JP2007061024A - Organism excellent in resistance to lactic acid and method for preparing organism excellent in resistance to lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare a microorganism having resistance to lactic acid without elucidating or genetically introducing a gene having resistance to lactic acid into a microorganism having no resistance to lactic acid or by introducing mutation into the microorganism having no resistance to lactic acid. <P>SOLUTION: Mutation is introduced into an organism by using a method and a system for rapidly imparting a desired character to the organism and the organism is cultured in the presence of a desired lactic acid to prepare the organism, preferably microorganism having resistance to the lactic acid. The organism has higher resistance than resistance which natural type of the organism has. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a method for producing a microorganism excellent in lactic acid resistance, and a microorganism excellent in lactic acid resistance produced by the method.

Accumulation of lactic acid produced by lactic acid fermentation decreases both the medium pH and intracellular pH, but microorganisms consume ATP to keep the intracellular pH constant and try to pump protons out of the cell. . For example, S.M. In the case of cerevisiae, when the extracellular pH is 3.0, the intracellular pH is 5.5 to 5.75 (Non-Patent Document 1 = Imai, T. et al. Appl. Environ. Microbiol. 61, 3604-3608, 1995), when the extracellular pH is 6.0 to 10.0, the intracellular pH is maintained at 5.9 to 6.75 (Non-Patent Document 2 = Imai, T. et al. J. Biotechnol. 38, 165-172, 1995). When microorganisms are unable to maintain intracellular pH reduction, cAMP protein kinase, which is a key enzyme in glycolysis and gluconeogenic pathway, suppression of intracellular cAMP concentration, trigger of other biological reactions such as heat shock, etc. In the end, it is thought that growth is suppressed.

  Various attempts have been made to produce lactic acid from unused biomass using lactic acid bacteria represented by Lactobacillus, but the pH at which the lactic acid fermentation is carried out is as high as 5.0 to 5.5, and the pKa of lactic acid (˜3. 8) Fermentation in the following is desired. The reason is that lactic acid below pKa (up to 3.8) exists in an undissociated state and can be extracted directly from the fermentation broth by lactic acid extraction. This means that the direct extraction using can not be performed and the cost of extraction and purification becomes high. It is very important for industrial production to produce a higher concentration of lactic acid by lactic acid fermentation.

  Lactococcus lactis cell growth under low pH is suggested to be related to 18 loci using insertion mutation analysis, and many resistance mechanisms against low pH are expected to be involved (Non-patent Document 3). = Rallu, F. et al. Mol. Microbiolo. 35, 517-528, 2000). Further, from the results of proteomic analysis of adaptation of Lactobacillus to acid, it has been reported that cooperative expression induction of 63 kinds of proteins occurs (Non-patent Document 4 = DeAngelis, M. et al. Microbiology 147, 1863- 1873, 2001). In addition, from the results of whole genome analysis of Lactobacillus acidophilus NCFM, nine kinds of regulatory systems composed of two components are estimated as genes related to acid resistance (Non-Patent Document 5 = Alternmann, E. et al. Proc). Natl.Acad.Sci.USA.102 (11), 3906-3912, 2005).

In the analysis of acid resistance of Lactobacillus sanfranciscensis, only GrpE was selectively increased in expression among the four heat shock proteins (Non-patent Document 6 = Maria DeAngelis et.al. Microbiol.147, 1863-1873, 2001). . As shown in the above examples, there is a lot of room for studying the lactate tolerance mechanism at the gene level.
Currently, the following methods are used as methods for imparting lactic acid tolerance or maintaining lactic acid production ability. The first method is a method of newly isolating a lactic acid resistant strain having a high degree of lactic acid resistance. In order to carry out genetic recombination for producing a high concentration of lactic acid using the obtained lactic acid resistant bacteria as a host, it is necessary to develop a vector system, and there is a long way to practical use. The second method is a method in which a lactic acid bacterium is treated with a chemical mutagen and a mutation is introduced to produce a lactic acid resistant mutant, but it has not succeeded in conferring significant lactic acid resistance. The third method is a method for producing a lactate-resistant mutant by cell fusion of lactic acid bacteria having different properties. As a specific example, a lactic acid resistant strain that produces lactic acid three times as much as a wild strain at pH 4.0 has been obtained (Non-patent Document 7 = Patnaik, R. et al. Nature Biotechnology 20.707-712, 2002). Furthermore, it is unclear whether a strain having a high tolerance to lactic acid can be produced. In the fourth method, a lactic acid tolerance-related gene isolated from a lactic acid resistant bacterium is introduced into a host bacterium by gene recombination to impart lactic acid resistance. As a specific example, three genes of orfC belonging to the multidrug resistance gene family isolated from wine yeast Oenococcus oeni, putative transcriptional regulator orfB of LysR family, and low molecular weight heat shock protein Lo18 are introduced into Escherichia coli. There is an example of imparting resistance (Non-patent Document 8 = Lett. Appl. Microbiol. 33 (2), 126-130, 2001). It is unclear whether this method can be applied to species that want to confer lactic acid tolerance. Even if it can be applied, there is no rational means other than increasing the expression of lactic acid resistance-related genes when it is desired to further enhance the lactic acid resistance level. The fifth method is a method of acclimatizing to acid using a chemostat. As a specific example, after pretreatment at a low pH (pH 5.0, 60 minutes) less than the lethal amount of the bacterium, treatment with lethal pH (pH 3.0) is induced to induce expression of the resistance gene and acclimatize to lactate resistance. There is a method (Non-Patent Document 9 = Lorca GL et.al.J.Mol.Microbiol.Biotechnol, 4 (6), 525-532, 2002). In this method, it is completely unclear whether the trait acquired by habituation is stably maintained, to which species, and what lactic acid concentration can be acclimated.
In addition, as a method for maintaining the lactic acid fermentation ability for a long period of time, in order to prevent the lactic acid fermentation ability of the lactic acid bacteria from decreasing in the later stage of the culture, the lactic acid bacteria are activated by applying a static magnetic field in a magnetic field formed by a superconducting magnet. A method is considered in principle (Patent Document 1 = Akira Miura, JP-A-2005-34040). However, in an industrial production situation, it is practically impossible to place a host bacterium in a superconducting magnetic field.
Imai, T .; et al. Appl. Environ. Microbiol. 61, 3604-3608, 1995 Imai, T .; et al. J. et al. Biotechnol. 38,165-172,1995 Rallu, F.M. et al. Mol. Microbiolo. 35,517-528,2000 DeAngelis, M.M. et al. Microbiology 147, 1863-1873, 2001 Altermann, E .; et al. Proc. Natl. Acad. Sci. USA. 102 (11), 3906-3912, 2005 Maria DeAngelis et. al. Microbiol. 147, 1863-1873, 2001 Patnaik, R.A. et al. Nature Biotechnology 20.707-712, 2002 Lett. Appl. Microbiol. 33 (2), 126-130, 2001 Lorca GL et. al. J. et al. Mol. Microbiol. Biotechnol, 4 (6), 525-532, 2002 JP 2005-34040 A

  An object of the present invention is to provide a method for imparting a trait excellent in resistance to lactic acid at a high concentration at low cost, in a short time, and efficiently to an experimental microorganism having low lactic acid resistance.

  Another object of the present invention is to provide a microorganism imparted with a trait excellent in lactic acid resistance.

  The above object of the present invention is to cultivate in the presence of a lactic acid concentration that does not normally grow using an organism subjected to accuracy in gene replication as a result of intensive studies, and to isolate the growing organism. This has been achieved by a method for producing resistant microorganisms.

  It is possible to produce microorganisms with excellent resistance to various lactic acids. Further, the present invention does not require the cost for elucidating the lactate tolerance mechanism, and the genomic information of experimental microorganisms and the developed vector system that have been elucidated so far will be effectively utilized. When lactic acid is produced industrially using the lactic acid resistant yeast produced in the present invention as a host, lactic acid fermentation corresponding to the increased degree of lactic acid resistance becomes possible, leading to cost reduction.

  Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

(the term)
Listed below are definitions of terms particularly used in the present specification.

  In this specification, “living organism” is used in the broadest sense in the field and refers to a living phenomenon, typically cell structure, proliferation (self-reproduction), growth, regulation, substance metabolism, It has various characteristics such as repair ability, and usually has inheritance governed by nucleic acids and growth involved in metabolism governed by proteins as basic attributes. Organisms include viruses, prokaryotes, eukaryotes (unicellular organisms such as yeast, multicellular organisms such as plants, animals, etc.) and the like. It will be appreciated that the methods of the invention can be applied to any organism, including Gram positive bacteria and higher organisms such as eukaryotes. In the present specification, a small-sized organism is also referred to as “microorganism”, but it is understood that the organism and the microorganism can be used interchangeably in this specification because they are the same in terms of lactic acid resistance.

  As used herein, “eukaryotic organism” is used in the same manner as commonly used in the art, and refers to an organism composed of cells having a nuclear membrane and a distinct nuclear structure. Examples of eukaryotes include unicellular organisms such as yeast, plants such as rice, wheat, corn and soybeans, animals such as mice, rats, cows, horses, pigs and monkeys, and insects such as flies and silkworms. But not limited to. In this specification, yeast, nematode, fruit fly, silkworm, rice, wheat, soybean, corn, Arabidopsis, human, mouse, rat, cow, horse, pig, frog, fish (eg, zebrafish), etc. are used as models. Can be, but is not limited to.

  As used herein, “prokaryote” is used in the same manner as commonly used in the art, and refers to an organism composed of cells that do not have a clear nuclear structure. Examples of prokaryotes include, but are not limited to, gram-negative bacteria such as E. coli and Salmonella, Gram-positive bacteria such as Bacillus subtilis, actinomycetes, and staphylococci, cyanobacteria, and hydrogen bacteria. In this specification, it can typically be used as a gram positive bacterium other than E. coli, but is not limited thereto.

  As used herein, “animal” is used in the broadest sense in the art, and includes vertebrates and invertebrates (eg, arthropods). Examples of animals include, but are not limited to, mammals, birds, reptiles, amphibians, fishes, insects, and worms. The animal may preferably be a vertebrate, such as, but not limited to, eel eels, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals and the like. In one embodiment, the animal is a mammal (e.g. single pore, marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, Cloven hoofs, rodents, scales, marine cattle, cetaceans, primates, rodents, rabbits, etc.) More preferably, it can be a primate (for example, chimpanzee, Japanese monkey, human) or other species that can be a model animal (for example, odd-hoofed, cloven-hoofed, rodents such as mice, rabbit eyes, etc.) It is not limited. Since it was first clarified in the present invention that the method of the present invention can be applied to any organism, it should be understood that any organism can be targeted. It is.

  As used herein, “plant” is a general term for organisms belonging to the plant kingdom, characterized by chlorophyll, hard cell walls, the presence of abundant permanent embryonic tissues, and organisms that are not capable of motility. . Typically, a plant refers to a flowering plant having cell wall formation and anabolism by chlorophyll. “Plant” includes both monocotyledonous and dicotyledonous plants. Preferred plants include, but are not limited to, useful plants such as monocotyledonous plants belonging to the family Gramineae such as wheat, corn, rice, barley and sorghum. Other examples of preferred plants include tobacco, pepper, eggplant, melon, tomato, sweet potato, cabbage, leek, broccoli, carrot, cucumber, citrus, Chinese cabbage, lettuce, peach, potato and apple. Preferred plants are not limited to crops, but also include flowers, trees, lawns, weeds and the like. Unless otherwise indicated, a plant means any plant, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include roots, leaves, stems and flowers. Examples of plant cells include callus and suspension culture cells. Since it was first clarified in the present invention that the method of the present invention can be applied to any organism, it should be understood that any organism can be targeted. It is.

  As used herein, “gene” refers to a sequence of a certain length of nucleic acid present in a cell. In the present invention, the gene may or may not define a genetic trait. In the present specification, a gene refers to a gene that is usually present in the genome, but is not limited thereto, and it is understood to include an extrachromosomal sequence, a mitochondrial sequence, and the like. Many genes are usually arranged in a certain order on the chromosome. Those that define the primary structure of a protein are called structural genes, and those that influence the expression are called regulatory genes (for example, promoters). In the present specification, genes include structural genes and regulatory genes unless otherwise specified. Thus, for example, a DNA polymerase gene usually includes both a structural gene for DNA polymerase and a transcriptional and / or translational regulatory sequence such as a promoter for DNA polymerase. In the present invention, it is understood that regulatory sequences such as transcription and / or translation as well as structural genes are also useful as the genes to which the present invention is directed. As used herein, “gene” refers to “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide”. Sometimes. As used herein, “gene product” also refers to “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and / or “protein” “polypeptide”, “oligopeptide” expressed by a gene. And “peptide”. A person skilled in the art can understand what a gene product is, depending on the situation.

  As used herein, “gene replication” refers to a new nucleic acid molecule having the same structure and function as a parent nucleic acid in DNA or RNA, which is genetic material, using the parent nucleic acid strand as a template (DNA, DNA, RNA if DNA). Then RNA). In eukaryotic cells, replication is initiated by the formation of a replication initiation complex containing a replication enzyme (DNA polymerase α) at a large number of replication initiation sites on a double-stranded DNA molecule. Proceed in both directions. Replication initiation is controlled by the cell cycle. A self-replicating sequence (ARS = autonomously replicating sequence) in yeast is regarded as a replication origin. In prokaryotic cells such as E. coli, there is one replication origin (ori) in the double-stranded circular DNA molecule of the genome, a replication initiation complex is formed in ori, and the reaction proceeds in both directions from ori. The replication initiator complex has a complex structure including 10 or more protein factors including a replication enzyme (DNA polymerase III). The replication reaction begins with partial unwinding of the double-stranded DNA helical structure, after which a short DNA primer is synthesized, a new DNA strand is extended from the 3'-OH group, and an Okazaki fragment is synthesized in the complementary strand template. The replication reaction proceeds through multi-step reactions such as linking Okazaki fragments and proofreading against a template (plough frieting).

  The mechanism of replication of genomic DNA, which is the genetic information of an organism, is described in Kornberg A. and Baker T .; "DNA Replication", New York, Freeman, 1992. Typically, an enzyme that synthesizes a complementary strand using one single-stranded DNA as a template and, as a result, produces one double-stranded DNA (an enzyme that performs DNA replication) is called a DNA polymerase. DNA replication requires at least two DNA polymerases. This is because usually the leading strand and the lagging strand must be synthesized simultaneously. DNA replication starts from a fixed position on DNA, and this position is called a replication start point (ori). For example, bacteria usually have at least one bi-directional origin of replication in circular genomic DNA. Taken together, usually one genomic DNA replication requires four DNA polymerases to act simultaneously. In the present invention, it may be advantageous that the replication error of only one of the leading strand and the lagging strand is preferably adjusted, or it may be advantageous that the frequency of replication errors between the two strands is different. .

As used herein, “replication error” refers to an error in nucleotide incorporation that occurs in the process of gene (DNA, etc.) replication. In general, the frequency of replication errors is very low in a living body, about once every 10 ⁸ to 10 ¹² times. The reason for the low frequency of replication errors is that the incorporation of nucleotides causes replication by forming complementary base pairs between the template DNA and the incorporated nucleotides, and the proofreading function of enzymes such as DNA polymerase δ and ε, ie 3 For example, there is a function that the '→ 5' exonuclease has a function of detecting and immediately cutting out a nucleotide that does not show complementarity to the template when it is mistakenly incorporated. Therefore, in the present invention, the frequency of error prone in replication can be adjusted by the failure of specific base pairing, the failure of the calibration function, or the like.

  In the present specification, “error-free” refers to a property in which there is almost no error in replication of a gene (DNA or the like), preferably substantially no error. Error free is mainly affected by the accuracy of the calibration function of an enzyme having a calibration function (for example, DNA polymerase δ, ε, etc.).

  As used herein, “error prone” refers to a property that is prone to error in replication of a gene (DNA or the like) (ie, replication error). Error prone is mainly affected by the accuracy of the calibration function of an enzyme having a calibration function (for example, DNA polymerase δ, ε, etc.).

  In this specification, error-prone and error-free can be classified absolutely (that is, determined by the level of error-prone frequency, etc.) or relative (factors responsible for replication of two or more genes (eg, , DNA polymerase, etc.) can be classified into error prone with a higher error prone frequency and error free with a lower error prone frequency.

  As used herein, “error-prone frequency” refers to the level of nature of error-prone. The error-prone frequency can be expressed by, for example, the absolute number of mutations in the gene sequence (the number of mutations themselves) or the relative number (ratio of the number of mutations in the full length). Alternatively, when referring to an organism or enzyme, the error-prone frequency may be expressed as the absolute or relative number of mutations in the gene sequence per reproduction or division of an organism. Unless otherwise stated, it is expressed as the number of errors per replication process in a gene sequence. Error-prone frequency is sometimes referred to herein as “accuracy” as an inverse measure. The error-prone frequency being uniform means that the error-prone frequencies are substantially equal to each other when referring to factors (polymerase or the like) responsible for the replication of a plurality of genes. On the other hand, when the error-prone frequency is non-uniform, it means that a significant difference exists in a factor (polymerase or the like) responsible for the replication of a plurality of genes.

  In this specification, “adjustment of error-prone frequency” means changing the error-prone frequency. Such adjustment of error-prone frequency includes increasing and decreasing error-prone frequency. Examples of methods for adjusting the error-prone frequency include modification of a DNA polymerase having a proofreading function, insertion of a factor that inhibits or suppresses a polymerization reaction or extension reaction during replication, and a factor that promotes these reactions Inhibition, suppression, deletion of single or multiple bases, deletion of DNA repair enzyme, removal of abnormal base, modification of enzyme having repair factor function, modification of mismatch base pair repair factor, reduction of accuracy of replication itself, etc. Is not limited to them. The error-prone frequency may be adjusted for both DNA double strands or only one of them. Preferably, it may be advantageous to do it only on one side. This is because harmful mutagenesis is reduced.

As used herein, “DNA polymerase” or Pol refers to an enzyme having a function of polymerizing DNA by releasing pyrophosphate from four types of deoxyribonucleoside 5′-triphosphates. For the DNA polymerase reaction, template DNA, primer molecules, Mg ^{2+ and the} like are required. A nucleotide complementary to the template is sequentially added to the 3′-OH end of the primer to extend the molecular chain.

  In Escherichia coli, at least three kinds of enzymes, DNA polymerases I, II and III are known. DNA polymerase I is involved in DNA damage repair, genetic recombination and DNA replication. DNA polymerases II and III are said to have auxiliary functions. This enzyme has a subunit structure composed of several kinds of proteins, and is divided into a core enzyme and a holoenzyme according to its structure. The core enzyme is composed of α, ε, and θ, and the holoenzyme has components of τ, γ, δ, and β in addition to the α, ε, and θ subunits. Eukaryotic cells are also known to have a plurality of DNA polymerases. In higher organisms, there are many types such as DNA polymerases α, β, γ, δ, and ε. In animals, DNA polymerase α (involved in replication of nuclear DNA, acts on DNA replication in cell growth phase), DNA polymerase β (involved in DNA repair in nucleus, repair of DNA damage in growth phase, arrest phase, etc.) ), DNA polymerase γ (involved in mitochondrial DNA replication and repair. Exonuclease activity), DNA polymerase δ (involved in DNA elongation. Exonuclease activity), DNA polymerase ε (lagging strand gap) Involved in replication. Has exonuclease).

  In DNA polymerases responsible for the calibration function (Gram positive bacteria, Gram negative bacteria, eukaryotes, etc.), the amino acid sequence for ExoI is assumed to be responsible for the 3 ′ → 5 ′ exonuclease activity center. It is believed to affect accuracy.

SEQ ID NO: 1: Pol III: 317-IMSFDIECAGRI-328 (Saccharomyces cerevisiae);
SEQ ID NO: 2: Pol II: 286-VMAFDIETTKPP-297 (Saccharomyces cerevisiae);
A mutant pol-3 gene expression strain (AMY128-1, hereinafter abbreviated as pol-3 mutant) is presumed to have this sequence. A representative example of AMY128-1 is a patent biological deposit center (National Institute of Technology and Evaluation) (2-5-8 Kazusa-Kamashita, Kisarazu-shi, Chiba). It has been deposited as a 3-gene expression strain (haploid strain) (AMY128-1)) (date of deposit, August 19, 2005). In addition, the mutant pol-3 gene expression strain (diploid strain) (BYD5) has been deposited under the deposit number NITE P-131 (deposit date August 19, 2005). The wild-type pol-3 gene expression strain (haploid strain) (AMY52-3D) has been deposited under the deposit number NITE P-129 (deposit date August 19, 2005).

  As is clear here, aspartic acid and glutamic acid are well preserved in the DNA polymerase having a proofreading function. In the present specification, such a region containing aspartic acid and glutamic acid may be referred to herein as a proofreading functional active site.

  In one preferred embodiment, the proofreading function is reduced by introducing such a mutation that disrupts the 3 ′ → 5 ′ exonuclease activity into a gene encoding DNA polymerase (DNA polymerase gene) ( That is, it is possible to produce nucleic acid molecules and polypeptides encoding DNA polymerase (with increased error-prone frequency). Note that the 3 ′ → 5 ′ exonuclease activity of the proofreading function is contained in a molecule responsible for DNA polymerization activity in a single DNA polymerase gene (PolC, POL2, CDC2, etc.) (for example, eukaryote, gram It is known that there are cases (for example, gram-negative bacteria) encoded by a gene (for example, dnaQ) different from a gene (for example, dnaE) encoding a DNA polymerization activity (for example, positive bacteria). A. and Baker T., “DNA Replication”, New York, Freeman, 1992), those skilled in the art can appropriately adjust the error-prone frequency in the present invention after understanding these characteristics. For example, in eukaryotes and the like, it is preferable to introduce a mutation that changes the proofreading function but hardly changes the DNA polymerization activity into DNA polymerase. In this case, two acidic amino acids (Derbyshire et.al., EMBO J.10, pp. 17-24, Jan. 1991; Fijalkowski and Schaaper, “Mutants in the Exo I mofof of the proofreading function as described above). Escherichia coli dnaQ: Defective proofreading and inability due to error catastrophe ", Proc. Natl. Acad. Sci. U.S.A. Vol.93, pp. Non-conservative substitutions (eg, substitutions with alanine, valine, etc.)), but are not limited thereto.

  In the present specification, the “proofreading function” refers to a function for detecting and repairing DNA damage and / or errors received by cells. Such a function is due to the insertion of a base as it is in the presence of depurination or depyrimidine, or 5 ′ after single-strand breakage with an AP endonucleic acid endonuclease. → can be achieved by removal with 3 'exonuclease. The removed portion is synthesized and supplemented with DNA polymerase, and ligation with normal DNA is performed by DNA ligase. Such a reaction is called removal repair. Chemical modification by an alkylating agent, DNA base damage due to abnormal base, radiation, ultraviolet light, etc. are repaired by the above reaction after removing the damaged part with DNA glycosidase (irregular DNA synthesis). Examples of the DNA polymerase having such a proofreading function include, but are not limited to, DNA polymerase δ and DNA polymerase ε in eukaryotes. In this specification, the term fidelity may also be used to describe the degree of calibration function. The term fidelity refers to the accuracy of DNA replication. A normal DNA polymerase is usually a high-fidelity DNA polymerase, and a DNA polymerase whose proofreading function has been reduced by modification can be a low-fidelity DNA polymerase.

  For the calibration function of such a DNA polymerase, see, for example, Kunkel, T .; A. : J Biol. Chem. 260, 12866-12874 (1985); Kunkel, T .; A. , Sabotino, R .; D. & Bambara, R.A. A. : Proc. Natl. Acad. Sci. USA, 84, 4865-4869 (1987); I. & Maeda, N .; : Nature, 327, 167-170 (1987); Roberts, J. et al. D. & Kunkel, T .; Al: Proc. Natl. Acad. Sci. USA, 85, 7064-7068 (1988); Thomas, D .; C. Fitzgerald, M .; P. & Kunkel, T .; A. : Basic Life Sciences, 52, 287-297 (1990); Trinh, T .; Q. & Siden, R .; R. Nature, 352, 544-547 (1991); Weston-Hafer, K .; , & Berg, D.M. E. Genetics, 127, 649-655 (1991); Beaute, X. et al. & Fuchs, R.A. p. p. Science, 261, 598-600 (1993); Roberts, J .; D. Izuta, S .; , Thomas, D .; C. & Kunkel, T .; A. : J Biol Chem. , 269, 1711-1717 (1994); Roche, W. et al. A. Trinh, T .; Q. & Siden, R .; R. J. et al. Bacteriol. , 177, 4385-4391 (1995); , Jawski, A .; Ohshirna, K .; & Wells, Nat. Genet. , 10, 213-218 (1995); Fijalkowska, I .; J. et al. Jonczyk, P .; Maliszewska-Tkaczzyk, M .; Bialoskorska, M .; & Schaper, R.A. M.M. Proc. NatJ. Acad. Sci. USA. 95, 10020-10025 (1998); Maliszewska-Tkaczzyk, M .; Jonezyk, P .; Bialoskorska, M .; Schaper, M .; & Fijalkowska, I .; : Proc. Natl. Acad. Sci. USA, 97, 12678-12683 (2000); Gwel, D .; Jonezyk, P .; Bialoskorska, M .; Schaper, R .; M.M. & Fijalkowska, I .; J. et al. : Mutation Research, 501, 129-136 (2002). Negative; Roberts, J .; D. , Thomas, D .; C. & Kunkel, T .; A. : Proc Nat]. Acad Sci. USA, 88, 3465-3469 (1991); Roberts, J. et al. D. Nguyen, D .; & Kunkel, T .; A. : Biochemistry; 32, 4083-4089 (1993); Francino, M .; P. , Chac, L .; Riley, M .; A. & Ochman, H .; : Science, 272, 107-109 (1996); A Boulet, M .; Simon, G.M. Faye, GA Bauer & PM Burgers. EMBO J, 8, 1849-1854 (1989); Morrison A, Araki H, Clark AB, Hamake RK, & Sugino A. See Cell, 62 (6), 1143-1151, (1990).

  In this specification, “DNA polymerase δ” refers to an eukaryotic enzyme that is involved in DNA elongation, has exonuclease activity, and is therefore said to have a proofreading function. The regulation of the proofreading function of DNA polymerase δ can be achieved by introducing a modification into the amino acid sequence related to the proofreading function. A typical DNA polymerase δ has a sequence described in pol δ: X61920 gi / 171411 / gb / M61710.1 / YSCDPB2 [171411] as a nucleic acid sequence and an amino acid sequence. DNA polymerase δ is described in Simon, M .; et al. , EMBO J. et al. 10, 2163-2170, 1991, the contents of which are incorporated herein by reference.

  In this specification, “DNA polymerase ε” refers to an enzyme involved in replication of a gap in a lagging strand when referring to a eukaryotic organism, and has an exonuclease activity and has a proofreading function due to this. It is said. A typical DNA polymerase ε has a sequence described in pol ε: M60416 gi / 171408 / gb / M60416.1 / YSC DNAPOL [171408] as a nucleic acid sequence and an amino acid sequence.

  According to the HUGO classification, DNA polymerases δ and ε are classified as POL1 / POL3 for epsilon and POLE / POL2 for epsilon, and any nomenclature can be used herein.

  For other DNA polymerases, see, for example, Lawrence C. et al. W. et al. , J .; Mol. Biol. 122, 1-21, 1978, Lawrence C.I. W. et al. , Genetics 92, 397-408, Lawrence C .; W. et al. MGG, 195, 487-490, 1984, Lawrence C.I. W. et al. MGG. 200, 86-91, 1985 (DNA polymerase β and DNA polymerase ζ); M.M. et al. Nature 261, 593-595, 1976, McGregor, W .; G. et al. Mol. Cell. Biol. 19, 147-154, 1999 (DNA polymerase η); et al. , Nature 365, 275-276, 1993, Prolla T. et al. A. , Et al. Mol. Cell. Biol. 15, 407-415, 1994, Kat A. et al. , Et al. , Proc. Natl. Acad. Sci. USA 90, 6424-6428, Bhattacharya N .; P. , Et al. , Proc. Natl. Acad. Sci. USA 91, 6319-6323, 1994, Faber F. et al. A. , Et al. , Hum. Mol. Genet. 3,253-256, 1994, Eshleman, J. et al. R. , Et al. Oncogene 10, 33-37, 1995, Morrison A. et al. , Et al. , Proc. Natl. Acad. Sci. USA 88, 9473-9477, 1991, Morrison A. et al. , Et al. , EMBO J. et al. 12, 1467-1473, 1993, Foury F. et al. , Et al. , EMBO J. et al. 11, 2717-2726, 1992 (DNA polymerase λ, DNA polymerase μ, etc.) and the like, the contents of which are incorporated herein by reference.

  As used herein, a “wild type” of a gene such as a DNA polymerase and an organism such as yeast, in its broadest definition, includes a gene such as a naturally occurring DNA polymerase and an organism such as yeast, and usually exists in nature. Among the genes such as DNA polymerase and organisms such as yeast, the most widely present in the species of origin. Therefore, genes such as DNA polymerases and organism species such as yeast that are first identified in a certain species are usually wild-type. The wild type is also referred to as “natural standard type”. If it is an organism, wild type can have normal enzyme activity, normal traits, normal behavior, normal physiology, normal reproduction, normal genome obtain.

  In this specification, when the proofreading function is “lower than that of the wild type”, when referring to an enzyme or the like having a certain proofreading function, the proofreading function is lower than that of the wild type of the enzyme (that is, The number of mutations remaining after the calibration process is larger than the number of mutations remaining after the wild type calibration process). Comparison with such wild type can be done by relative or absolute indications. Such a comparison can also be made, such as by error-prone frequency.

  As used herein, “mutation” refers to a state of a gene (nucleic acid or amino acid) sequence that causes a change in the sequence of the gene or a gene caused by the change. As used herein, for example, mutations are used for gene sequence changes that occur for the proofreading function. In this specification, unless otherwise stated, mutation is used synonymously with modification.

  It is most common to perform mutagenesis in an organism to create useful mutants. The mutation usually means a change in the base sequence encoding the gene, and includes a change in the DNA sequence. Mutations can be broadly divided into the following three types according to the effects on the individuals in which they occur: A) Neutral mutations: This mutation applies to most mutations and has almost no effect on the growth and metabolism of organisms. There is no. B) deleterious mutation: This mutation is less frequent than the neutral mutation. Inhibit the growth or metabolism of an organism. The harmful mutation includes a lethal mutation that destroys a gene essential for growth. In the case of microorganisms, although it varies depending on the species, the ratio of harmful mutations to all mutations is usually about 1/10 to 1/100. C) Beneficial mutation: This mutation is beneficial for the breeding of organisms. Its frequency of occurrence is very low compared to neutral mutations. Therefore, in order to obtain an organism individual into which a beneficial mutation has been introduced, a large organism population and a long time are required. In addition, a sufficient effect of breeding an organism rarely appears with only a single mutation, and it is often necessary to accumulate a plurality of beneficial mutations. Therefore, the present invention has been provided for the first time as a technique for imparting only lactic acid resistance.

  As used herein, “growth (or growth)” refers to a quantitative increase of an organism as an individual when referring to the organism. More specifically, the growth can recognize the quantitative increase of the individual by the increase in the measured values such as the body length (height) and the body weight. An individual's quantitative increase depends on cell growth and cell number increase.

  As used herein, “substantially the same growth (or growth)” refers to an organism in which the organism grows compared to the organism to be compared (eg, the organism before genetic trait conversion). It means that the speed hardly changes. Examples of such a range in which the growth rate hardly changes include, but are not limited to, entering within one deviation in the normal statistical distribution of growth. In the organism of the present invention, for example, (1) the number of children does not change; (2) the form changes, but unlike normal artificial mutations, it is not disabling and has a very high mutation rate. Nevertheless, it is expected to be “beautiful”. (It is not directly related to growth, but is considered to be a feature of the mutant created by the method of the present invention); (3) The acquired trait or genotype or phenotype does not return. .

  As used herein, “production” of an organism refers to the creation of an individual of the organism when referring to the organism.

  As used herein, “reproduction” of an organism refers to the creation of a new individual of the next generation from a parent individual when referring to an organism. Reproduction includes, but is not limited to, those due to natural phenomena such as reproduction and reproduction, and those due to artificial techniques such as clone (nuclear transfer) technology. As a technique used for reproduction, for example, in the case of a plant, an individual can be made from one cultured cell; Organisms produced by reproduction usually have a genetic trait derived from the parent. In an organism that is reproduced by sexual reproduction, the reproduced organism usually has genetic traits derived from two sexes, respectively. Usually, such inherited traits have approximately equal proportions from two sexes. In organisms that are regenerated by asexual reproduction, the normally regenerated organism has a genetic trait derived from the parent.

  As used herein, a “cell” is defined in the same way as the broadest meaning used in the art, and is a structural unit of a tissue of a multicellular organism that is enclosed in a membrane structure that isolates the outside world, Refers to a living organism having self-regenerative ability and having genetic information and its expression mechanism. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells). The source of cells can be, for example, a single cell culture, or such as cells from a normally grown transgenic animal embryo, blood, or body tissue, or a normally grown cell line Examples include but are not limited to cell mixtures.

  The cells used in the present invention can be cells from any organism (eg any type of unicellular organism (eg bacteria, yeast) or multicellular organism (eg animals (eg vertebrates, invertebrates), plants). (For example, monocotyledonous plants, dicotyledonous plants, etc.))). For example, cells derived from vertebrates (eg, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) are used, and more particularly mammals (eg, single pores, Marsupials, rodents, wings, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates , Rodents, rabbit eyes, etc.). In one embodiment, cells derived from primates (eg, chimpanzees, Japanese monkeys, humans), particularly cells derived from humans, are used, but are not limited thereto. The cells used in the present invention may be stem cells or somatic cells. Such cells may be used for transplantation purposes. Alternatively, as a plant cell, a cell derived from a flowering plant (monocotyledonous or dicotyledonous) is preferably used, more preferably a dicotyledonous plant cell is used, and more preferably a grass family, solanaceae, cucurbitaceae, Cells derived from plants of the Brassicaceae, Aceraceae, Rosaceae, Legumes, and Muraceae are used. More preferably, wheat, corn, rice, barley, sorghum, tobacco, bell pepper, eggplant, melon, tomato, strawberry, sweet potato, rape, cabbage, leek, broccoli, soybean, alfalfa, flax, carrot, cucumber, citrus, Chinese cabbage, Cells derived from lettuce, peach, potato, purple, auren, poplar and apple are used. Plant cells can be plant parts, organs, tissues, cultured cells, and the like. Methods for transforming cells, tissues, organs or individuals are well known in the art. Such techniques are sufficiently described in the literature cited in the present invention. Introduction of a nucleic acid molecule into a biological cell may be transient or constitutive. Transient or homeostatic gene transfer techniques are each well known in the art. Techniques for producing transformed plants by differentiating cells used in the present invention are also well known in the art, and it is understood that such techniques are well described in the literature cited in the present invention. . Techniques for obtaining seeds from transformed plants are also well known in the art, and such techniques are described in the literature cited in the present invention.

  As used herein, “isolated” refers to a substance that is at least reduced, preferably substantially free of naturally associated substances in a normal environment. Thus, an isolated cell refers to a cell that is substantially free of other substances (eg, other cells, proteins, nucleic acids, etc.) that accompany it in its natural environment. When referring to a nucleic acid or polypeptide, “isolated” means, for example, substantially free of cellular material or culture medium when made by recombinant DNA technology, and precursor when chemically synthesized. Refers to a nucleic acid or polypeptide that is substantially free of somatic or other chemicals. An isolated nucleic acid preferably includes sequences that are naturally flanking in the organism from which the nucleic acid is derived (ie, sequences located at the 5 'and 3' ends of the nucleic acid). Absent.

  As used herein, “established” or “established” cells maintain certain properties (eg, pluripotency) and the cells continue to grow stably under culture conditions. State.

  In this specification, “lactic acid” is α-hydroxypropionic acid, and L-form is a final product of glycolysis, which is produced by reduction of pyruvic acid by the action of lactate dehydrogenase.

  As used herein, “lactic acid resistance” refers to resistance to lactic acid. Such resistance means at least not killing in the presence of trace amounts of lactic acid, but more preferably may mean growing in the presence of lactic acid.

  Examples of lactic acid that can be used in the present specification include L form, D form, racemic form and the like, which can exist in nature, but L form is preferred. These may be used alone or in combination of two or more.

  Lactic acid resistance is determined as follows. A microorganism whose lactic acid tolerance is to be evaluated is cultured in a medium not containing lactic acid, and an overnight culture is prepared. A medium containing various concentrations of lactic acid is prepared, and an overnight culture is added at 0.1 to 10% (v / v) and cultured overnight. The volume of bacteria to be added depends on the purpose and type of bacteria. The bacterial concentration after overnight culture is determined by a method using an absorptiometer, an agar plate method, viability measurement by trypan blue staining, and the like. The value of the lactic acid concentration at which the growth ability equivalent to that in the case of using a medium near neutrality is evaluated as having the lactic acid resistance at that concentration.

  In the method of the present invention, the step of selectively culturing microorganisms by adding lactic acid may be carried out by coating lactic acid-sensitive microorganisms that have been subjected to mutation treatment on an agar medium, and laminating lactic acid at a desired concentration ratio, or in liquid culture. The treated lactic acid sensitive bacteria and the target lactic acid may be put together and cultured. In addition, conditions such as pH, temperature, and aeration rate (when aerated) can be easily set by those skilled in the art. However, it is needless to say that conditions under which the microorganisms grow are preferable.

(General biochemistry / molecular biology)
(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associate ES and Wiley-Interscience; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer and Expression Analysis”, Yodosha, 1997, etc., which are related parts in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R .; L. etal. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T. T. et al. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  The description of the preferred embodiment is described below, but it should be understood that this embodiment is an illustration of the present invention and that the scope of the present invention is not limited to such a preferred embodiment.

  In one aspect, the present invention provides an organism having lactic acid resistance, wherein the organism has a higher tolerance than that of the natural form of the organism.

  In one embodiment, the lactic acid targeted in the present invention may be a type selected from the group consisting of L-type lactic acid, D-type lactic acid, and racemic isomers of L-type and D-type lactic acid.

  In one embodiment, the present invention provides an organism having a concentration of at least 1% resistance to lactic acid. To date, there have been few organisms that are resistant to lactic acid to concentrations of at most 1%. In particular, other than lactic acid bacteria, only resistance of less than 1% has been reported, and it was considered impossible to produce organisms having resistance of 2% or more. In addition, there is no report that lactate tolerance was obtained by crossover. Therefore, the present invention has a remarkable effect of providing an organism that has not existed conventionally. In a preferred embodiment, the present invention provides at least 2%, preferably at least 3%, more preferably at least 4%, more preferably at least 5% and even more preferably at least 6% of lactic acid, More preferably at a concentration of at least 7%, more preferably at least 8%, more preferably at least 9%, even more preferably at least 10%, more preferably at least 15%, most preferably at least 20%. Provide organisms that are resistant. Here, the tolerance to lactic acid is at least 18 hours, usually at least 1 day, preferably 2 days, preferably 3 days, preferably 4 days, preferably 4 days, more preferably 6 days in the environment to be resistant. Surviving for a day (at least survival rate is 10% or more, preferably at least survival rate is 20%. The present invention generally has survival rate of 80% or more and exhibits excellent survival rate.) Means that.

  In another embodiment, the organism of the present invention is any microorganism, more preferably a eukaryote, even more preferably a yeast. In particular, Saccharomyces common organisms are preferred, and Saccharomyces cerevisiae is more preferred. In another embodiment, the organism of the present invention may be a prokaryotic organism. For yeast, tolerance could not be obtained even with 1% lactic acid.

  A representative example of the strain obtained in the present invention is 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818, Japan. Dated July 29, 2005; can be grown in YPD medium containing 7% lactic acid).

In another aspect, the present invention provides a method for making an organism with improved resistance to lactic acid. This method includes: A) culturing the target organism for the lactic acid at a maximum allowable concentration in the normal state, preferably about 1 day; B) the target organism for the lactic acid, for the desired concentration for improvement. And C) selecting and reproducing a living organism. It is understood that a technique known in the art can be applied to such a reproduction method. By culturing once at a concentration lower than the maximum permissible concentration, and then culturing at a concentration higher than the maximum permissible concentration of the network, it was possible to efficiently obtain a resistant organism at a desired concentration more efficiently. There is a special feature. More specifically, in one embodiment of the present invention, the method of the present invention comprises: A) culturing for 1 day at the maximum allowable concentration in a normal state; B) 1 day at a lactic acid concentration 1% higher than A. The step of culturing, C) the step of culturing for 1 day at a lactic acid concentration 1% higher than B, and the steps of B) to C) are repeated for 1 day at the concentration of lactic acid to be finally given resistance. It is characterized by the ability to efficiently acquire organisms that are resistant at concentrations. As shown in Table 3 and FIG. 4, the growth rate of the 5% lactic acid resistant strain increases with repeated passage at the same lactic acid concentration. Between the culture step A) and the culture step B), A ′) a step of culturing in lactic acid at a concentration 1% higher than the maximum allowable concentration, and if necessary, A ″) from the concentration in the culture step A ′) Perform a step of culturing in 1% higher concentration of lactic acid, and repeat the step of culturing in 1% higher concentration of lactic acid in A ″) as necessary to achieve culturing at the desired concentration. In a preferred embodiment, the method of the invention further comprises the step of D) adjusting the error prone frequency in the replication of the organism's genes.

  In another embodiment, the step of adjusting the error-prone frequency used in the present invention preferably includes adjusting the error-prone frequency of the DNA polymerase of the organism. The DNA polymerase used in the present invention is at least one selected from the group consisting of DNA polymerase α, DNA polymerase β, DNA polymerase γ, DNA polymerase δ and DNA polymerase ε and their corresponding DNA polymerases in eukaryotic cells. Contains a polymerase.

  In a preferred embodiment, the DNA polymerase used in the present invention includes at least one polymerase selected from the group consisting of DNA polymerase δ and DNA polymerase ε and its corresponding DNA polymerase in eukaryotic cells.

  In a preferred embodiment, the organism used in the present invention is a eukaryote (eg, an animal, plant, fungus or yeast cell), more preferably a yeast. Yeast has a feature that it is difficult to acquire resistance to various environmental factors, and the present invention is remarkable in that it is linked to the production of yeast, which was impossible in the past in the form of acquiring resistance to lactic acid. is there.

  In another embodiment, the organism used in the present invention exists in a state of separated cells.

  In a preferred embodiment, the organism obtained in the present invention shows substantially the same growth as the wild type even after transformation of the desired trait. Classical mutagenesis methods rarely yielded the same growth as wild-type. Therefore, the present invention could not be provided in the prior art in that it provides a living organism having a resistance to lactic acid and having the same growth characteristics as the wild type (substantially the same as the normal type). There is an effect of providing living creatures.

  In another embodiment, the desired concentration used in the methods of the present invention is preferably a concentration that is higher than the maximum allowable concentration. The maximum allowable concentration may be actually measured immediately before carrying out the method of the present invention, or a known numerical value may be obtained from the literature and used.

  In another aspect, the present invention relates to an organism produced by the method of the present invention. Such an organism has an increased resistance to lactic acid compared to the prior art, and may be an organism having lactic acid resistance that was not available in the prior art.

  In another aspect, the present invention provides a method for evaluating the lactic acid tolerance performance of an organism. The method comprises: A) growing the organism under conditions in which the organism normally grows; B) growing the organism in the presence or absence of the lactic acid; and C) killing the organism. Determining the concentration of lactic acid.

  In one embodiment, determining whether an organism is dead can be used as a criterion based on whether the cell remains in shape. Since it is unclear whether cells that only maintain their shape are alive or not, it has been conventionally necessary to perform further measurement by trypan blue staining or the like. It has become clear that maintaining itself is an important indicator of survival. Therefore, the evaluation method using the maintenance of this shape as an index provides a remarkably simple determination method as compared with the prior art.

  Here, in determining whether or not the shape is maintained, the criterion for determining that the shape is maintained (the cell is alive) is a case where the cell outline is clear and the cell outline is clear. The criterion for determining that the shape cannot be maintained is a case where the inside of the cell is thin and transparent due to a thin outline and a wrinkle rather than a beautiful spherical shape.

  In another aspect, the present invention provides a method for evaluating the lactic acid tolerance performance of an organism. The method comprises: A) growing the organism under conditions in which the organism normally grows; B) growing the organism in the presence or absence of the lactic acid; and C) killing the organism. Determining the concentration of lactic acid.

  In one embodiment, the determination of whether or not the organism is dead may use as a criterion whether or not the cell maintains its shape.

  In another aspect, the invention also provides an organism whose bacteriological properties are the same as the wild type except for lactate resistance. Such organisms were not available with classical mutation methods.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. With the exception of the reagents and reagents used in the following examples, those commercially available from Sigma (St. Louis, USA, Wako Pure Chemical (Osaka, Japan), etc. were used. In the following, the present invention will be described in more detail with reference to examples.

(Example 1: Examination of change in pH of medium and effect on cell growth by addition of lactic acid)
(Yeast strains and reagents)
Mutant pol-3 gene expression strain (BYD5, hereinafter abbreviated as pol-3 mutant strain) donated by Shimoda et al., Osaka City University. BYD5 is accession number NITE P-131 (date of deposit August 19, 2005) 2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818, and the wild-type pol-3 gene expression strain (AMY52-3D, below) AMY52-3D has the deposit number NITE P-129 (date of deposit: August 19, 2005) 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818 It has been deposited at the Japan Patent Organism Depositary. The genotype of BYD5 is [pol3-01 / pol3-01, MATa / MATα, lys1-1 / lys1-1], and the genotype of AMY52-3D is [MATα, ura3-52, leu2-1, lys1]. -1, ade2-1, his1-7, hom3-10, trp1-289, canR].

  The liquid medium YPD medium (cat. # 630409) and the agar medium YPD agar medium (cat. # 630410) were purchased from BD Biosciences Clontech and sterilized by autoclaving (120 ° C., 15 minutes). The added L-lactic acid (hereinafter abbreviated as lactic acid, cat. # 129-02666) was purchased from Wako Pure Chemical Industries.

(Method)
The solution was added to the liquid medium so that the lactic acid concentration was 0% to 20% (v / v), and each pH was measured.

  The pol-3 mutant and wild type colonies developed on the agar plate were scraped with a platinum loop and inoculated into a test tube (diameter 18 mm) containing 4 mL of YPD medium. These were cultured with shaking at 30 ° C. and 180 rpm overnight (16 hours or longer).

  A part of each culture solution was taken, the cell density was measured using a hemocytometer, and diluted with a liquid medium so that the cell densities of both strains were equal.

Each culture solution was placed in a microtube so that the cell density at the start of culture was about 2.0 × 10 ⁶ cells / mL, and the cells were collected by centrifugation at 5000 × g for 3 minutes.

  Liquid with lactic acid concentration of 1%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 13%, 15%, 17%, 19%, 20% (v / v) The cells collected in 4 mL of the medium were resuspended and cultured with shaking at 30 ° C. and 180 rpm for 18 hours. In addition, the culture tube used the test tube of diameter 18mm.

  After culturing, the cell density was measured using a hemocytometer, and the influence of lactic acid addition on cell proliferation was compared.

(result)
Examination of medium pH by addition of lactic acid Changes in pH (Table 1 and FIG. 2) and changes in the medium (FIG. 1) when lactic acid is added to the liquid medium are described below.

  It was revealed that the pH of the YPD medium was significantly reduced by adding lactic acid (Table 1).

  Table 1 Changes in pH of YPD medium by addition of lactic acid

.

  It was found that by adding lactic acid, some of the medium components were precipitated and the medium became cloudy (FIG. 1).

Examination of the influence which it has on the cell growth by the addition of lactic acid The result of measuring the cell density when the wild strain and the pol-3 mutant strain were cultured in a lactic acid-containing medium for 18 hours is shown below (Table 2 and FIG. 2).

  Table 2 Comparison of cell density of wild-type and pol-3 mutants with lactic acid addition (18 hours culture)

.

  The cell density of the wild strain and the pol-3 mutant was reduced by the addition of lactic acid. In particular, it became clear that when the amount of lactic acid added exceeds 5%, almost no growth was possible.

  A correlation was observed between the change in the pH of the medium due to the addition of lactic acid and the cell growth of both strains, indicating that the cell growth of both strains was inhibited by a decrease in the pH of the medium.

(Example 2: Method for producing lactate-resistant yeast)
In this example, a method for imparting lactic acid resistance to the pol-3 mutant is described below.

(Yeast strain used)
Similarly to Example 1, a pol-3 mutant (BYD5) was used.

(Method)
(1) A colony of the pol-3 mutant strain was scraped off, inoculated into a test tube (diameter 18 mm) containing 4 mL of a liquid medium, and cultured overnight at 30 ° C. and 180 rpm.

  (2) The culture solution was placed in a 15 mL centrifuge tube (cat. # 430791 / Corning) and centrifuged at 1000 × g for 5 minutes to collect all cells.

  (3) All the cells collected in a 300 mL Erlenmeyer flask containing 100 mL of liquid medium were resuspended and cultured overnight at 30 ° C. and 180 rpm.

  (4) The culture solution was placed in a 50 mL centrifuge tube (cat. # 4300829 / Corning) and centrifuged at 1000 × g for 5 minutes to collect all cells.

  (5) All the collected cells were suspended in a 300 mL Erlenmeyer flask containing 150 mL of 3% (v / v) lactic acid medium, and cultured overnight at 30 ° C. and 180 rpm.

  (6) A part of the 3% lactic acid culture solution was taken and mixed with an equal amount of 0.1% trypan blue solution (cat. # 345-07421 / DOJINDO) to stain the cells. Since the cells stained blue were dead cells and the cells not stained were viable cells, the ratio of viable cells was observed.

  (7) After confirming the presence of viable cells, 1/3 volume (50 mL) of 3% lactic acid culture solution was placed in a 50 mL centrifuge tube, and centrifuged at 1000 × g for 5 minutes to collect the cells.

  (8) The collected cells were suspended in a 300 mL Erlenmeyer flask containing 150 mL of 4% (v / v) lactic acid medium, and cultured overnight at 30 ° C. and 180 rpm.

  (9) A part of the 4% lactic acid culture solution was taken and the cells were observed by trypan blue staining. After confirming the viable cells, 1/3 volume (50 mL) of the culture solution was placed in a 50 mL centrifuge tube and centrifuged at 1000 × g for 5 minutes to collect the cells.

  (10) The collected cells were suspended in a 300 mL Erlenmeyer flask containing 150 mL of 5% (v / v) lactic acid medium, and cultured overnight at 30 ° C. and 180 rpm.

  (11) A part of 5% lactic acid culture solution was taken, and viable cells were confirmed by trypan blue staining.

  (12) A 1/10 volume (15 mL) of 5% lactic acid culture solution was taken, and the cells were collected by centrifugation at 1000 × g for 5 minutes. The cells collected were suspended in a 200 mL shake flask (cat. # 4070FK 200 / IWAKI) containing 70 mL of 5% lactic acid medium, and then the cell density was measured. After the measurement, these cultures were cultured with shaking at 30 ° C. and 180 rpm.

  (13) The cell density of the culture solution was measured every 24 hours, and the cells were cultured until the cell density was about 5 to 10 times the cell density immediately after the start of the culture.

  (14) A 1/10 volume (7 mL) was taken from the culture medium in which growth was confirmed in a 5% lactic acid medium, and the cells were collected by centrifugation at 1000 × g for 5 minutes. These were again suspended in 5% lactic acid medium and cultured with shaking at 30 ° C. and 180 rpm.

  Subculture was performed by repeating the operations (11) to (14). As a reference for subculture, 1. 1. Confirm viable cells by trypan blue staining. The above two points were set such that the cells grew about 5 to 10 times the cell density immediately after the start of culture.

(result)
The cell density when the lactic acid concentration of the medium is gradually increased and finally subcultured in a 5% (v / v) lactic acid medium is shown below (Table 3 and FIG. 4).

  (1) As a result of culturing the pol-3 mutant strain in a 3% lactic acid medium, the cell density increased and many viable cells were observed.

  (2) As a result of transplanting a strain grown in 3% lactic acid culture to 4% lactic acid medium, an increase in cell density and viable cells were observed.

  (3) As a result of transplanting a strain grown in 4% lactic acid culture to a 5% lactic acid medium, an increase in cell density was observed on the fourth day from the start of the culture.

  (4) As a result of trypan blue staining, many viable cells were confirmed in 5% lactic acid culture (FIG. 3).

(5) The strain grown in 5% lactic acid culture was transplanted again into 5% lactic acid medium, and subculture was repeated in the same medium. As a result, it was recognized that the rate of cell proliferation increased as the number of passages increased (Table 3 and FIG. 4).
As a result, it was shown that 5% lactic acid resistance can be imparted to the pol-3 mutant by this method.
Table 3 Cell growth of pol-3 mutant in 5% lactic acid culture

(Example 3: Culture characteristics of 5% lactic acid resistant strain)
A method for examining the culture characteristics of a pol-3 mutant strain that has acquired resistance to 5% lactic acid (referred to as a 5% lactic acid resistant strain) is described below.

(Yeast strain used)
Similarly to Example 1, the wild type strain and the 5% lactic acid resistant strain obtained in Example 2 were used.

(Method)
The 5% lactic acid resistant strain obtained in Example 2 is subcultured in a 200 mL baffled Erlenmeyer flask containing 70 mL of 5% lactic acid medium. The culture is performed at 30 ° C. and 200 rpm, and about 1/10 of the preculture solution is passaged.

  (1) A colony of a wild strain was scraped, inoculated into a test tube (φ18 mm) containing 4 mL of a liquid medium, and cultured overnight at 30 ° C. and 180 rpm.

  (2) The culture solution of the wild strain was centrifuged at 5000 × g for 3 minutes to recover the cells, and suspended in a 200 mL baffled Erlenmeyer flask containing 70 mL of liquid medium. This was cultured with shaking at 30 ° C. and 200 rpm.

  (3) A 5% lactic acid resistant strain passaged in a 5% lactic acid medium and a precultured wild strain were placed in a 50 mL centrifuge tube, and centrifuged at 1000 × g for 5 minutes to collect cells.

  (4) The collected cells were resuspended in a few mL of liquid medium without lactic acid, and the cell density was measured with a hemocytometer.

  (5) Based on the measured cell density, the cells were diluted with a liquid medium without addition of lactic acid so that the cell densities of the wild strain and the 5% lactic acid resistant strain were equal.

(6) The cell dilution was placed in a 15 mL centrifuge tube so that the final cell density was about 2.0 × 10 ⁷ cells / mL, and the cells were collected by centrifugation at 1000 × g for 5 minutes. The collected cells were suspended in a 200 mL baffled Erlenmeyer flask containing 70 mL of 5%, 6% and 7% lactic acid medium, and cultured with shaking at 30 ° C. and 200 rpm. Note that only 5% of the wild strain was cultured. These lactic acid media were used after being filtered with a 0.45 μm sterilizing filter (ADVANTEC / cat. # 25CS045AS).

  (7) The cell density for 3 days was measured every 24 hours on the 0th day immediately after suspension. The measurement with a hemocytometer was performed 3 times per measurement, and the average value and standard deviation were calculated.

(result)
Changes in cell density when wild strains and 5% lactic acid resistant strains are cultured in each lactic acid medium are described below (FIG. 5 and Tables 4 and 6).

  (1) As a result of 5% lactic acid culture, it was found that the 5% lactic acid resistant strain showed remarkable growth compared to the wild strain, and the wild strain hardly grew (FIG. 5).

  (2) The 5% lactic acid resistant strain proliferated rapidly under the culture conditions containing 5% and 6% lactic acid, and grew about 10 times on the third day of culture (Table 4, FIG. 6).

  (3) A 5% lactic acid resistant strain was cultured in a 7% lactic acid medium for 3 days, but no significant growth was observed (Table 4, FIG. 6).

As a result, it was found that the 5% lactic acid resistant strain obtained by acclimatizing the pol-3 mutant strain to lactic acid has acquired resistance to 5% and 6% lactic acid.
Table 4 Effect of lactic acid addition on cell growth of wild strain and 5% lactic acid resistant strain

(Example 4: Production of 7% lactic acid resistant strain)
The methods and results of imparting resistance to 7% lactic acid to the 5% lactic acid resistant strains described in Examples 2 and 3 are described below.

(Method)
The 5% lactic acid resistant strain (pol-3 mutant strain) obtained by Examples 2 and 3 was prepared at 30 ° C. and 200 rpm using a 200 mL baffled Erlenmeyer flask containing 70 mL of 5% (v / v) lactic acid medium. Subculture is performed. Passage to a new medium is performed every 2 to 3 days, and 1/10 amount of cells are collected and transplanted with respect to the whole culture solution.

(1) A 1/5 volume of the whole culture solution was taken from the subculture of a 5% lactic acid resistant strain, and the cells were collected by centrifugation at 1000 × g for 5 minutes. The recovered cells were suspended in a 200 mL baffled Erlenmeyer flask containing 70 mL of 7% (v / v) lactic acid medium, and cultured overnight at 30 ° C. and 200 rpm. (First passage)
(2) A 1/5 volume of the whole culture solution was taken, and the cells were collected by centrifugation at 1000 × g for 5 minutes. The collected cells were suspended in 70 mL of fresh 7% lactic acid medium and cultured at 30 ° C. and 200 rpm for 5 days. (Second passage)
(3) A 1/10 volume of the whole culture solution was taken and centrifuged at 1000 × g for 5 minutes to collect cells. The collected cells were suspended in 70 mL of fresh 7% lactic acid medium and cultured at 30 ° C. and 200 rpm for 5 days. (3rd passage)
(4) A 1/10 volume of the whole culture was taken and centrifuged at 1000 × g for 5 minutes to collect cells. The collected cells were suspended in 70 mL of fresh 7% lactic acid medium, and cultured at 30 ° C. and 200 rpm for 4 days. (4th passage)
(5) A part of the fourth subculture strain and a wild strain pre-cultured overnight in a liquid medium were transplanted into a 7% lactic acid medium and cultured with shaking at 30 ° C. and 200 rpm overnight. These cells were stained with trypan blue, and the survival cells were observed under a microscope to confirm resistance to 7% lactic acid.

(result)
Changes in cell density when a 5% lactic acid resistant strain is subcultured in a 7% lactic acid medium are shown below (Table 5, FIG. 7).

  (1) After culturing for 5 days in the second subculture, about 6.7 times the proliferation was observed compared to immediately after the subculture (Table 5, FIG. 7).

  (2) When cultured for 5 days in the third passage, the growth was about 66.9 times that immediately after the passage (Table 5, FIG. 7).

  (3) When cultured for 4 days in the 4th subculture, about 310-fold proliferation was observed compared to immediately after the subculture (Table 5 and FIG. 7).

  (4) As a result of trypan blue staining, although almost all cells were killed in the wild strain, many viable cells were observed in the lactic acid resistant strain (FIG. 8).

As a result, the 5% lactic acid resistant strain obtained from the pol-3 mutant strain was able to grow rapidly by acclimatization to 7% lactic acid culture. That is, this strain acquired resistance to 7% lactic acid.
Table 5 Changes in cell density when 5% lactic acid resistant strains were subcultured in 7% lactic acid medium

A representative example of the strain obtained in this example is 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818, Japan. It has been deposited (July 29, 2005) as a resistant strain (BYD5-LA7) accession number that can be grown in a YPD medium containing 7% lactic acid.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Lactic acid resistant yeast is used as a host for producing a high concentration of lactic acid. Lactic acid, sodium lactate, and calcium lactate are already used as raw materials for pharmaceuticals such as infusions, food additives, and cosmetics. In addition, polylactic acid, which is a polymer of lactic acid, has attracted attention as a biodegradable polymer, and applications are being developed in many fields such as films, resin products, medical materials, and the like.

  In addition, the method of the present invention that enhances the lactic acid resistance of microorganisms is applied to lactic acid bacteria to enhance the acid resistance that reaches the small intestine or colon without being killed by gastric acid after oral ingestion. As a result, it can be used as a functional food with an enhanced probiotic characteristic (Europe, No. 9281516-2) of a protective function against intestinal pathogen infection and an effect on immune response. In addition, lactic acid bacteria are expected to have medical applications such as the possibility of preventing ulcerative colitis, the possibility of preventing diabetes, the effect of reducing infectious diseases, the effect of increasing the influenza antibody titer, and the effect of suppressing Helicobacter pylori.

FIG. 1 shows changes in culture medium due to the addition of lactic acid (left: YPD medium without addition, right: YPD medium with 3% lactic acid added). FIG. 2 ¥ is a comparison of cell growth of the wild strain and the pol-3 mutant strain by addition of lactic acid (green circle: cell density of the wild strain, red circle: cell density of the pol-3 mutant strain, blue line: medium pH). . FIG. 3 is an observation of 5% lactic acid resistant strain by trypan blue staining (second day of 5% lactic acid culture, blue cells: dead cells, colorless cells: viable cells). FIG. 4 shows cell growth of the pol-3 mutant strain in 5% lactic acid culture (from Table 3). FIG. 5 is a comparison of cell growth in 5% lactic acid culture (1st day, left: wild strain, right: 5% lactic acid resistant strain). FIG. 6 shows the effect on cell growth of lactic acid addition of wild strain and 5% lactic acid resistant strain (from Table 3). FIG. 7 shows changes in cell density (from Table 5) when a 5% lactic acid resistant strain was subcultured in a 7% lactic acid medium. Fig. 8 shows trypan blue-stained photographs of wild and lactic acid resistant strains cultured in 7% lactic acid medium (left: wild strain, right: 7% lactic acid resistant strain) Shows dead cells).

Claims (31)

An organism having lactic acid resistance, wherein the organism has a higher tolerance than the natural type of the organism has. The organism according to claim 1, wherein the lactic acid is a type selected from the group consisting of D-type, L-type and racemic type. The organism of claim 1, wherein the resistance is resistance to lactic acid at a concentration of at least 1%. The organism of claim 1, wherein the tolerance is a tolerance to lactic acid at a concentration of at least 3%. The organism of claim 1, wherein the resistance is resistance to lactic acid at a concentration of at least 5%. The organism of claim 1, wherein the resistance is resistance to a concentration of at least 7% against lactic acid. The organism of claim 1, wherein the resistance is resistance to lactic acid at a concentration of at least 10%. The organism according to claim 1, wherein the resistance includes survival in a lactic acid environment for at least 18 hours or longer. The organism according to claim 1, wherein the tolerance includes survival in a lactic acid environment for at least 4 days. The organism according to claim 1, wherein the organism is a yeast. The organism according to claim 1, wherein the organism is an organism belonging to Saccharomyces. The organism according to claim 1, wherein the organism is Saccharomyces cerevisiae. A method for producing an organism with improved resistance to lactic acid, the method comprising:
A) culturing the target organism with the maximum allowable concentration in the normal state for the lactic acid;
B) culturing the organism of interest with the lactic acid at a desired concentration for improvement; and C) selecting and regenerating a living organism;
Including the method. 14. The method according to claim 13, wherein the culturing step A) is performed for 1 day. Between the culturing step A) and the culturing step B), A ′) culturing in lactic acid at a concentration 1% higher than the maximum allowable concentration, and if necessary A ″) culturing step A ′) The step of culturing in lactic acid at a concentration 1% higher than the concentration in step 1) is repeated, and if necessary, the culturing step in lactic acid at a concentration higher by 1% is repeated in A ″) to culture at the desired concentration. 14. The method of claim 13, comprising achieving:   14. The method of claim 13, further comprising the step of D) adjusting the error-prone frequency in gene replication of the organism.   The method of claim 16, wherein the step of adjusting the error prone frequency comprises adjusting the error prone frequency of the DNA polymerase of the organism.   The DNA polymerase comprises at least one polymerase selected from the group consisting of DNA polymerase α, DNA polymerase β, DNA polymerase γ, DNA polymerase δ and DNA polymerase ε and their corresponding DNA polymerases in eukaryotic cells. Item 18. The method according to Item 17.   16. The method of claim 15, wherein the DNA polymerase comprises at least one polymerase selected from the group consisting of DNA polymerase δ and DNA polymerase ε in eukaryotic cells and the corresponding DNA polymerase. 14. The method of claim 13, wherein the organism is a eukaryote. The method according to claim 13, wherein the organism exists in a state of separated cells. 14. The method of claim 13, wherein the organism is an animal, plant, fungus or yeast cell. The method according to claim 13, wherein the organism is a yeast. The method according to claim 13, wherein the organism is an organism belonging to Saccharomyces. 14. The method of claim 13, wherein the organism is Saccharomyces cerevisiae. 14. The method of claim 13, wherein the organism exhibits substantially the same growth as the wild type after transformation of the desired trait. The method of claim 13, wherein the desired concentration is higher than the maximum allowable concentration. An organism produced by the method according to any one of claims 13 to 27. A method for evaluating the lactic acid resistance of a living organism
A) growing the organism under conditions under which the organism normally grows;
B) growing the organism in the presence or absence of the lactic acid; and C) determining the concentration of the lactic acid at which the organism dies.
Including the method.   30. The method of claim 29, wherein determining whether the organism is dead uses the cell as a criterion for determining whether the cell maintains its shape. Mycological properties are the same as the wild type except for lactic acid resistance.