing the first reorganization step for the homothalismous yeast, performing a selection step including spore breeding to select the homotismalism yeast having the reorganized genome set under selective pressure for a first trait, Further, the method according to (12), wherein the second said rearrangement step is performed on said selected homothalismic yeast.
(14) A method of producing a eukaryotic organism comprising a rearranged genome set, comprising:
1 or 2 or more steps of genome rearrangement in which a protein having DNA double-strand breaking activity is allowed to temporarily act in a eukaryotic cell under the conditions of the growth environment of the cell to perform genetic recombination;
A method.
(15) Furthermore, one or more selection steps of selecting intended eukaryotic organisms based on any index from the population of eukaryotic organisms holding the rearranged genome set,
The production method according to (14), comprising:
(16) The production method according to (14) or (15), wherein the eukaryotic organism is a homothalmic yeast.
(17) the eukaryotic organism is a homothalmic yeast;
The production method according to (15), wherein the selection step includes a spore breeding step.
(18) A method of producing a population of eukaryotic organisms, comprising:
1 or 2 or more steps of genome rearrangement in which a protein having DNA double-strand breaking activity is allowed to temporarily act in a eukaryotic cell under the conditions of the growth environment of the cell to perform genetic recombination;
A method.
(19) A breeding material that is a homothalmic yeast that expressably retains a gene encoding a protein having DNA double-strand breaking activity.

FIG. 2 is a diagram showing the outline of the structure of a TaqI gene yeast expression vector. FIG. 3 shows construction of a plasmid containing a GF-FP reporter gene for evaluation of genome rearrangement efficiency. FIG. 1 shows an outline of a genome rearrangement evaluation method using a GF-FP reporter gene. FIG. 10 is a diagram showing a comparison of TaqI expression level at each induction temperature using BY4741+GFP(HIS) strain. It is a figure showing the genome rearrangement efficiency by high-temperature and short-time heat treatment using the BY4741 + GFP (HIS) strain, A shows the rearrangement efficiency immediately after heat treatment, and B shows the rearrangement efficiency after recovery culture. FIG. 10 shows genome rearrangement efficiency by treatment at cell growth temperature with strain BY4741+GFP(HIS). FIG. 1 shows a comparison of genome rearrangement efficiency using haploid, diploid and tetraploid yeast by treatment at cell growth temperature. FIG. 4 shows ploidy histograms by TaqI treatment time using haploid, diploid and tetraploid yeasts by treatment at cell growth temperature. FIG. 2 shows an evaluation system (A and B) for interchromosomal genetic recombination (genome rearrangement) and an evaluation result (C) by treatment at cell growth temperature. FIG. 10 is a diagram showing the evaluation results of interchromosomal genome rearrangement efficiency by genetic recombination at high temperature for a short period of time. FIG. 4 is a diagram showing changes in the amount of bacterial cells in the enrichment culture process. FIG. 10 is a diagram showing fermentation test results (5% xylose fermentation medium) on xylose assimilation capacity of strain OC2A-C5. Fig. 2 is a diagram showing fermentation test results (high sugar concentration glucose + xylose mixed fermentation medium (80 g/L glucose and 100 g/L xylose)) on xylose assimilation capacity of strain OC2A-C5. Fig. 2 is a diagram showing fermentation test results (glucose + xylose mixed fermentation medium (30 g/L glucose, 30 g/L xylose) for heat resistance (40°C) of OC2A-TT strain. Fig. 2 is a diagram showing fermentation test results (glucose + xylose mixed fermentation medium (50 g/L glucose, 50 g/L xylose)) for heat resistance (40°C) of OC2B-TT1 strain and OC2B-TT2 strain. FIG. 10 is a diagram showing evaluation results of genome rearrangement efficiency (whole cell) by treatment at cell growth temperature. FIG. 10 is a diagram showing evaluation results of genome rearrangement efficiency (living cells) by treatment at cell growth temperature.

The present disclosure relates to genome rearrangement methods and uses thereof. According to the genome reorganization method disclosed in the present specification (hereinafter also simply referred to as the present reorganization method), a protein having DNA double-strand breaking activity, such as a restriction enzyme, is added to a cell in a eukaryotic cell. It is possible to easily control the level (degree) of genome rearrangement and achieve a higher efficiency of genome rearrangement than ever before by temporarily acting under the growth environment conditions of . According to this reorganization method, it is possible to improve not only the efficiency of various types of genome reorganization within chromosomes, but also the efficiency of interchromosomal genome reorganization, which was low in conventional methods.

In conventional genome rearrangement, genetic recombination is performed by allowing restriction enzymes to act in cells for a short period of time under conditions that are not general cell growth environmental conditions applied to cells, for example, at temperatures higher than general culture temperatures. and rearranged the genome. Thereafter, the temperature is returned to normal culture temperature to stop the action of restriction enzymes, thereby completing the genome rearrangement, and creating a cell library comprising various genome sets generated by various genetic recombination in individual cells. was building.

However, according to the studies of the present inventors, it was found that restriction enzymes are capable of causing DNA double-strand breaking activity to act on chromosomal DNA at a sufficiently low temperature in cells, unexpectedly. In addition, it was found that under high-temperature and short-time action conditions, the DNA repair action originally possessed by the cells was not exerted at the same time as the DNA breakage. As a result, it was difficult to promote genetic recombination under high-temperature and short-time action conditions, and sufficient genome rearrangement efficiency could not be achieved.

Although not binding to the disclosure of this specification, the present inventors can infer the superiority of this reorganization method as follows. That is, according to this reorganization method, by adopting cell growth environmental conditions, it is possible to exert not only the DNA double-strand breaking action but also the DNA repair action inherent in the cell. Through DNA double-strand break action, DNA repair action, and cell growth and eventually proliferation, as a result, functional damage to chromosomes and genes due to DNA breakage can be effectively avoided or suppressed, and genetic recombination can be achieved. The result is that different rearranged genome sets will be tolerated within the cell. From the above, it is considered possible to obtain proliferative cells that have various rearranged genome sets.

Furthermore, according to this restructuring method, the DNA double-strand breaking action and the DNA repair action are controlled by controlling the temperature, time, etc. under the environmental conditions for cell growth, thereby promoting genetic recombination and damaging cells and the like. can be adjusted. As a result, it is thought that excellent genome rearrangement efficiency can be obtained while controlling the level of genome rearrangement.

As a result, according to this reorganization method, it is possible to efficiently construct a population of eukaryotic organisms rich in genome set compositional diversity. The resulting eukaryote population rich in genome set compositional diversity simultaneously becomes a eukaryote population rich in trait diversity.

This eukaryote population includes eukaryotes that have acquired new traits that may arise in the process of evolution, eukaryotes that have lost or deteriorated traits, and eukaryotes that have improved traits. It is believed that there are populations of eukaryotic organisms that can have enhancements, losses, degradation, alterations, etc. in various ways.

Therefore, the intended eukaryotic organisms can be obtained efficiently by selecting the eukaryotic organism population obtained by the present reorganization method based on the desired index.

As used herein, the term "genome set" refers to DNA that exists as chromosomal DNA in eukaryotic cells, is capable of self-replication in eukaryotic cells, and is transmitted to daughter cells.

In addition, the term "genetic recombination" as used herein means, in a broad sense, a DNA cleavage/recombination phenomenon that occurs between DNAs. Therefore, "genetic recombination" as used herein includes homologous recombination and non-homologous recombination. Furthermore, the term "genetic recombination" as used herein refers to genetic mutations, chromosomal inversions, unequal crossovers, crossovers, translocations, duplications, and deletions due to substitutions, insertions and deletions of one or more bases. It includes chromosomal mutations such as loss, copy number loss, copy number gain, chromosomal polyploidy and chromosomal aneuploidy. In addition, "genetic recombination" as used herein includes both genetic recombination within the same chromosome and genetic recombination between different chromosomes.

Various embodiments of the present disclosure are described in detail below.

(Method for genome rearrangement in eukaryotic organism)
(Genome rearrangement process)
This reorganization method comprises a genome reorganization step (hereinafter simply referred to as a reorganization step) in which a protein having DNA double-strand breaking activity is allowed to act temporarily in eukaryotic cells under the growth environmental conditions of said cells. can be provided. The rearrangement process is applied to cells of eukaryotic organisms. The restructuring process can be repeated one or more times as necessary. Moreover, various selection processes such as enrichment culture can be performed after the restructuring process. Moreover, such a reorganization process and a selection process can also be repeatedly implemented.

(eukaryotic organism)
This reorganization method is applicable to any eukaryotic organism. Eukaryotic organisms applicable to this restructuring method include animals, plants, and eukaryotic microorganisms. Animals include, but are not limited to, mammals and non-mammals such as various fish. In addition, the animal to be applied to this reorganization method may be derived from an animal, and may be in any form such as a cell, tissue, organ, or fertilized egg. Anything that retains the ability to regenerate into a whole animal, such as a fertilized egg, is convenient for obtaining a modified animal.

Plants to be applied to this reorganization method are not particularly limited, but for example, dicotyledonous plants, monocotyledonous plants, such as plants belonging to the family Brassicaceae, Poaceae, Solanaceae, Leguminosae, Salicaceae, etc. (see below) ).

Brassicaceae: Arabidopsis thaliana, Brassica rapa, Brassica napus, Brassica oleracea var. capitata, Chinese cabbage (Brassica rapa var. pekinensis), Bok choy (Brassica rapa var. rapa), Nozawana (Brassica rapa var. hakabura), Mizuna (Brassica rapa var. lancinifolia), Komatsuna (Brassica rapa var. peruviridis), Pakuchoi (Brassica rapa var. chinensis), Japanese radish (Brassica Raphanus sativus), Wasabi (Wasabia) japonica), etc.
Solanaceae: Nicotiana tabacum, Solanum melongena, Solaneum tuberosum, Lycopersicon lycopersicum, Capsicum annuum, Petunia, and the like.
Legumes: soybean (Glycine max), pea (Pisum sativum), broad bean (Vicia faba), wisteria (Wisteria floribunda), peanut (Arachis. hypogaea), japonicus (Lotus corniculatus var. japonicus), kidney bean (Phaseolus vulgaris), adzuki bean (Vigna angularis), Acacia, etc.
Asteraceae: chrysanthemum (Chrysanthemum morifolium), sunflower (Helianthus annuus), etc.
Palm family: Elaeis guineensis, Elaeis oleifera, Cocos nucifera, Phoenix dactylifera, Copernicia, and the like.
Anacardiaceae: Rhus succedanea, Anacardium occidentale, Toxicodendron vernicifluum, Mangifera indica, Pistacia vera, and the like.
Cucurbitaceae: Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, Cucumis sativus, Trichosanthes cucumeroides, gourds (Lagenaria siceraria var. gourda), etc.
Rosaceae: almonds (Amygdalus communis), roses (Rosa), strawberries (Fragaria), cherry blossoms (Prunus), apples (Malus pumila var. domestica) and the like.
Caryophyllaceae: Carnation (Dianthus caryophyllus), etc.
Salicaceae: Poplar (Populus trichocarpa, Populus nigra, Populus tremula), etc.
Gramineae: Zea mays, Oryza sativa, Hordeum vulgare, Triticum aestivum, Phyllostachys, Saccharum officinarum, Pennisetum pupureum, Erianthus ravenae ), Miscanthus virgatum, Sorghum, Panicum, etc.
Liliaceae: Tulipa, Lilium, etc.
Myrtaceae: Eucalyptus (Eucalyptus camaldulensis, Eucalyptus grandis), etc.

The plant to be applied to this reorganization method may be derived from a plant, but any plant that retains the ability to regenerate into a complete plant is convenient for obtaining a genome-modified plant. . Therefore, plants may be in any form such as cells, tissues, organs, seeds, callus, and the like.

Microorganisms are also not particularly limited, but microbial cells such as molds such as Aspergillus oryzae and yeast can be used in consideration of substance production and the like. Aspergillus genus, such as Aspergillus aculeatus (Aspergillus aculeatus) and Aspergillus oryzae (Aspergillus orizae), is mentioned as koji mold. As the yeast, various known yeasts can be used, and yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces pombe, Candida Candida yeast such as Candida shehatae, Pichia yeast such as Pichia stipitis, Hansenula yeast, Klockckera yeast, Schwanniomyces yeast and yeast of the genus Yarrowia, yeast of the genus Trichosporon, yeast of the genus Brettanomyces, yeast of the genus Pachysolen, yeast of the genus Yamadazyma, yeast of the genus Kluyveromyces marxianus ), yeast of the genus Kluyveromyces such as Kluveromyces lactis, and yeast of the genus Isatokenkia such as Issatchenkia orientalis. Among them, yeast of the genus Saccharomyces is preferable from the viewpoint of industrial usability. Among them, Saccharomyces cerevisiae is preferable.

Moreover, the yeast may be homothalismic yeast as well as heterothalismic yeast. Homotarhythmic yeasts can undergo spore breeding to enhance positive (excellent) genetic recombination results and eliminate the effects of negative genetic recombination when the selection process is performed after the restructuring process. can. In addition, because yeast carrying genome sets with negative genetic recombination can be efficiently eliminated to yield homothalmic yeast populations carrying genome sets with positive genetic recombination results, further one Alternatively, even if two or more rearrangement steps are repeated, excellent genome rearrangement efficiency can be obtained.

Eukaryotic organisms that are polyploid beyond intrinsic ploidy can also be used in the present reorganization methods. By using such polyploids as eukaryotic organisms, it is possible to suppress or avoid damage caused by various genetic recombination caused by DNA double-strand breaks, and dramatically and efficiently increase the diversity of genome set composition and traits. , can build populations that are favorable for proliferation.

As a polyploid that exceeds the intrinsic polyploidy, for example, since the intrinsic polyploidy of animals is diploid, a polyploid that exceeds the diploid can be used as a eukaryotic organism. In addition, since the proper ploidy of plants varies, polyploids exceeding the proper ploidy can be used as eukaryotic organisms. In microorganisms, such as yeast, polyploids greater than diploid can be used as eukaryotic organisms.

A polyploid that exceeds intrinsic polyploidy may be a wild type. For example, while diploids such as wheat exist as wild types, wild-type wheat exceeding diploids, that is, tetraploid wheat and hexaploid wheat also exist as wild types. of wheat may be used. Eukaryotic organisms obtained by artificially increasing the genome size can also be used as eukaryotic organisms.

The eukaryotic organism is preferably a tetraploid or higher polyploid. Tetraploids and higher polyploids include, for example, tetraploids, pentaploids, hexaploids, heploids, octoploids, and the like. According to the present disclosure, even if the chromosome ploidy is high, genetic recombination of the genome set via DNA double-strand breaks avoids the disadvantages related to the size of the genome and takes advantage of the genome size advantage. Diverse populations of eukaryotic organisms can be obtained. Therefore, when the intrinsic ploidy of eukaryotic organisms is tetraploid or higher, the efficiency of genetic recombination is increased, and it becomes possible to construct eukaryotic organism populations that are remarkably superior in diversity. It is more preferably pentaploid, still more preferably hexaploid, still more preferably heploid, and even more preferably octoploid or higher.

The eukaryotic organism may be a eukaryotic organism that has been artificially engineered to increase genome size to exceed intrinsic ploidy. When such a eukaryotic organism is used, a previously obtained eukaryotic organism or an already existing eukaryotic organism obtained by artificially increasing the genome size may be subjected to the rearrangement process. In addition, a step of subjecting a eukaryotic organism to be a eukaryotic organism to an operation for increasing the genome size to stably obtain a eukaryotic organism with polyploidy exceeding the intrinsic ploidy is performed, and the eukaryotic organism obtained by this step is performed. It may be in a form in which the reorganization process is performed on the nuclear organism.

Artificial genome manipulations include cell fusion using various cells, suppression of meiosis by temperature and pressure in fertilized and unfertilized eggs in animals, mating in plants, and supply of chromosome doubling inducers such as colchicine. etc. Furthermore, in the case of yeast, selective isolation of low-frequency zygotes between general heterothalism strains, methods applying mating type conversion systems, cell fusion methods, etc. (Experimental Methods for Biochemistry Vol. 39, Experimental Methods for Yeast Molecular Genetics (Taiji Oshima, Gakkai Publishing Center) Incidentally, a person skilled in the art can appropriately apply known methods to eukaryotic organisms in order to obtain polyploidy eukaryotic organisms exceeding proper ploidy.

(Present protein: protein having DNA double-strand breaking activity)
Although the present protein is not particularly limited, for example, a known DNA double-strand cleavage enzyme can be used. In this reorganization method, the DNA double-strand break activity is allowed to act while the DNA repair activity possessed by cells is activated under cell growth environmental conditions. The double-strand cleavage activity is not particularly limited. Therefore, any restriction enzyme or the like can be selected from known restriction enzymes and the like and used as the present protein.

The cleavage site (recognition site) possessed by the DNA double-strand cleavage enzyme is not particularly limited. Restriction enzymes generally have various recognition sites, and different recognition sites may result in different genome rearrangement effects. From the viewpoint of the efficiency of genetic recombination, DNA double-strand cleavage enzymes called so-called multiple restriction enzymes, which recognize about 4 to 6 bases on DNA, are preferred. Since the number of cleavage sites in the genome contributes to genome rearrangement efficiency, chromosomal DNA can be cleaved at a preferred frequency by using recognition sites with such base numbers. For example, a DNA double-strand cleavage enzyme with a 4-base or 5-base recognition site is more preferable, and a 4-base recognition site is even more preferable. Examples of such DNA double-strand cleavage enzymes include, but are not limited to, ApeKI, BsrI, BssKI, BstNI, BstUI, BtsCI, FatI, FauI, PhoI, PspGI, SmlI, TaqI, TfuI, TseI, Tsp45I, and TspRI. be done. In addition, various known multi-frequency enzymes such as Sse9I, MseI, DnpI and CviAII can be mentioned.

Different recognition sites for DNA double-strand breaking enzymes enable different genetic recombination, ie, different genome rearrangements. Therefore, different rearrangement results can be obtained by appropriately selecting the recognition site of the DNA double-strand breaking enzyme used in the rearrangement step. Therefore, the rearrangement step may be performed using one type of DNA-cleaving enzyme or using DNA-cleaving enzymes having two or more different recognition sites. Furthermore, when the rearrangement step is performed multiple times, different rearrangement steps may be performed using one or more DNA double-strand cleavage enzymes having different recognition sites.

As a restriction enzyme, a thermophile-derived restriction enzyme (so-called heat-stable DNA It is more preferred to use a double-strand-cleaving enzyme). This is because such a protein can exert a relatively mild DNA double-strand breaking activity at the cell growth temperature.

As such a present protein, for example, a restriction enzyme having an optimum temperature for DNA double-strand cleavage activity of 50° C. or higher and 80° C. or lower can be used. For example, the optimum temperatures are 50° C., 55° C., 60° C., 65° C., and 75° C. (all catalog values). The optimum temperature for the restriction enzyme can be selected based on catalogs (catalog values) of various sales companies. When the optimum temperature is less than 50°C, the optimum temperature is often close to the temperature of the cell growth environment conditions (referred to as the cell growth temperature) described later, and the DNA double-strand break activity becomes stronger at the cell growth temperature. sometimes too much. If the optimum temperature exceeds 80°C, the DNA double-strand breaking activity may become too weak at the cell growth temperature. In general, the optimum temperature is preferably 55°C or higher, more preferably 60°C or higher, and may be 62°C or higher, or about 65°C. In general, the optimum temperature is preferably 75°C or lower, more preferably 70°C or lower, and may be 68°C or lower.

In addition, as the present protein, if the DNA double-strand cleavage activity is too strong, cell damage is large. is preferably used. By doing so, a mild cutting action can be produced and the cutting action can be easily adjusted.

In general, it is preferred that the optimal temperature for the DNA double-strand breaking activity of the present protein is 10° C. or more higher than the upper limit of the cell growth temperature of the eukaryote to be used or a temperature close to it. It is more preferably 15° C. or higher, and still more preferably 20° C. or higher.

For example, since the cell growth temperature for yeast is 20°C or higher and 40°C or lower, the optimum temperature for the DNA double-strand cleavage enzyme used in the yeast restructuring step is preferably 50°C or higher, more preferably 55°C. °C or higher, may be 60 °C or higher, or may be about 65 °C.

In addition, the activity of the present protein at 37°C is preferably, for example, 10% or more and 30% or less when the DNA double-strand breaking activity at the optimum temperature is taken as 100%. This is because even with such a low activity, genetic recombination can be promoted and cell damage can be suppressed.

For example, the present protein can be appropriately selected and used from the following known restriction enzymes.

In this reorganization method, as described above, the DNA double-strand breaking activity possessed by this protein is not particularly limited, and any restriction enzyme or the like can be selected and used. For example, it can be selected from BclI, Sse9I, BstUI, TfiI, TseI, Tsp45I, TaqI and PhoI. These restriction enzymes have an optimum temperature of 50° C. or higher and 75° C. or lower, and a base length of the recognition site of 4 or higher and 6 or lower.

In addition, for example, from the viewpoint of optimum temperature and recognition site, ApeKI, BsrI, BssKI, BstNI, BstUI, BtsCI, FatI, FauI, PhoI, PspGI, SmlI, Sse9I, TaqI, TfuI, TseI, Tsp45I, and TspRI. Examples of suitable restriction enzymes for proteins include: Further, from the activity ratio between the optimum temperature and the cell growth temperature, for example, ApeKI, BsaBI, BsaJI, BsaWI, BsIEI, BslI, BsmBI, BsmI, BspQI, BsrDI, BsrI, BssKI, BstAPI, BstBI, BstNI, BstUI, BstYI, FatI, FauI, MwoI, Nb.BsmI, Nb.BsrDI, PspGI, SfiI, SmlI, TaqI, TfiI, TliI, TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I and the like.

In addition to the relationship between the present protein, the optimum temperature, and the cell growth temperature, the frequency and mode of cleavage (the number of recognized bases and the characteristics of the recognition sequence) are also taken into account to select the one suitable for use in the reorganization step. Regarding the optimum temperature, the restriction reaction conditions generally applied to restriction enzymes (10 units of restriction enzyme, 1 μg of DNA, buffer recommended for the restriction enzyme, BSA (100 μg/ml if necessary), reaction time (typically 1 hour) can be obtained by performing a restriction reaction at a plurality of different reaction temperatures, or it can be obtained from a catalog or the like of the supplier of the restriction enzyme. In addition, the ratio of DNA double-strand breaking activity at cell growth temperature (vs. activity at optimum temperature) can also be obtained from catalogs, etc. It can be obtained by performing a restriction reaction under the same conditions except for changing .

In order for the present protein to act within the cells of eukaryotic organisms, at least the present protein must be present within the cells. The protein is naturally present in the cell, but is preferably externally supplied from the viewpoint of optimum temperature and control of temporal action. When externally supplying the protein, the protein may be directly supplied to the eukaryotic cell, or an expression vector capable of expressing the gene encoding the protein may be supplied to the eukaryotic cell. may be transformed. Preferably, the protein is induced to act in cells of eukaryotic organisms. By doing so, the action of the present protein can be expressed at the intended timing.

A vector for expressing the present protein in a eukaryotic cell can be constructed by a person skilled in the art by conventionally known techniques depending on the cell type and transformation technique. Nucleotide sequences encoding this protein can be obtained from various databases based on the names of restriction enzymes. Moreover, a desired expression cassette can be constructed by suitably selecting a suitable promoter, terminator, enhancer, etc., in addition to appropriately obtaining a vector suitable for the cell. Although not particularly limited, it preferably accompanies a nuclear localization signal that is useful in the eukaryotic organism to be used.

For example, various conventionally known vectors can be used as a parent vector of an expression vector for expressing a protein in a plant cell. For example, a plasmid, phage, cosmid, or the like can be used, and can be appropriately selected according to the plant cell to be introduced and the method of introduction. Specific examples include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, and pBI-based vectors. In particular, when the method of introducing the vector into the plant body is a method using Agrobacterium, it is preferable to use a pBI-based binary vector. Specific examples of pBI-based binary vectors include pBIG, pBIN19, pBI101, pBI121, and pBI221.

The promoter is not particularly limited as long as it is capable of expressing the restriction enzyme gene in plants, and known promoters can be suitably used. Such promoters include, for example, cauliflower mosaic virus 35S promoter (CaMV35S), various actin gene promoters, various ubiquitin gene promoters, nopaline synthase gene promoter, tobacco PR1a gene promoter, tomato ribulose 1,5-bisphosphate carboxylase. - Oxidase small subunit gene promoter, napin gene promoter and the like can be mentioned. As will be described later, it is also preferable to use a promoter whose expression strength is lower than that of the 35S promoter, such as the SIG2 (AtSIG2) promoter derived from Arabidopsis thaliana sigma factor.

The expression vector may optionally contain other DNA segments in addition to the promoter and the restriction enzyme gene. The other DNA segment is not particularly limited, but includes terminators, selection markers, enhancers, base sequences for enhancing translation efficiency, and the like. In addition, the recombinant expression vector may further have a T-DNA region. The T-DNA region can enhance the efficiency of gene transfer, particularly when Agrobacterium is used to transfer the above recombinant expression vector into a plant.

The transcription terminator is not particularly limited as long as it functions as a transcription termination site, and may be a known one. For example, specifically, the transcription termination region (Nos terminator) of the nopaline synthase gene, the transcription termination region of cauliflower mosaic virus 35S (CaMV35S terminator), and the like can be preferably used. Among these, the Nos terminator can be used more preferably.

In addition, known elements can be appropriately selected and used as the selection marker and the nucleotide sequence for enhancing translation efficiency. The method for constructing the expression vector is not particularly limited either, and the necessary elements may be appropriately introduced into an appropriately selected parent vector.

Such expression vectors are introduced into plant cells to permit transient or constitutive expression of such proteins. To transiently express proteins, for example, the PEG method, electroporation method, and particle gun method are used to physically introduce an expression vector as a plasmid or the like into plant cells. For constant expression, the gene is integrated into the plant genome using the Agrobacterium method or the like.

The use of the Agrobacterium method is advantageous in that the diversity of the genome set can be ensured because the death rate of plant cells caused by the introduction of the gene encoding the present protein can be reduced. be. Also, the Agrobacterium method is preferably applied to dicotyledonous plants, particularly Arabidopsis thaliana.

Conventionally known methods can be applied to regenerate plants from transformed plant cells or the like.

In addition, in order to express the present protein in cells such as yeast, an expression vector suitable for yeast may be similarly constructed and introduced into yeast. An expression vector can be constructed by a person skilled in the art using a known method using a promoter, a terminator, and an appropriate enhancer. The expression cassette can be configured in a chromosomally integrated form or in a form that is retained extrachromosomally.

When constructing an expression vector, it is preferable to use an inducible promoter capable of intentionally determining the timing of protein expression. Moreover, in order to set the expression intensity to a desired level, it is preferable to appropriately set other control regions such as promoters and terminators.

For example, inducible promoters include galactose-inducible promoters such as GAL1 and GAL10, promoters used in induction systems by addition/removal of doxycycline such as the Tet-on system/Tet-off system, heat such as HSP10, HSP60 and HSP90. A promoter of a gene encoding a shock protein (HSP) can be used, but the CUP1 promoter, which is activated by the addition of copper ions, is preferably used. By using the CUP1 promoter, cells are cultured in a medium containing a carbon source such as glucose and not containing copper ions, and then a copper ion compound is added to the medium and cultured to induce the expression of a DNA double-strand breaking enzyme. be able to. The concentration of copper ions to be added can be appropriately set, and can be, for example, about 50 μM or more and 300 μM or less. In addition, the culture time can be about 1 hour to 6 hours. Furthermore, in order not to act on the DNA double-strand cleavage enzyme at the same time as the induction of expression, the cells should be heated at a temperature that does not correspond to the action conditions of the DNA double-strand cleavage enzyme (for example, about 25°C or lower, preferably about 20°C or lower). is preferably cultured. The CUP1 promoter has the advantage of being able to intentionally induce the expression of a DNA double-strand breaking enzyme simply and quickly.

A person skilled in the art can use conventionally known methods to transform yeast so that such an expression vector is introduced into yeast and retained intrachromosomally or extrachromosomally.

In this reorganization method, the gene encoding the present protein can be retained and expressed as a eukaryotic cell body, and has a desired polyploidy exceeding the specific polyploidy of the eukaryotic cell body. Eukaryotic cell bodies can be used. In order to obtain such a eukaryotic cell body, a candidate eukaryotic organism having the desired polyploidy is transformed with a gene encoding the present protein to obtain a eukaryotic organism having the desired polyploidy. can. A eukaryotic organism candidate having such a desired ploidy is obtained by repeating an artificial genome expansion operation once or twice or more for a eukaryotic organism having a ploidy exceeding the intrinsic ploidy. good too.

In addition, after transformation by introducing such a gene into a eukaryotic organism having intrinsic ploidy, the desired ploidy is obtained by repeating the artificial genome expansion operation on the transformant one or more times. A eukaryotic organism can be obtained.

As described above, a eukaryotic organism that expressably retains a gene encoding the present protein is also a breeding material for producing a eukaryotic organism or a eukaryotic population for breeding or the like. Therefore, according to the disclosure of the present specification, a eukaryotic organism useful as a breeding material that can be suitably used in the present restructuring method and a method for producing the same are also provided.

Also, for example, a production method particularly suitable for producing breeding material with a desired polyploidy is to subject eukaryotic organisms that are polyploids with intrinsic ploidy to a single artificial genome expansion operation. Alternatively, a step of obtaining a desired polyploid eukaryote by repeating two or more times, and transforming the desired polyploid eukaryote to express a gene encoding a protein having DNA double-strand breaking activity. and converting. In addition, this production method comprises the steps of transforming a polyploid eukaryotic organism having intrinsic polyploidy so that a gene encoding a protein having DNA double-strand breaking activity can be expressed; and B. repeating the artificial genome augmentation operation on the karyotes one or more times to obtain the desired ploidy transformed eukaryotes.

In such a eukaryotic organism and its production method, the eukaryotic organism, the protein having DNA double-strand breaking activity, the polyploidy or polyploidy, the protein having DNA double-strand breaking activity, and the gene encoding the protein Various embodiments of the present reorganization method already explained can be applied to expression, artificial manipulation of genome expansion, and the like.

(Embodiments in which the present protein acts in eukaryotic cells)
Next, the mode of action of the present protein in eukaryotic cells will be described. In the following description, the term "inside the cell of a eukaryotic organism" does not particularly limit the embodiment of the eukaryotic organism. For example, when the eukaryotic organism is a plant body, cells, tissues, and organs as a part of the plant body, as well as seeds, seedlings, or subsequently grown plant bodies, regardless of the form of the plant body. In addition, it means inside the cell in the callus. Moreover, when the eukaryotic organism is a microorganism such as yeast, it means that the present protein acts within the cell.

The rearrangement step allows the protein to act transiently within the cells of eukaryotic organisms under cell growth environmental conditions. By doing so, the DNA double-strand breaking activity of the protein and the DNA repair activity inherent in the cell can be caused to act, and the conditions can be controlled to balance these activities.

In order to make the present protein act temporarily in the cell, the present protein can be temporarily supplied to the cell from the outside, or the gene encoding the present protein can be provided under the control of an inducible promoter. If the cassette is to be retained on the chromosome, the induction conditions should be temporarily applied.

For example, in order to allow the present protein to act temporarily under cell growth environment conditions in cells that retain a gene encoding the present protein so that the expression can be induced, expression of the present protein is previously induced, and then the expression is induced in the cell growth environment. Although it may be applied under conditions, it may be applied under cell growth environmental conditions while inducing the expression of the present protein. The latter method is convenient because the expression of the present protein can be induced during the rearrangement process. When the former method is employed, the expression induction step is performed at a temperature sufficiently lower than the optimum temperature for the DNA double-strand breaking activity of the present protein in order to suppress the action of the DNA double-strand breaking activity. is preferred. For example, the expression induction step can be performed at about 15°C or higher and 25°C or lower.

(Environmental conditions for cell growth)
Environmental conditions for cell growth refer to conditions including a temperature (cell growth temperature) at which eukaryotic cells exhibit sufficient growth potential. More specifically, it refers to conditions including at least a temperature suitable for the growth of eukaryotic cells or a temperature generally used. The cell growth temperature may vary depending on the eukaryotic organism or organisms used. For example, for yeast such as Saccharomyces cerevisiae, it is generally from about 20°C to about 40°C. Therefore, the cell growth temperature (range) in yeast can be 20°C or higher and 40°C or lower. Further, for example, when the eukaryotic organism to be used is a plant body or its partial organ, tissue, cell, or the like, the normal growth or culture temperature applied to these corresponds to the cell growth temperature (range).

The cell growth temperature in the reorganization step is preferably a temperature at which the DNA double-strand breaking activity of the present protein at the optimal temperature is 100%, and more preferably about 5% or more and 30% or less. It is preferable that the temperature be about 5% or more and 20% or less. This is because even with such low activity, genetic recombination can be sufficiently promoted, and damage to cells and the like can be suppressed.

Although the cell growth temperature depends on the type of the present protein and the type of eukaryotic organism, it can generally be, for example, 18°C or higher and 45°C or lower. More preferably, for example, the lower limit is 20°C or higher, more preferably 22°C or higher, still more preferably 25°C or higher, still more preferably 30°C or higher, and even more preferably 35°C or higher. . Further, for example, the upper limit is preferably 45° C. or lower, more preferably 42° C. or lower, still more preferably 40° C. or lower, still more preferably 38° C. or lower, and still more preferably 37° C. or lower, More preferably, it is 36° C. or lower. This is because at such a temperature, damage to the cells themselves can be suppressed, and the DNA repair activity inherent in the cells can be expressed, making it easier to tolerate genome rearrangement by promoting genetic recombination. In particular, the above cell growth temperature is suitable for microorganisms such as yeast and plants.

Environmental conditions for cell growth can include temperature as well as light, gas, moisture, nutrient requirements, etc., depending on the eukaryotic organism, and these conditions are known to those skilled in the art. It can be appropriately set based on various publicly known information.

The time for which the present protein is allowed to act in the cell is not particularly limited, but considering the expression of the DNA repair action, it is preferably longer than the doubling time of eukaryotic cells. The action time is appropriately set so that suitable genome rearrangement and rearrangement efficiency can be obtained according to the DNA double-strand breaking activity (optimal temperature, breaking frequency, etc.) of the present protein and the type of eukaryotic organism. . Generally, for example, usually 1 hour or more than 1 hour, preferably 2 hours or more, more preferably 3 hours or more, even more preferably 4 hours or more, still more preferably 6 hours or more, and even more preferably 8 hours. Above, more preferably 10 hours or more. Also, it may be 12 hours or more, 18 hours or more, 24 hours or more, 48 hours or more, 60 hours or more, or 72 hours or more. Although the upper limit of the action time is not particularly limited, it can be about 168 hours or less, and may be 144 hours or less, 120 hours or less, 96 hours or less, or 72 hours or less.

The reorganization step is not particularly limited as long as it causes the present protein to act temporarily in the cell, and the form of the temporary action is not particularly limited. For example, the inducible promoter may be induced only at the beginning of the restructuring process and left to deplete the inducer, or the inducer may be continuously induced by supplementing the inducer. , multiple inductions may be performed discontinuously.

For example, with respect to TaqI described above, although it depends on the type and timing of the parent eukaryotic organism, the cells expressing TaqI are preferably heated at 20° C. or higher and 45° C. or lower, more preferably at 25° C. or higher and 42° C. or lower, even more preferably at can be cultured or grown at a temperature of 30°C or higher and 42°C or lower, more preferably 30°C or higher and 40°C or lower, more preferably 35°C or higher and 40°C or lower, still more preferably about 37°C. The duration of action is preferably 2 hours or longer, more preferably 4 hours or longer, still more preferably 5 hours or longer, still more preferably 6 hours or longer, still more preferably 12 hours or longer, still more preferably 24 hours or longer, and even more preferably Growing or culturing for 36 hours or more, even more preferably 48 hours or more, even more preferably 60 hours or more, even more preferably 72 hours or more. Such conditions are particularly suitable for microorganisms such as yeast and plants.

The restructuring step in which the present protein acts in the cells of eukaryotic organisms includes, for example, in plants, seeds before sowing harvested from eukaryotic organisms that are transformed plants capable of expressing the present protein, and seeds after sowing. Until germination, it is carried out for a certain period of time for seedlings after germination and more grown plants. In addition, for example, in yeast, the present protein is expressed for a certain period of time in yeast that has been transformed so that the present protein can be expressed.

Such rearrangement steps are preferably seeds or seedlings when the eukaryotic organism is a plant. This is because it facilitates processing of multiple specimens and is convenient for obtaining a population of eukaryotic organisms having a rearranged genome set. Also, in the case of yeast, it is also preferable to use a cycle in which budding is actively performed (that is, a cycle in which the polyploidy of the eukaryotic organism is maintained), such as the somatic cell division period.

For example, when the eukaryotic organism is a plant, the reorganization step is performed by growing seeds, seedlings, grown plants, etc. under a temperature condition of 37 ° C. for 24 hours, and then growing under lower temperature conditions (for example, Arabidopsis thaliana , the temperature can be returned to about 20° C. to 25° C.).

In addition, in the reorganization step when the eukaryotic organism is yeast, the yeast culture conditions are maintained at 37° C. for 24 hours, and then returned to the normal culture temperature (approximately 25° C. to 30° C.). mentioned.

In addition, action conditions such as temperature and time for the action of the present protein can be determined by those skilled in the art, such as the expression state of the present protein, the growth (proliferation) state of eukaryotic organisms, and the evaluation of genome rearrangement efficiency (using a reporter gene). It can be determined as appropriate by considering the present protein to be used, the eukaryotic organism, and the intended efficiency of genome rearrangement, using the evaluation by quantification, evaluation by ploidy histogram, or the like.

The restructuring process can be terminated as appropriate. The rearrangement process can be terminated by intentionally stopping the action of the present protein or by making the state in which the expression of the present protein is not induced. For example, an inducer of an inducible promoter controlling expression of the gene encoding the present protein may be removed.

By carrying out the reorganization step, the DNA double-strand breaking activity of this protein acts to break the chromosomal DNA in the cell, and the DNA repair activity inherent in the cell also acts to cause damage to the cell or gene. is suppressed or avoided, genetic recombination within chromosomes and between chromosomes is promoted. As a result, even if eukaryotic organisms have the intrinsic polyploidy of cells, damage to cells and genes is suppressed, and large-scale (diverse) genome rearrangements are tolerated within proliferative cells. The Rukoto. As a result, genome rearrangement efficiency can be dramatically improved. In addition, as a result of allowing large-scale genome rearrangement, it is possible to obtain a proliferative cell population (library) that retains a diverse genome set, resulting in diverse traits and sufficient resistance to selective pressure. A cell population that is advantageous for breeding by enrichment culture or the like can be obtained. Such an effect could not have been expected from the conventional operating conditions of high temperature and short time.

In addition, the DNA double-strand breaking activity of this protein (optimal temperature, recognition sequence, etc.) can be activated because the DNA repair activity of the cell can be activated by allowing this protein to act in cells under cell growth environmental conditions. , it is possible to freely control the genome rearrangement level and genome rearrangement efficiency by controlling the action temperature and action time. Such an effect could not have been expected from the conventional working conditions of high temperature and short time.

In addition, according to this restructuring method, the balance between DNA double-strand breaking activity and DNA repair activity can be controlled. It became possible to use this protein, and not only one type of restriction enzyme, but also a restructuring process using a combination of two or more types of restriction enzymes, and restriction enzymes with different recognition characteristics, different optimum temperatures, etc. Multiple restructuring steps can be combined. That is, according to this reorganization method, the diversity of usable restriction enzymes and other proteins of the present invention can also be expanded, so that it becomes possible to obtain cells having a highly diverse genome set.

Furthermore, in the present restructuring method, when homothalmic yeast is used as the eukaryotic organism, after performing the first restructuring step on the homothalmic yeast, the homothalmic yeast having the rearranged genome set is selected for the first trait. A selection step comprising spore breeding with selection under pressure may be carried out, and a second rearrangement step may also be carried out on said selected homothalmic yeast. By performing a selection process involving spore breeding on a library of homothalmic yeasts, it is possible to obtain homothalmic yeasts in which the positive effects of genetic modification are enhanced and the negative effects are eliminated. By performing a second rearrangement step on these homothalmic yeasts, a yeast library with a genome set of better genetic makeup can be obtained.

According to the rearrangement process, genetic recombination occurs in each eukaryotic organism subjected to the rearrangement process, and the genome set rearranged by the genetic recombination is maintained. As a result, a population of eukaryotic organisms comprising the rearranged genome set can be obtained. The individual eukaryotic organisms of this population of eukaryotic organisms comprise different "rearranged genome sets" resulting from the genetic recombination that occurred in the original population of eukaryotic organisms. The genome set composition of eukaryotic organisms that have undergone the rearrangement process is rich in diversity compared to conventional methods due to the action of DNA double-strand break activity and DNA repair activity.

For example, a genome set of a eukaryotic organism that constitutes a newly constructed eukaryotic organism population obtained by the present reorganization method has or has increased chromosomal aneuploidy as a result of genetic recombination. tend to Also, the genome set of such eukaryotic organisms is prone to having deletions and/or duplications in portions of the chromosomes as a result of genetic recombination. Furthermore, the genome set of such eukaryotic organisms tends to have a reduced genome size due to the loss of some regions of the genome set as a result of genetic recombination, or part of the genome set overlaps. tend to have an increased size over time. Also, genome sets of such eukaryotic organisms tend to have one or more mutations. Moreover, the genome sets of such eukaryotic organisms tend to have interchromosomal genetic recombination.

As described above, according to the present reorganization method, a high efficiency of genome reorganization is realized, so that a eukaryotic cell population having a highly diverse genome set can be obtained. Such cell populations have excellent adaptability in screening such as enrichment culture, and efficient breeding is possible.

In this reorganization method, one or more eukaryotes selected from the eukaryote population are used as eukaryotes, and the reorganization step and the selection step are repeated, such as further performing the reorganization step. You can also

In view of the above, the present rearrangement method can also be implemented as a method for producing a population of eukaryotic organisms comprising a rearranged genome set, which includes the rearrangement step.

(Method for producing a eukaryotic organism having a rearranged genome set)
A method for producing a eukaryotic organism having a rearranged genome set disclosed herein comprises: one or more of the above-described rearrangement steps; , and one or more steps of selecting the intended eukaryotic organism based on any indicator. According to this method, one or more intended eukaryotic organisms or a group thereof are selected from a population of highly diverse eukaryotic organisms, so the intended eukaryotic organisms can be efficiently obtained. . Moreover, according to this method, efficient breeding is possible using the selected eukaryotic organisms. Therefore, this production method can also be implemented as a method for breeding eukaryotes such as plants and yeast. In addition, conventionally known breeding techniques can be applied to the breeding of further progeny after obtaining useful plants, yeast, and the like.

For the restructuring step in this method, various embodiments of the restructuring step in the restructuring method already described can be applied as they are. In addition, in this method, the reorganization step and the selection step may be repeated. That is, the reorganization step and the selection step may be performed using one or more selected eukaryotic organisms or a population thereof as eukaryotic organisms.

In addition, according to this production method, the primary library obtained by genome rearrangement in the rearrangement process is subjected to the selection process by applying the selective pressure of the first trait to obtain useful eukaryotic cells (populations). ) can be obtained. Then, a secondary library obtained by further performing genome rearrangement on such cells (population) can be selected by applying selective pressure of a second trait. It is possible to efficiently obtain cells (populations) that are additionally provided with different traits.

In addition, when the present production method is applied to a homothalmic yeast as a eukaryotic organism, it is preferable to carry out spore breeding as a selection step. That is, in the selection process, through the process of forming spores, the heterozygous mutation introduced in the rearrangement process is homogenized. By going through a selection process such as enrichment culture from a homogenized yeast population, negative genetic recombination can be eliminated and positive genetic recombination can be doubled, resulting in a genome with a better genetic composition. Yeast with a set can be obtained.

As used herein, homothalismic yeast refers to not only wild-type homothallism yeast having an intrinsically active HO gene, but also heterothalism yeast, which is transformed into homothalism by introducing the HO gene from the outside. Yeast is also included.

Examples embodying the disclosure of the present specification are described below. It should be noted that the following examples are intended to illustrate the present disclosure and not to limit its scope.

(Preparation of TaqI gene yeast expression vector)
As a TaqI gene yeast expression vector, as shown in FIG. 1, a TaqI gene (TtTaqI opt) optimized for yeast codons downstream of a galactose-inducible promoter (pGAL1), 3'UTR (tDIT1) of DIT1 protein derived from Saccharomyces cerevisiae pORF-pGAL1-TtTaqI-tDIT1(AUR) was used.

(Generation of genome rearrangement evaluation yeast)
As shown in Fig. 2, the GF-FP reporter gene for evaluating genome rearrangement efficiency is flanked by an antibiotic Nourseothricin resistance marker (nat1) between approximately 600 bp on the N-terminal side and 600 bp on the C-terminal side of green fluorescent protein (GFP). is designed to By using this cassette, when the double-stranded DNA cleavage by TaqI causes homologous recombination between GF-FP and the full-length GFP gene is reconstructed, the yeast emits green fluorescence. Genome rearrangement can be detected by using, etc. (see FIG. 3).

S. cerevisiae BY4741 and BY4742 strains, in which the GF-FP reporter gene was introduced into the ADH3 gene upstream region (3000 bp to 2000 bp upstream from the transcription initiation site), were designated BY4741+GFP strain and BY4742+GFP strain, respectively. In addition, BY4741 + GFP (LEU) strain, BY4741 + GFP (HIS) strain, and BY4742 + GFP (LEU) strain in which the leu2 gene and his3 gene of each strain were complemented with S. cerevisiae S288C-derived LEU2 gene and HIS3 gene, A BY4742+GFP(HIS) strain was generated.

(Examination of TaqI expression induction conditions)
The TaqI gene yeast expression vector pORF-pGAL1-TtTaqIopt-tDIT1 (AUR) prepared in Example 1 was transformed into the BY4741 + GFP (HIS) strain prepared in Example 2 (BY4741 + GFP (HIS) + TaqI). . Culture overnight at 30°C using YPD medium (10 g/L Yeast extract, 20 g/L Peptone, 20 g/L Glucose) with glucose as sugar source + 0.5 mg/L Aureobasidin A (AbA). After that, the medium was changed to YPG (10 g/L Yeast extract, 20 g/L Peptone, 20 g/L Galactose) with galactose as a sugar source + 0.5 mg/L AbA medium. TaqI expression was induced by overnight culture at 30°C. In order to measure the TaqI expression level at each temperature, SDS-PAGE was performed with the same amount of cells, and Western blotting was performed using an anti-TaqI antibody. The results are shown in FIG.

As shown in Fig. 4, it was found that the expression level of TaqI was highest when the expression was induced at 20°C. In addition, as a result of comparing the viable cell rate, it was found that the viable cell rate decreased as the temperature increased, and TaqI was activated even at 30°C, causing cell death due to genome cleavage.

(Evaluation of genome rearrangement efficiency by high-temperature and short-time heat treatment)
Expression of TaqI was induced at 20° C. in the same manner as in Example 3 using the BY4741+GFP(HIS)+TaqI strain. After heat treatment at 42°C for 30 or 60 minutes to transiently promote TaqI activation, the percentage of cells with GFP fluorescence among 1 x 10 ⁵ cells was measured using a flow cytometer. Genome rearrangement efficiency was calculated. The results are shown in FIG. 5A. As a result, the genome rearrangement efficiency was 0.03-0.04%. On the other hand, as shown in FIG. 5B, the genome rearrangement efficiency increased to 0.06-0.07% when the medium was replaced with YPD medium after heat treatment and recovery culture was performed for 18 hours.

(Evaluation of genome rearrangement efficiency by treatment at cell growth temperature)
Using BY4741 + GFP (HIS) + TaqI strain, after changing the medium to YPG + 0.5 mg / L AbA medium in the same manner as in Example 3, gently while inducing TaqI expression at 20, 25, 30, 35 ° C. Activation of TaqI was performed. After 5, 23, and 46 hours, the yeast was collected, and the efficiency of genome rearrangement was measured using a flow cytometer in the same manner as in Example 4. The results are shown in FIG.

As a result, as shown in FIG. 6, the efficiency of genome rearrangement increased over time at any temperature. In particular, at 30°C or higher, the genome rearrangement efficiency increased significantly, reaching about 10-20 times higher than that of high-temperature, short-time heat treatment.

(Generation of diploid and tetraploid yeast)
BY4741 + GFP (LEU) strain and BY4742 + GFP (LEU) strain, BY4741 + GFP (HIS) strain and BY4742 + GFP (HIS) strain were joined according to a standard method, and cultured on an SD-Met-Lys agar plate at 30°C for 5 days. After the grown colonies were singled, the amount of genomic DNA was measured with a flow cytometer, and strains confirmed to be diploid were designated BY4743+GFP(LEU) strain and BY4743+GFP(HIS) strain.

BY4743+GFP(LEU) strain and BY4743+GFP(HIS) strain were fused to produce tetraploid yeast. Cell fusion was performed using the protoplast-PEG method. Colonies grown on SD-Leu-His agar plates were used as cell fusion candidate strains. After the grown colonies were singled, the amount of genomic DNA was measured with a flow cytometer, and the strain confirmed to be tetraploid was designated as the BY4744+GFP(LH) strain.

(Measurement of genome rearrangement efficiency using doubled yeast by treatment of cell growth temperature)
The TaqI gene yeast expression vector pORF-pGAL1-TtTaqIopt-tDIT1 (AUR) prepared in Example 1 was transformed into each doubled yeast prepared in Examples 2 and 6. Using the transformants, the medium was changed to YPG+0.5 mg/L AbA medium in the same manner as in Example 3, and TaqI was activated at the cell growth temperature while TaqI expression was induced at 35°C. After 22 hours, the yeast was collected, and the efficiency of genome rearrangement was measured using a flow cytometer in the same manner as in Example 4. The results are shown in FIG.

As shown in Fig. 7, the genome rearrangement efficiency of haploid yeast was 0.5-0.75%, whereas it increased to 1.0% in diploid yeast and 2.5% in tetraploid yeast. It has been shown to improve restructuring efficiency.

(Ploidy change due to genome rearrangement of doubled yeast by treatment of cell growth temperature)
As in Example 7, each doubled yeast was used to induce TaqI expression at 35° C., and TaqI was activated at the cell growth temperature. After 0, 16, and 46 hours, the yeast was recovered, fixed with 70% ethanol, and the genomic DNA was fluorescently stained by DAPI staining. Nuclear phase analysis was performed with a flow cytometer using each doubling yeast that was fluorescently stained. The results are shown in FIG. As shown in FIG. 8, in doubled yeast, an increased frequency of individuals with increased or decreased ploidy was observed.

(Generation of yeast for evaluation of genome rearrangement between chromosomes)
After amplifying the GF-nat1 region of pRS436 (NAT) prepared in Example 2 by PCR, δ sequences were added to both ends using overlapping PCR. The BY4741 strain was transformed with the amplified PCR product, and a yeast library was constructed in which GF-nat1 was randomly introduced at multiple sites in the genome by the delta integration method. Similarly, a yeast library was prepared by delta-integrating the nat1-FP region into the BY4742 strain, and the two libraries were randomly spliced to prepare a yeast library for assessing genome rearrangement between chromosomes (Fig. 9A). As shown in FIG. 9B, in this evaluation system, homologous recombination occurs in the interchromosomal GF and FP regions, and yeast emits green fluorescence when the full-length GFP gene is reconstituted. Genome rearrangement can be detected by using it.

The prepared interchromosomal genomic rearrangement evaluation yeast library was transformed with pORF-pGAL1-TtTaqIopt-tDIT1 (AUR) (+Taq). As a control, a yeast library for evaluating interchromosomal genome rearrangement was transformed with a vector in which the TaqI gene was deleted from pORF-pGAL1-TtTaqIopt-tDIT1 (-Taq). Using the prepared interchromosomal genome rearrangement evaluation yeast library, TaqI was activated at 35° C. in the same manner as in Example 7 while TaqI expression was induced. After 0, 24, 48 and 72 hours, the yeast was harvested and the genome rearrangement efficiency was calculated by calculating the ratio of cells with GFP fluorescence among 1×10 ⁶ cells using a flow cytometer. The results are shown in Figure 9C.

As shown in FIG. 9C, the genome rearrangement efficiency increased up to 15-fold when TaqI was expressed (+Taq) compared to when TaqI was not expressed (-Taq) (FIG. 9C).

Furthermore, similar to Example 4, induction culture was performed at 20°C, followed by heat treatment at 42°C for 30 minutes or 60 minutes to transiently activate TaqI. Genome rearrangement efficiency was calculated by calculating the percentage of cells with GFP fluorescence in 1×10 ⁶ cells with a flow cytometer. The results are shown in FIG.

As shown in FIG. 10, the efficiency of genome rearrangement did not increase regardless of the presence or absence of TaqI expression. After the heat treatment, the medium was changed to YPD medium and recovery culture was performed for 18 hours, but the efficiency of genome rearrangement did not increase. From the above, it was found that the efficiency of genetic recombination between chromosomes is low in the method in which restriction enzymes act at high temperature for a short period of time.

(Evolutionary breeding of xylose utilization ability by genome doubling and genome rearrangement)
(Acquisition of OC2-A (CF) shares)
OC2-A strain was constructed by introducing xylose utilization gene into wine yeast OC-2 strain. That is, based on the OC700 strain (JP 2014-193152) in which the xylose assimilation gene was introduced into the wine yeast OC-2 strain, the ADH2 gene was disrupted, the ADH1 gene was enhanced, and the mhpF gene (derived from E. coli) was added. The introduced OC2-A strain was obtained.

(Acquisition of OC2-A (CF) shares)
Using the OC2-A strain, evolutionary breeding for xylose assimilation ability was performed by genome rearrangement as follows. An OC2-A (KlURA3) strain, in which the URA3 gene derived from Kluyveromyces lactis was introduced into the OC2-A strain, and an OC2-A (KmTRP1) strain, in which a TRP1 gene derived from Kluyveromyces marxianus was introduced, were prepared. Using both strains, cell fusion was performed by the protoplast-PEG method to prepare a cell fusion strain OC2-A (CF) strain.

OC2-A(CF) strain was transformed with TaqI gene yeast expression vector pORF-pGAL1-TtTaqIopt-tDIT1(AUR) to obtain OC2-A(CF)+TaqI strain. Using the OC2-A(CF)+TaqI strain, the medium was changed to YPG+0.5 mg/L AbA medium in the same manner as in Example 3, and cultured overnight at 20° C. to induce expression of TaqI. After that, TaqI was activated at 35° C. for 8 to 24 hours, and recovery culture was performed in YPD medium+0.5 mg/L AbA medium.

(Acquisition of OC2A-C5 shares)
Enrichment culture (35° C.) was performed by repeating culture and subculture in a medium containing xylose as a main sugar source. After starting enrichment culture, the cells were cultured in YPX medium (10 g/L Yeast extract, 20 g/L Peptone, 1 g/L Glucose, 20 g/L Xylose) slightly supplemented with glucose for about 300 hours. Thereafter, the medium was changed to YPX medium (10 g/L Yeast extract, 20 g/L Peptone, 20 g/L Xylose) containing only xylose as a sugar source, and enrichment culture was further performed for about 200 hours. FIG. 11 shows changes in the amount of cells in these culture steps. The culture medium after the enrichment culture was streaked on a YPD agar plate, and the single strain was designated as the OC2A-C5 strain.

(Xylose assimilation capacity test of OC2A-C5 strain)
The xylose assimilation ability of strain OC2A-C5 was evaluated by fermentation test. OC2-A strain, OC2-A(CF) strain, and OC2A-C5 strain were inoculated into 5% xylose fermentation medium (10 g/L yeast extract, 50 g/L Xylose) at OD600 = 1.0, and incubated at 32°C. A fermentation test was performed. Samples were taken at 0, 16, 24, 48 and 72 hours and xylose and ethanol were measured by HPLC. The results are shown in FIG.

As shown in FIG. 12, the OC2A-C5 strain has significantly improved xylose assimilation ability compared to the parent strain OC2-A strain and its cell fusion strain OC2-A (CF) strain. It completely consumed 50 g/L of xylose and produced 21.3 g/L of ethanol in 48 hours.

Furthermore, except for using a high sugar concentration glucose + xylose mixed fermentation medium (10 g/L Yeast extract, 80 g/L Glucose, 100 g/L Xylose), evaluation of fermentation ability was performed in the same manner as the above assimilation test. gone. The results are shown in FIG.

As shown in FIG. 13, the OC2A-C5 strain has an improved ability to assimilate xylose compared to the parent strain OC2-A strain and its cell fusion strain OC2-A (CF) strain, completely consumed 80 g/L glucose and 100 g/L xylose and produced 76.2 g/L ethanol.

As shown in FIGS. 12 and 13, it was found that the OC2A-C5 strain remarkably increased the ability to assimilate xylose and, as a result, also increased the ability to produce and ferment ethanol and the like. In particular, even in a fermentation test at a high sugar concentration, which is a realistic fermentation condition, the fermentation ability was about 1.3 times that of the OC2-A (CF) strain and about 1.4 times that of the OC2-A strain. It was found to be excellent in practical xylose assimilation performance.

Based on the above results, it was concluded that enrichment culture of the OC2-A(CF)+TaqI strain into which the TaqI gene was introduced under the evolutionary pressure of a xylose-containing medium after genome rearrangement in which the TaqI gene was expressed resulted in an evolutionary change. And it was found that the OC2A-C5 strain, which is a useful strain, can be obtained by selection. That is, it was found that by growing cells in which a DNA double-strand breaking enzyme is expressed and activated under arbitrary conditions, cells that meet the conditions can be efficiently obtained.

(Evolutionary breeding of heat resistance by genome rearrangement)
Using the OC2A-C5 strain prepared in Example 10, TaqI treatment was performed in the same manner as in Example 10, and the genome rearrangement step was further performed to prepare a genome-rearranged yeast library. Next, YPX medium (10 g/L Yeast extract, 20 g/L Peptone, 20 g/L Xylose) was cultured at 39 to 41° C., and subculture was repeated to enrich culture for about 500 hours. The culture solution after the enrichment culture was streaked on a YPD agar plate, and the single strain was designated as the OC2A-TT strain.

Using the OC2A-TT strain and the same, heat resistance was evaluated by a fermentation test at 40°C. OC2-A strain, OC2-A(CF) strain, OC2A-C5 strain, and OC2A-TT strain were added to a mixed fermentation medium containing glucose and xylose (10 g/L Yeast extract, 30 g/L Glucose, 30 g/L Xylose). Each was inoculated at OD600 = 1.0 and a fermentation test was performed at 40°C. Sampling was performed at 0, 16, 24, 48 and 65 hours, and glucose, xylose and ethanol were measured by HPLC. The results are shown in FIG.

As shown in FIG. 14, the OC2A-TT strain was found to have increased growth ability (heat resistance) at 40° C. compared to other strains. As shown in FIG. 14, the heat resistance of the OC2A-TT strain is remarkably increased, and it exhibits good xylose assimilation ability even in good high-temperature culture. As a result, it was found that the ability to produce and ferment ethanol etc. was also increased. In particular, even in fermentation tests at a realistic sugar concentration, fermentation is about 1.2 times that of OC2-A strain, and about 3 times that of OC2-A (CF) and OC2A-C5 strains. It has been found to be excellent in practical use because it specializes in heat resistance.

Based on the above results, the OC2-A (C5) + TaqI strain into which the TaqI gene was introduced was enriched and cultivated under the evolutionary pressure of high temperature after reorganization by expressing the TaqI gene. could obtain a useful strain, the OC2A-TT strain, through different evolutionary changes and selection. According to this reorganization method, the following things were found. That is, a useful secondary library can be obtained by further performing genome rearrangement on the cells (population) selected by applying the selective pressure of the first trait to the primary library obtained by genome rearrangement. Then, selection pressure of a second trait can be applied to this secondary library for selection. It is possible to efficiently obtain cells (populations) that are additionally provided with different traits.

(Improvement of thermotolerance by genome rearrangement and spore breeding)
Based on the OC700 strain (Japanese Patent Laid-Open No. 2014-193152) in which xylose assimilation genes were introduced into the wine yeast OC-2 strain, the ADH2 gene was disrupted, the ADH1 gene was enhanced, and the mhpF gene (derived from E. coli) was introduced, followed by acclimation. Using the OC2-B strain, whose xylose assimilation ability was improved by culture, evolutionary breeding for thermostability was carried out by genome rearrangement and spore breeding. The OC2-B strain was transformed with the TaqI gene yeast expression vector pORF-pGAL1-TtTaqIopt-tDIT1 (AUR) prepared in Example 1 to obtain the OC2-B+TaqI strain. Using the OC2-B+TaqI strain, the medium was changed to YPG+0.5 mg/L AbA medium in the same manner as in Example 3, and cultured overnight at 20° C. to induce expression of TaqI. Then, TaqI was activated at 35°C for 8 to 24 hours, and the sample was recovered in YPD medium + 0.5 mg/L AbA medium and used as the genome-rearranged yeast library (1 ^st -TAQing). . YPX medium (10 g/L Yeast extract, 20 g/L Peptone, 20 g/L Xylose) was cultured at 40 to 42°C, and enrichment culture was performed by repeating subculture. On the way, TaqI treatment was performed again (2 ^nd -TAQing), and enrichment culture was further performed at 42-43°C. The culture solution after the enrichment culture was streaked on a YPD agar plate, and the single strain was designated as the OC2B-TT1 strain.

In addition, the culture solution after enrichment culture was applied to a sporulation agar plate (10 g/L Potassium acetate, 1 g/L Yeast extract, 0.5 g/L Glucose, 20 g/L Agar) to perform sporulation. The cells were recovered from the sporulation agar plate, and after extracting the spores by a conventional method, they were diluted and spread on a YPD agar plate. Since the OC2 strain is a homothalism strain, it is expected that heterozygous mutations introduced by genome rearrangement will homogenize during automatic diploidization from spores. About 10,000 clones grown on the YPD agar plate were collected and enriched in YPX medium at 42-44°C. The culture medium after the enrichment culture was streaked on a YPD agar plate, and the single strain was designated as the OC2B-TT2 strain.

A fermentation test at 40°C was performed using the OC2B-TT1 strain and the OC2B-TT2 strain. OC2-B strain, OC2B-TT1 strain, and OC2B-TT2 strain were inoculated into glucose + xylose mixed fermentation medium (10g/L Yeast extract, 50g/L Glucose, 50g/L Xylose) at OD600 = 5.0, and incubated at 40℃. A fermentation test was performed on Sampling was performed at 0, 17, 24 and 48 hours and measured by HPLC. The results are shown in FIG.

As shown in FIG. 15, both the OC2B-TT1 strain and the OC2B-TT2 strain improved in xylose assimilation capacity at 40° C. compared to the parent strain OC2-B strain. Notably, after genome rearrangement, the spore-breeding strain OC2B-TT2 completely consumed 50 g/L glucose and 50 g/L xylose and produced 40.0 g/L ethanol in 48 hours). From the above results, the OC2B-TT2 strain that underwent spore breeding exhibited a higher xylose assimilation ability than the OC2B-TT1 strain that did not. Based on the above, in homothalismous yeast with a rearranged genome set, homogenization of heterozygous mutations through spore breeding enhances the positive effects of genetic recombination and reduces the negative effects. found to be

(Evaluation of genome rearrangement efficiency by treatment at cell growth temperature)
Using BY4741 + GFP (HIS) + TaqI strain, after changing the medium to YPG + 0.5 mg / L AbA medium in the same manner as in Example 3, TaqI TaqI was activated for a long period of time while inducing the expression of . After 0, 5, 24, 48, 72 and 147 hours, the yeast was harvested and PI-stained to determine viability. In living cells, PI does not permeate into the cell due to the cell membrane, but in dead cells, the cell membrane is destroyed, so PI enters the cell and stains the nucleic acid, making it possible to distinguish between life and death. The stained yeast was analyzed with a flow cytometer in the same manner as in Example 4 to measure genome rearrangement efficiency. The results are shown in FIGS. 16 and 17. FIG.

As shown in FIG. 16, when all cells were analyzed, the genome rearrangement efficiency was the highest at 0.8 to 1.0% in the temperature range of 35-39°C. Moreover, as shown in FIG. 17, when living cells were analyzed, the genome rearrangement efficiency was the highest at about 0.5% at 35-37°C. From the above results, it was concluded that high genome rearrangement efficiency can be obtained by applying DNA double-strand breaking activity at a relatively weak level under general culture conditions (culturing temperature of 40°C or less) applied to yeast. I found out. In addition, by appropriately setting the DNA double-strand breaking activity and the temperature at which it acts from the culture temperature generally applied to eukaryotic organisms and the optimum temperature of the protein having DNA double-strand breaking activity, It was found that genome rearrangement that is advantageous for improving genome rearrangement efficiency and that has diversity is possible.

Claims (14)

A method for genome rearrangement in eukaryotic cells (but excluding yeast cells, plant cells, embryos and germ cells that retain the ability to regenerate into a complete human being including human fertilized eggs, and human cells within a human body), ,
A thermophile-derived protein having thermostable DNA double-strand breaking activity is inducibly expressed at a temperature of 30° C. or higher and 40° C. or lower or at the growth temperature of the eukaryotic cell, and the genome in the eukaryotic cell. 1 or 2 or more genome rearrangement steps for genetic recombination by acting on
with
The method, wherein in the step of genome rearrangement, the protein is inducibly expressed and the rearrangement is allowed to act on the genome at the same time for 18 hours or more and 168 hours or less. 2. The method of claim 1, wherein the restructuring time is 24 hours or more. 3. The method according to claim 1 or 2, wherein the restructuring time is 24 hours or more and 72 hours or less. The method according to any one of claims 1 to 3, wherein the restructuring time is 24 hours or more and 48 hours or less. The method according to any one of claims 1 to 4, wherein the protein is one or more selected from BclI, Sse9I, BstUI, TfiI, TseI, Tsp45I, TaqI and PhoI. The method according to any one of claims 1 to 5, wherein the protein is one or more selected from Sse9I, BstUI, TfiI, TseI, Tsp45I and TaqI. The method according to any one of claims 1 to 6, wherein said protein is TaqI. The temperature at which the protein is allowed to act on the genome is a temperature of 30° C. or higher and 40° C. or lower, or within the range of the growth temperature of the eukaryotic cell, which is the optimum temperature for the DNA double-strand breaking activity of the protein. The method according to any one of claims 1 to 7, wherein the temperature is about 5% or more and 20% or less when taken as 100%. The method of any of claims 1-8, wherein said eukaryotic cell is polyploid above intrinsic ploidy. 10. The method of claim 9, wherein the polyploid is a polyploid due to artificial genome size expansion engineering. The method according to any one of claims 1 to 10, wherein said eukaryotic cells are animal cells. Eukaryotic cells with a rearranged genome set, excluding yeast cells, plant cells, embryos and germ cells retaining the ability to reproduce into a complete human being, including human fertilized eggs, and human cells in vivo. A method of production,
A thermophile-derived protein having thermostable DNA double-strand breaking activity is inducibly expressed at a temperature of 30° C. or higher and 40° C. or lower or at the growth temperature of the eukaryotic cell, and the genome in the eukaryotic cell. 1 or 2 or more genome rearrangement steps for genetic recombination by acting on
with
The method, wherein in the step of genome rearrangement, the protein is inducibly expressed and the rearrangement is allowed to act on the genome at the same time for 18 hours or more and 168 hours or less. Furthermore, one or more selection steps of selecting intended eukaryotic cells based on any index from the population of eukaryotic cells holding the rearranged genome set,
13. The method of production of claim 12, comprising: A method for producing a population of eukaryotic cells ( but excluding yeast cells, plant cells, embryos and germ cells retaining the ability to regenerate into a complete human being, including human fertilized eggs, and human cells within a human body). hand,
A thermophile-derived protein having thermostable DNA double-strand breaking activity is inducibly expressed at a temperature of 30° C. or higher and 40° C. or lower or at the growth temperature of the eukaryotic cell, and the genome in the eukaryotic cell. 1 or 2 or more genome rearrangement steps for genetic recombination by acting on
with
The method, wherein in the step of genome rearrangement, the protein is inducibly expressed and the rearrangement is allowed to act on the genome at the same time for 18 hours or more and 168 hours or less.

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