JP2010115112A - Method of preparing yeast, the yeast, and method of producing lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of preparing a yeast having an ability to synthesize lactic acid, and to provide a yeast by using the production method and also a method of producing lactic acid using the yeast. <P>SOLUTION: The method of preparing a yeast having an ability to synthesize lactic acid includes the step of culturing a yeast variant, which controls a proofreading function of DNA polymerase, at a lactic acid concentration of at least 1% (v/v) and then separating the survival cells, to obtain a yeast having lactic acid tolerance, and transferring a lactic acid synthase gene into this yeast to prepare a yeast having an ability to synthesize lactic acid. The yeast is prepared by such method and the method of producing lactic acid uses the yeast. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a yeast excellent in lactic acid resistance and lactic acid synthesis ability, such a yeast, and a method for producing lactic acid using such a yeast.

  Conventionally, lactic acid has been preferably used as a raw material for medical products such as infusions, food additives, and cosmetic raw materials. Polylactic acid has been attracting attention as a polymer that is synthesized from lactic acid, a bio-derived material, and has excellent properties such as biodegradability, and is expected to be used in many fields such as films, resin products, medical materials, etc. It is growing. In this way, lactic acid is used for various purposes, and the demand is rapidly increasing. Therefore, it is required to produce lactic acid as a raw material at low cost.

  Lactic acid is mainly produced using lactic acid bacteria represented by the genus Lactobacillus. Since these lactic acid bacteria do not grow under acidic conditions, in order to accumulate a large amount of lactic acid in the culture solution, culture with a neutralizing agent such as calcium carbonate, ammonia or sodium hydroxide must be performed. , A large cost is required for the neutralization process and the lactic acid purification process. In addition, when lactic acid is produced using lactic acid bacteria, it is necessary to perform fermentation at a pKa (˜3.86) or less of lactic acid, so complicated steps must be taken to extract and purify lactic acid from the culture solution. I must.

On the other hand, a plurality of methods for producing organic acids using yeast strains that can grow under acidic conditions have been proposed (see, for example, Patent Documents 1 to 3 and Non-Patent Documents 1 and 2 below). According to these proposals, by introducing a lactic acid synthase gene into a yeast strain that does not originally have lactic acid production ability and producing lactic acid, the neutralization operation can be reduced and lactic acid production can be constructed at low cost. Is possible. Furthermore, by improving the production capacity of lactic acid and other organic acids by yeast strains using techniques such as genetic recombination, a cheaper method for producing organic acids can be constructed.
In addition, when acid accumulates in the medium, the intracellular pH also decreases as the medium pH decreases, but the microorganism activates proton ATPase (H ⁺ -ATPase) localized in the cell membrane to activate cells. It tries to keep the intracellular pH constant by pumping protons out.

However, in yeast cells, H ⁺ -ATPase, which should be activated under acidic conditions, is inhibited when lactic acid is present in the culture medium, and the intracellular pH can be suitably maintained. However, it has been reported that the survival rate of yeast cells decreases (see Non-Patent Document 3 below). In other words, in order to improve the production capacity of lactic acid to a higher level, it is a very important issue in the industry to improve the ability of yeast cells to resist lactic acid and produce a higher concentration of lactic acid. .

Currently, the following methods are used as methods for imparting lactic acid tolerance or maintaining lactic acid production ability.
As a first method, a method of isolating a novel lactic acid resistant strain having high lactic acid resistance and producing lactic acid using the obtained lactic acid resistant bacterium as a host can be mentioned. However, in order to carry out genetic recombination to produce a high concentration of lactic acid using the obtained lactic acid resistant bacteria as a host, it is necessary to develop an easy-to-handle vector system.
The second method is to introduce a mutation by treating a lactic acid bacterium with a mutagenic agent to produce a lactic acid resistant mutant, but it has not yet been able to confer significant lactic acid resistance. .
As a third method, a method (genome shuffling method) for producing a lactic acid resistant mutant by fusing lactic acid bacteria having different properties into cells can be mentioned. Specifically, for example, an example in which a lactic acid resistant strain producing lactic acid three times as much as a wild strain at pH 4.0 has been reported (see Non-Patent Document 4 below). However, this method has a problem that it takes a long time to produce a strain having a higher resistance to lactic acid.

  As a fourth method, there can be mentioned a method of imparting lactic acid resistance by introducing a lactic acid resistance-related gene isolated from a lactic acid resistant bacterium into a host bacterium by gene recombination. Specifically, for example, three genes, orfC belonging to the multidrug resistance gene family isolated from wine yeast Oenococcus oeni, putative transcription regulatory factor orfB belonging to LysR family, and low molecular weight heat shock protein Lol18 are introduced into Escherichia coli. An example of imparting acid resistance has been reported (see Non-Patent Document 5 below). However, it is unclear whether this method can be applied to host microorganisms that want to confer lactic acid resistance. Even if this method can be applied, there is no reasonable means other than increasing the expression of the lactic acid tolerance-related gene in order to further enhance the lactic acid tolerance level.

As a fifth method, there is a method of acclimatizing with acid using a chemostat. Specifically, for example, the target bacteria are pretreated at low pH (pH 5.0) for 60 minutes and then treated with lethal pH (pH 3.0) to induce expression of the resistance gene and acclimatize to lactate resistance. Has been reported (see Non-Patent Document 6 below). However, with this method, it is uncertain whether the mutation will be stably maintained, and other factors such as variability by species and acclimated lactic acid concentration are mixed. Absent.
Therefore, a yeast having excellent lactic acid tolerance, a production method for efficiently obtaining such yeast, and a method for producing lactic acid with excellent productivity using yeast have not yet been realized, and the prompt provision thereof is not possible. What is desired is the current situation.

JP 2001-204468 A Special table 2001-51658 pamphlet JP 2003-259878 A Applied and Environmental Microbiology, 71, 1964 (2005) Applied and Environmental Microbiology, 71, 2789 (2005) The Journal of the American Society of Brewing Chemists (The Journal of the American Society of Brewing Chemistry), 59, 187 (2001) Nature Biotechnology (Nature Biotechnology), 20, 707-712, 2002 Letters in Applied Microbiology, 33, 126-130, 2001 Journal of Molecular Biology and Biotechnology (Journal of Molecular Biology and Biotechnology), 4,525-532, 2002

  An object of this invention is to provide the manufacturing method of the yeast excellent in lactic acid tolerance and lactic acid synthesis ability.

  An object of the present invention is to provide a yeast excellent in lactic acid resistance and lactic acid synthesis ability.

  An object of the present invention is to provide an effective method for producing lactic acid using yeast.

  The present invention is basically obtained by culturing a yeast mutant strain in which the proofreading function of DNA polymerase is controlled under a lactic acid concentration of at least 1% (v / v) or more and then isolating viable cells. It is based on the knowledge that a yeast capable of synthesizing lactic acid can be obtained by introducing a lactic acid synthase gene into the obtained yeast.

  In addition, the present invention basically cultivates a yeast mutant strain into which a lactic acid synthase gene has been introduced and whose DNA polymerase proofreading function is controlled at a lactic acid concentration of at least 1% (v / v) or more. Then, it is based on the knowledge that a yeast capable of efficiently synthesizing lactic acid can be obtained by isolating viable cells.

  The present invention is based on the knowledge that lactic acid can be produced effectively by using such yeast.

  The present invention is basically obtained by culturing a yeast mutant strain in which the proofreading function of DNA polymerase is controlled under a lactic acid concentration of at least 1% (v / v) or more and then isolating viable cells. This is based on the knowledge that a yeast capable of efficiently synthesizing lactic acid can be obtained by introducing a lactic acid synthase gene into the obtained yeast.

  In addition, the present invention basically cultivates a yeast mutant strain into which a lactic acid synthase gene has been introduced and whose proofreading function of DNA polymerase is controlled at a lactic acid concentration of at least 1% (v / v) or more. Then, it is based on the knowledge that a yeast capable of efficiently synthesizing lactic acid can be obtained by isolating viable cells.

  The present invention is based on the knowledge that lactic acid can be effectively synthesized by using such a yeast.

  The first aspect of the present invention is obtained by culturing a yeast mutant strain in which the proofreading function of DNA polymerase is controlled under a lactic acid concentration of at least 1% (v / v) or more and then isolating viable cells. The present invention relates to a method for producing a yeast having the ability to synthesize lactic acid by introducing a lactic acid synthase gene into the obtained yeast.

  A preferred embodiment of the first aspect of the present invention relates to the yeast production method described above, wherein the yeast mutant is a strain into which a gene encoding mutant DNA polymerase δ has been introduced.

  A preferred embodiment of the first aspect of the present invention relates to the yeast production method according to any one of the above, wherein the yeast mutant is a budding yeast mutant in which the amino acid sequence of Pol3 is modified.

  In a preferred embodiment of the first aspect of the present invention, after culturing under a predetermined lactic acid concentration, the step of isolating viable cells is repeated, and the predetermined lactic acid concentration is 1% (v / v) or more and 7% (v / V) The method for producing yeast according to any one of the above, wherein the lactic acid concentration is increased as the culture and isolation steps are repeated.

  In a preferred embodiment of the first aspect of the present invention, a yeast mutant strain into which a lactic acid synthase gene has been introduced and the proofreading function of DNA polymerase is controlled can be obtained under a lactic acid concentration of at least 1% (v / v) or more. The present invention relates to a method for producing a yeast having the ability to synthesize lactic acid, comprising a step of isolating viable cells after culturing.

  The second aspect of the present invention comprises a step of isolating viable cells after culturing under a lactic acid concentration of at least 1% (v / v) or more using a yeast mutant strain in which the calibration function of DNA polymerase is controlled. It is obtained by introducing a lactic acid synthase gene after obtaining yeast having lactic acid resistance by repeating while increasing the lactic acid concentration within the range of 1% (v / v) to 7% (v / v). It is related with the yeast which has the ability to synthesize lactic acid.

  A preferred embodiment of the second aspect of the present invention relates to the yeast as described above, wherein the mutant strain of yeast is a strain into which a gene encoding mutant DNA polymerase δ has been introduced.

  A preferred embodiment of the second aspect of the present invention uses a yeast mutant strain into which a lactic acid synthase gene is introduced and the proofreading function of DNA polymerase is controlled, and at a lactic acid concentration of at least 1% (v / v) or more. It has the ability to synthesize lactic acid obtained by repeating the step of isolating viable cells after culturing in, while increasing the lactic acid concentration within the range of 1% (v / v) to 7% (v / v). Related to yeast.

  A preferred embodiment of the second aspect of the present invention relates to the yeast as described above, wherein the mutant strain of yeast is a strain into which a gene encoding mutant DNA polymerase δ has been introduced.

  A preferred embodiment of the second aspect of the present invention relates to the yeast according to any one of the above, wherein the yeast belongs to the genus Saccharomyces.

  A preferred embodiment of the second aspect of the present invention relates to the yeast according to any one of the above, wherein the yeast is Saccharomyces cerevisiae.

  A preferred embodiment of the second aspect of the present invention is deposited with the Patent Microorganism Depositary of the National Institute of Technology and Evaluation as a deposit number “NITE AP-319”, “NITE AP-320”, or “NITE AP-320”. The present invention relates to a yeast having the ability to synthesize lactic acid obtained by introducing a lactic acid synthase gene into yeast.

  According to a third aspect of the present invention, a yeast obtained by any one of the yeast production methods described above or the yeast described above is cultured in the presence of a carbohydrate to produce lactic acid, The present invention relates to a method for producing lactic acid by collecting lactic acid.

  According to the present invention, by culturing a yeast mutant strain in which the DNA polymerase proofreading function is controlled under a high lactic acid concentration, a yeast having excellent lactic acid resistance can be obtained, and a gene having lactic acid synthesis ability is introduced. By using a mutant strain capable of synthesizing lactic acid, a method for producing yeast capable of synthesizing lactic acid can be provided.

  According to the present invention, a yeast having a lactic acid resistance can be obtained by culturing a mutant strain of yeast with a controlled DNA polymerase proofreading function at a high lactic acid concentration. A yeast capable of synthesizing lactic acid can be obtained by introducing a gene having lactic acid or obtaining a yeast having lactic acid resistance using a mutant having lactic acid synthesis ability.

  According to the present invention, a yeast having high lactic acid tolerance and lactic acid synthesizing ability as described above can be obtained, so that an effective method for producing lactic acid using such yeast can be provided.

-Method for producing yeast having lactic acid synthesis ability-
In the yeast production method according to the first aspect of the present invention, basically, a yeast mutant strain in which the DNA polymerase proofreading function is controlled is cultured under a lactic acid concentration of at least 1% (v / v) or more. Then, it is related with the manufacturing method of the yeast which has a lactic acid synthesis ability which introduce | transduces a lactic acid synthase gene into the yeast obtained by isolating a living cell. That is, according to the first aspect of the present invention, basically, a yeast mutant strain in which the proofreading function of DNA polymerase is controlled is cultured under a lactic acid concentration of at least 1% (v / v) or more, and then a living cell. Is obtained by obtaining a yeast having lactic acid resistance by introducing a lactic acid synthase gene into the yeast having lactic acid resistance.

  Examples of “yeast mutants with controlled DNA polymerase calibration function” include yeast mutants in which the DNA polymerase error-prone frequency is adjusted. Examples of "yeast mutant strains that control the proofreading function of DNA polymerase" include strains into which a gene encoding mutant DNA polymerase δ has been introduced. More specifically, a mutant DNA polymerase δ expression plasmid is used. Examples include transformed strains and mutants of budding yeast in which the amino acid sequence of Pol3 has been modified. More specifically, a budding yeast obtained by transforming a DNA in which the 962nd base in the POL3 gene represented by SEQ ID NO: 4 is substituted from A to C and the 968th base is substituted from A to C Mutants. By culturing such a mutant strain of yeast with a controlled function of DNA polymerase under a high lactic acid concentration, a yeast having high lactic acid resistance can be obtained as demonstrated by the examples described later. .

  Having “lactic acid resistance” means having at least resistance to lactic acid, that is, not dying in an environment of a predetermined lactic acid concentration. A specific method for evaluating lactic acid resistance is as described later. It should be noted that known lactic acid can be used as appropriate as the lactic acid in the “environment of a predetermined lactic acid concentration”, but L-lactic acid is preferably used.

  In this specification, “1% (v / v) or more lactic acid concentration” is 1% or more and 15% or less, and may be 1% or more and 10% or less, or 1% or more and 7% or less. In addition, before culturing the strain in an environment where the lactic acid concentration is 1%, the yeast strain may be cultured at a concentration of 1% or less to be accustomed to the lactic acid environment. In addition, unless otherwise indicated, the lactic acid concentration in this specification is equivalent to the stock solution equivalent of the L-lactic acid solution (manufactured by Wako Pure Chemical Industries, Ltd., lactic acid content 85.0-92.0%) with respect to the volume of the medium (volume ratio ( v / v)). In a preferred embodiment of the present invention, the step of culturing under a predetermined lactic acid concentration and the step of isolating viable cells are repeated. Thus, by culturing under a predetermined lactic acid concentration and repeating the step of isolating viable cells, a yeast strain adapted to the lactic acid concentration can be effectively cultured.

  In the present invention, yeast is cultured in an environment with a predetermined lactic acid concentration. Known culture conditions can be appropriately employed as the culture conditions other than the lactic acid concentration. One culturing time is 1 hour to 1 week, and may be 6 hours or more and 2 days or less, or 1 day. A well-known thing can also be used suitably for the culture medium in the case of culture | cultivation. In the present invention, cultured cells are appropriately isolated. In order to isolate viable cells, a known isolation method may be used as appropriate.

  A preferred embodiment of the present invention is a method for producing yeast comprising a step of culturing under a predetermined lactic acid concentration and repeating a step of isolating viable cells, and a step of increasing the lactic acid concentration as the culturing and isolation steps are repeated. That is, this preferred embodiment of the present invention does not suddenly lead to an environment with a target lactic acid tolerance concentration, but gradually increases the lactic acid concentration to culture it so that it becomes accustomed to a high lactic acid concentration environment, and finally the object. Yeast having lactic acid resistance is obtained. Specifically, each culture is cultured at a lactic acid concentration of 0.1% or more and 2% or less, and cultivated at a lactic acid concentration of 0.5% or more and 1.5% or less. It may be. When repeating the culture, a step of culturing the yeast strain in the same environment as the lactic acid concentration in the previous culture or a step of culturing the yeast strain in an environment lower than the lactic acid concentration in the previous culture may be included as appropriate. You may obtain the yeast which has the target lactic acid tolerance by culture | cultivating a yeast mutant under the lactic acid concentration which has the target lactic acid tolerance density | concentration, and isolating a living cell.

  Specifically, after culturing under a predetermined lactic acid concentration, the step of isolating viable cells is repeated, and the predetermined lactic acid concentration is 1% or more and 7% or less, and as the culturing and isolation steps are repeated, The yeast production method according to any one of the above, wherein the lactic acid concentration is increased.

  A preferred embodiment of the present invention is a method for producing a yeast having the ability to synthesize lactic acid, comprising the step of introducing a lactic acid synthase gene into the lactic acid resistant yeast obtained as described above. As a method for introducing a gene, a known method may be adopted as appropriate. It should be noted that the lactic acid synthase gene is directly applied after conferring lactic acid resistance by culturing at a predetermined lactic acid concentration using a mutant having the property of inducing a mutation having lactic acid resistance by controlling the calibration function of DNA polymerase. The introduction of can lead to unwanted mutations in the introduced lactic acid synthesis gene and other genes as a result of subsequent unwanted mutations. Therefore, for yeast mutant strains that have been imparted with a property of inducing mutations having lactic acid resistance by controlling the proofreading function of the DNA polymerase, after cultivating at a predetermined lactic acid concentration to give lactic acid resistance, once the DNA polymerase It is preferable to introduce an acid synthase gene after the step of returning the proofreading function. As a step of returning the proofreading function of the DNA polymerase, there is a process in which a gene encoding a DNA polymerase having a proofreading function, such as a gene encoding a mutant DNA polymerase δ existing on a chromosome, is removed by homologous recombination. In addition, removing a plasmid from a cell with a DNA polymerase having a proofreading function, such as a mutant DNA polymerase δ existing on the plasmid, may also prevent subsequent unnecessary mutations from occurring after the target trait has been acquired. it can.

  The yeast used in the present invention is not particularly limited as long as it is generally called yeast, and budding yeast, fission yeast, and the like can be appropriately used. Representative yeasts include those belonging to the family Saccharomycesaceae or Schizosaccharomycesaceae, and among these, those belonging to the genus Saccharomyces are preferred. As a more specific yeast, Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is widely used as a eukaryotic model organism and is a kind of budding yeast is preferable. Among these, the budding yeast demonstrated in the examples is preferable, but the present invention is not particularly limited to the budding yeast and can be widely applied to all yeasts.

  As the yeast used in the present invention, yeast having lactic acid synthesis ability is preferable. That is, a preferred embodiment of the present invention includes a step of culturing under a lactic acid concentration using a mutant strain of yeast into which a lactic acid synthase gene is introduced and a DNA polymerase proofreading function is controlled, and a step of isolating viable cells. Are repeated as appropriate. By using such a mutant strain, it is not necessary to introduce a lactate synthase gene after producing a yeast having lactate tolerance. The yeast having the ability to synthesize lactic acid is not particularly limited and may be appropriately selected depending on the intended purpose. However, a yeast having a lactic acid synthase gene is preferable. Specifically, for example, a lactic acid synthase gene is introduced. Therefore, yeast that has acquired the ability to synthesize lactic acid is particularly preferred. Examples of such lactic acid synthase include lactate dehydrogenase. As the yeast having the ability to synthesize lactic acid, yeast belonging to the genus Saccharomyces is preferably used, and among these, Saccharomyces cerevisiae is more preferably used.

  In the case of imparting lactic acid synthesizing ability by introducing a gene, the gene encoding the lactic acid synthase to be introduced is not particularly limited as long as it is DNA encoding lactic acid synthase that can be produced in yeast. Although it can be selected, DNA encoding lactic acid synthase with enhanced enzyme activity is preferred. Examples of DNA encoding lactic acid synthase with enhanced enzyme activity include DNA encoding lactic acid synthase with strong enzyme activity obtained by a mutation technique using site-directed mutagenesis, and a strong promoter. Examples include DNA linked with DNA encoding lactic acid synthase under control.

  The yeast having the lactic acid synthase gene can be produced by introducing a gene encoding lactic acid synthase (lactate dehydrogenase) using a technique such as genetic recombination. On the other hand, when the yeast already has a gene encoding the target lactic acid synthase, it is not always necessary to introduce the gene. Specific examples of the lactic acid synthase include an enzyme consisting of a protein encoded by an L-lactic acid synthase gene (L-ldh-b) derived from Bos taurus. FIG. 13 is a construction diagram of plasmid pLDH-b used in the present invention for the gene recombination.

  According to the present invention, for example, by introducing a mutation by controlling the proofreading function of DNA polymerase as described above, it exhibits excellent lactic acid resistance not found in wild strains or poor in wild strains. be able to. In addition, lactic acid can be synthesized using yeast by introducing a gene having not only lactic acid resistance but also lactic acid synthesis ability into yeast.

Mutagenesis is most commonly used to produce useful mutants of yeast. The mutation usually means a change in the base sequence encoding the gene, and includes a change in the DNA sequence. Mutations are broadly classified into the following three types according to the effects on the individuals in which they occur.
(1) Neutral mutation: Most mutations fall under this neutral mutation, and the neutral mutation has little effect on the growth and metabolism of the organism.
(2) Deleterious mutation: This mutation is less frequent than a neutral mutation and inhibits the growth or metabolism of an organism. The harmful mutation includes a lethal mutation that destroys a gene essential for growth.
(3) Beneficial mutation: This mutation means a mutation that is beneficial to the breeding of an organism, but its frequency of occurrence is extremely low compared to a neutral mutation. The breeding effect of beneficial mutations rarely appears with a single mutation, and accumulation of multiple beneficial mutations is necessary. Therefore, in order to obtain an individual organism into which a beneficial mutation has been introduced, a larger mutant population and a longer time for selection are required.

In order to generate a mutation, for example, by introducing a mutation that destroys the 3 ′ → 5 ′ exonuclease activity into a gene encoding DNA polymerase (DNA polymerase gene), the proofreading function is lower than the wild type. , Nucleotides and polypeptides encoding DNA polymerases can be produced.
The calibration function refers to a biological function that detects and repairs DNA damage or errors received by cells. As a specific repair, if there is depurination or depyrimidine, a base is inserted as it is, or a single-strand break is inserted with an AP endonuclease (apuric-apirimidinic endonuclease), and then 5 ′ → 3 'It can be removed by endonuclease. The removed portion is synthetically supplemented with DNA polymerase, and the cleaved portion is ligated with DNA ligase. Such a reaction is called removal repair.
Examples of the DNA polymerase having such a proofreading function include DNA polymerase δ (delta) and DNA polymerase ε (epsilon) in eukaryotes. Among these, DNA polymerase δ is more preferable, and the adjustment of the proofreading function can be achieved by modifying the amino acid sequence related to the proofreading function.

  The lactic acid resistant yeast obtained by the present invention has a lactic acid resistance to a lactic acid concentration of at least 1%, preferably at least 3%, more preferably at least 4%, more preferably at least 5%, Even more preferably, the yeast is resistant to lactic acid concentrations of at least 7% or more, particularly preferably at least 10% or more. Yeast having tolerance to lactic acid concentration of 4% to 7% from the viewpoint of imparting lactic acid tolerance suitable for production of lactic acid and balancing with the number of steps and cost of production of yeast having lactic acid tolerance Is most preferred.

  In the present invention, lactic acid resistance is determined, for example, as follows. That is, a yeast strain whose lactic acid tolerance is to be evaluated is cultured overnight in a medium not containing lactic acid. Next, media containing various concentrations of lactic acid are prepared, and 1% of the overnight culture is added to each media and cultured overnight. After overnight culture, the bacterial concentration is measured by, for example, absorbance measurement, agar plate method, viability measurement by trypan blue staining, and the like. Evaluate that the lactate concentration is the same as that in the case of using a medium in the vicinity of neutral, and that it has tolerance to lactic acid at that concentration.

  As used herein, “error-prone frequency” refers to the level of error-prone nature. 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, error-prone frequency 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 is uniform when referring to factors responsible for the replication of multiple genes (such as polymerases), the error-prone frequencies are substantially equal to each other. 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 multiple 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, but preferably increasing error-prone frequency. Methods for adjusting the error-prone frequency include, for example, 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 enzymes, removal of abnormal bases modification of enzymes with repair factor function, modification of mismatched base pair repair factors, reduction of the 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, only one of the DNA duplexes is performed because harmful mutagenesis is reduced.

In this specification, “error prone” refers to a property that is likely to be erroneous in replication of a gene (DNA, etc.) (that is, replication error). Error prone is mainly affected by the accuracy of the calibration function of an enzyme having a calibration function (for example, a DNA polymerase). As used herein, “replication error” refers to an error in nucleotide incorporation that occurs during the process of gene (DNA, etc.) replication. The frequency of replication errors is usually very low in living organisms, about once every 10 ⁸ to 10 ¹² times. The reason for the low frequency of replication errors is that nucleotide incorporation causes replication by forming a complementary base pair with the nucleotide into which the template DNA is incorporated, and the proofreading function of an enzyme such as DNA polymerase, ie 3 ′ → 5 'Exonuclease has a function to detect when a nucleotide that does not complement the template is mistakenly incorporated, and to cut it out immediately. Therefore, in the present invention, the error prone frequency in replication can be adjusted by the failure of specific base pairing or the failure of the calibration function.

  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. As used herein, error-prone and error-free can be classified absolutely (ie, determined by the level of error-prone frequency, etc.) or relative (factors responsible for the 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.

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

  In the present invention, in order to control the proofreading function of DNA polymerase, any mutation may be introduced into the DNA polymerase gene according to a known method. For example, since the nucleotide sequence of a yeast DNA polymerase gene is known, the method for introducing a gene mutation is, for example, 1 to 100, preferably 1 to 10, more preferably 1 to Substitution with three other bases is sufficient. For example, the proofreading function of DNA polymerase can be controlled by expressing a mutant protein in which the proofreading function of polymerase 3 (Pol3), which is considered to be involved in lagging strand replication during DNA replication, is lost in the target yeast cell. That is, as a method for expressing mutant Pol3 in which the proofreading function has been lost in the target yeast cell, the mutant pol3 gene is artificially obtained and transformed into the target yeast. Those that can be expressed and function are included. That is, as a method for creating a mutant Pol3 that has lost its proofreading function, a part of the amino acid sequence of the proofreading function active site is artificially replaced using the previously cloned wild-type POL3 gene as a template to control the proofreading function. do it. Alternatively, a method may be used in which the gene is cloned from a strain having the same trait as the pol3 mutant and already identified as a natural mutant. The DNA polymerase gene may include not only a DNA polymerase structural gene but also both transcriptional and / or translational regulatory sequences such as a DNA polymerase promoter.

  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 of the enzyme is lower than that of the wild type (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). Such comparison with the wild type can be done by relative or absolute labeling. 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, mutation is used for gene sequence changes that occur with respect to the proofreading function. In this specification, unless otherwise stated, mutation is used synonymously with modification.

  The yeast having the ability to synthesize lactic acid according to the second aspect of the present invention is a yeast having the ability to synthesize lactic acid and having the ability to synthesize lactic acid with respect to a lactic acid concentration of at least 1%, which is produced by the above yeast production method. Yeast having the ability to synthesize lactic acid can be obtained by the method described in the above yeast production method. The yeast having lactic acid resistance of the present invention can be suitably used in the method for producing lactic acid of the present invention, which will be described later, and in particular, efficiently producing a high concentration of lactic acid by using a yeast having the ability to synthesize lactic acid. Can do.

—Method for Producing Lactic Acid— The third aspect of the present invention is a method for cultivating yeast obtained by the above-described method for producing yeast in the presence of carbohydrates, producing lactic acid, and collecting the lactic acid from the culture. It relates to the manufacturing method. In order to produce the lactic acid, it is preferable to use a yeast having the ability to synthesize lactic acid. As the yeast having the ability to synthesize lactic acid, yeast having a lactic acid synthase gene is preferable, and yeast into which a lactic acid synthase gene has been introduced is more preferable. The details of the yeast having the ability to synthesize lactic acid and the method for introducing a lactic acid synthase gene are as described in the above-mentioned yeast production method.

Culture of yeast used in the method for producing lactic acid according to the present invention can be carried out according to the usual method described below.
A medium for culturing yeast used in the method for producing lactic acid of the present invention contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the yeast, and can efficiently culture the yeast. If it is, there is no restriction | limiting in particular, According to the objective, it can select suitably, Either a natural culture medium and a synthetic culture medium may be used.
The carbon source is not particularly limited as long as the yeast strain can be assimilated, and can be appropriately selected according to the purpose. However, glucose, fructose, sucrose, molasses containing these, starch, And carbohydrates such as starch hydrolysates can be used.
The nitrogen source is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include ammonia, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, and ammonium acetate, peptone, meat extract, and yeast extract. , Corn steep liquor, casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermentation cell digests, and the like.
The inorganic salt is not particularly limited and may be appropriately selected depending on the intended purpose. However, magnesium phosphate, magnesium sulfate, sodium chloride, monopotassium phosphate, dipotassium phosphate, ferrous sulfate, Manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.

The carbon source may be added all at once at the start of the culture, may be divided during the culture, or may be added continuously, and is preferably used at a concentration of 50 g / L to 150 g / L.
The culture in the present invention is preferably performed by shaking culture, stirring culture, or the like, and can be performed under aerobic conditions, microaerobic conditions, or anaerobic conditions. The culture temperature is preferably 25 to 35 ° C., and the culture time is usually preferably 24 hours to 5 days. Moreover, pH may be adjusted as necessary, and the preparation can also be performed using an alkaline solution, ammonia, calcium carbonate, or the like.

  The concentration of lactic acid in the culture broth can be quantified by a method using high performance liquid chromatography (HPLC). For example, the culture broth is centrifuged to prepare a culture supernatant, and the culture supernatant is analyzed. By using an anion exchange column for organic acid analysis as a sample and measuring the electrical conductivity, the concentration of organic acid such as lactic acid or acetic acid in the culture solution can be quantified.

The method for collecting lactic acid produced in the culture solution is to remove precipitates such as bacterial cells from the culture after completion of the culture, and combine the ion exchange treatment method, concentration method, salting-out method, etc. The target lactic acid can be isolated and purified from the culture. For example, it can be purified by an electrodialysis method or a distillation method as disclosed in JP 2001-204464 A, JP 9-135698 A, and the like, but is not limited thereto. . Moreover, various lactic acid derivatives can be obtained by performing esterification etc. with respect to the said crude extraction fraction and its refined | purified material as needed.
Lactic acid obtained by the lactic acid production method of the present invention using lactic acid resistant yeast is suitably used for industrial applications such as polymer raw materials, food additives, cosmetic materials, pharmaceutical raw materials and the like.

Description of Deposited strain BYD5 (mutant pol3 gene expression strain) used in the examples described later is a patent microorganism deposit center of the National Institute of Technology and Evaluation, Japan from August 19, 2005 (Kisarazu, Chiba Prefecture 292-0818) It is deposited with the receipt number NITE P-131 in Kazusa City Kamashita 2-5-8). AMY52-3D (wild-type pol3 gene expression strain) used in the examples described later has been deposited as a receipt number NITE P-129 from August 19, 2005 to the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation. Has been.
BYD5-LA7 (lactic acid resistant strain) obtained in the examples described later has been deposited as an accession number NITE P-122 at the National Institute of Technology and Evaluation for Microorganisms on July 29, 2005. Yes.

TBSCR8 (lactic acid resistant strain) obtained in the examples described later has been deposited as an accession number “NITE AP-319” with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, February 22, 2007. .
HRSC 207 (lactic acid resistant strain) obtained in the examples described later has been deposited as an accession number “NITE AP-320” with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, February 22, 2007. .
HRSC 208 (lactic acid resistant strain) obtained in the examples described later has been deposited as an accession number “NITE AP-321” with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, February 22, 2007. .

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these. That is, the present invention includes those appropriately modified within the scope obvious to those skilled in the art from the matters disclosed in the present specification and the matters proved in the following examples. The reagents used in the following examples are those commercially available from Sigma (St. Louis, USA), Wako Pure Chemical Industries (Osaka, Japan), etc., except those specifically described in the specification notes. It was.

Reference Example 1 Examination of changes in medium pH and cell growth caused by addition of lactic acid-Yeast strain-As a yeast strain, mutant pol3 gene expression strain (BYD5, hereinafter referred to as pol3 mutant strain) and wild-type pol3 gene expression A strain (AMY52-3D, hereinafter referred to as a wild strain) was used. The BYD5 genotype is (pol3-01 / pol3-01, MATa / MATα, lys1-1 / lys1-1), and the AMY52-3D genotype is (MATα, ura3-52, leu2-1). Lys1-1, ade2-1, his1-7, hom3-10, trp1-289, canR).

  -Medium and Reagents-YPD medium (manufactured by BD Biosciences Clontech) was used as the liquid medium, and YPD agar medium (manufactured by BD Biosciences Clontech) was used as the agar medium. These were all purchased from BD Biosciences Clontech. The liquid medium and agar medium were sterilized at 120 ° C. for 15 minutes by an autoclave. L-lactic acid (Wako Pure Chemical Industries, Ltd.) was used as lactic acid.

-Experimental Method- (1) Experiment on measurement of medium pH by addition of lactic acid Liquid prepared by adding lactic acid to the liquid medium so that the lactic acid concentration is 1% to 20% in increments of 1% and without adding lactic acid The pH of the medium and the liquid medium of each lactic acid concentration was measured.

(2) Experiments on the effect of lactic acid on cell growth (A) The pol3 mutant and wild type colonies developed on the agar medium were scraped with a platinum loop and placed in a test tube (diameter 18 mm) containing 4 mL of liquid medium. Inoculated. Each test tube was cultured with shaking at 30 ° C. and 180 rpm overnight (16 hours or more). (B) A part of each culture solution was collected, the cell density was measured using a hemocytometer, and each culture solution was diluted with a liquid medium so that the cell densities of both strains were equal. (C) Each culture solution was collected in a microtube so that the cell density at the start of culture was 2.0 × 10 ⁶ cells / mL, and the cells were collected by centrifugation at 5,000 × g for 3 minutes. (D) 1%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 13%, 15%, 17%, 19% lactic acid concentration prepared as described above The cells collected in (C) were resuspended in 4 mL each of 20% liquid medium, and cultured with shaking at 30 ° C. and 180 rpm for 18 hours. In this culture, a test tube having a diameter of 18 mm was used. (E) After culture, the cell density of each culture solution was measured using a hemocytometer, and the effects of lactic acid addition on cell proliferation were compared.

-Experimental results- (1) Examination of medium pH by addition of lactic acid Changes in pH when lactic acid is added to the liquid medium are shown in Table 1 and FIG. Moreover, the change of the culture medium in that case is shown in FIG. From the results shown in Table 1 below, it was clarified that the pH of the YPD liquid medium was remarkably lowered by adding lactic acid. Further, as shown in FIG. 1, it was also clarified that by adding lactic acid, some of the medium components were precipitated and the medium became cloudy.

(2) Examination of influence on cell proliferation by addition of lactic acid The results of measuring the cell density when the wild strain and pol3 mutant strain were cultured for 18 hours in a medium containing the lactic acid prepared above at each lactic acid concentration, It shows in following Table 2 and FIG. From the results in Table 2 below, it was found that the cell density of the wild strain and the pol3 mutant was decreased by the addition of lactic acid. In particular, when the lactic acid concentration exceeded 5%, it became clear that most could not grow. In addition, since a correlation was observed between the pH of the medium and the cell growth of both strains due to the addition of lactic acid, it was shown that the cell growth of both strains was inhibited by a decrease in the pH of the medium.

Reference Example 2 Production of Yeast Using Method for Producing Yeast with Lactic Acid Resistance In Reference Example 2, lactic acid tolerance was imparted to the pol3 mutant using the method for producing a yeast with lactic acid resistance of the present invention. The procedure is shown below.

-Yeast strain-The same pol3 mutant (BYD5) as in Reference Example 1 was used. -Medium, etc.-The same liquid medium and lactic acid as in Reference Example 1 were used.

-Experimental method-(1) The colon of the pol3 mutant strain was scraped off, inoculated into a test tube (diameter 18 mm) containing 4 mL of liquid medium, and cultured overnight at 30 ° C. and 180 rpm.
(2) The culture solution was collected in a 15 mL centrifuge tube and centrifuged at 1,000 × g for 5 minutes to collect all cells.
(3) All the cells collected above were resuspended in a 300 mL Erlenmeyer flask containing 100 mL of liquid medium and cultured overnight at 30 ° C. and 180 rpm.
(4) The culture solution cultured as described above was placed in a 50 mL centrifuge tube (manufactured by Corning) and centrifuged at 1,000 × g for 5 minutes to collect all cells.
(5) All the cells collected above are suspended in a 300 mL Erlenmeyer flask containing 150 mL of a liquid medium (hereinafter referred to as 3% lactic acid medium) prepared to have a lactic acid concentration of 3%, and 30 ° C., 180 rpm In-night culture.
(6) A part of the 3% lactic acid culture medium cultured as described above was collected and mixed with an equal volume of 0.1% trypan blue solution (manufactured by DOJINDO) to stain the cells. Since the cells stained blue are dead cells and the cells not stained are viable cells, the ratio of viable cells was observed using this as a mark.
(7) After confirming the presence of viable cells, 1/3 volume (50 mL) of 3% lactic acid culture was collected in a 50 mL centrifuge tube and centrifuged at 1,000 × g for 5 minutes to recover the cells.

(8) The collected cells were suspended in a 300 mL Erlenmeyer flask containing 150 mL of 4% lactic acid medium, and cultured overnight at 30 ° C. and 180 rpm.
(9) A part of the 4% lactic acid culture medium cultured as described above was collected, and the cells were observed by trypan blue staining similar to the above. After confirming the viable cells, 1/3 volume (50 mL) of the culture solution was collected in a 50 mL centrifuge tube, and centrifuged at 1,000 × 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% lactic acid medium, and cultured overnight at 30 ° C. and 180 rpm.
(11) A part of the 5% lactic acid culture solution cultured as described above was collected, and viable cells were confirmed by trypan blue staining similar to the above.
(12) After confirming the presence of viable cells, 1/10 volume (15 mL) of 5% lactic acid culture solution was collected and centrifuged at 1,000 × g for 5 minutes to recover the cells. The collected cells were suspended in a 200 mL shake flask (manufactured by IWAKI) containing 70 mL of 5% lactic acid medium, and the cell density was measured. After the measurement, these culture solutions were cultured with shaking at 30 ° C. and 180 rpm.
(13) The cell density of the culture solution was measured every 24 hours and cultured until the cell density immediately after the start of the culture was about 5 to 10 times.
(14) A 1/10 volume (7 mL) was collected from the culture medium in which growth was confirmed in the above-mentioned 5% lactic acid medium, and centrifuged at 1,000 × g for 5 minutes to collect cells. These cells were suspended again in 5% lactic acid medium and cultured with shaking at 30 ° C. and 180 rpm.

  (15) Subculture was performed by repeating the operations (11) to (14). In addition, as a reference for subculture, (i) to confirm viable cells by trypan blue staining, (ii) to grow about 5 to 10 times the cell density immediately after the start of culture, Two points were set.

-Experimental results- As described above, the cell density when the lactic acid concentration in the liquid medium was gradually increased and finally subcultured in a lactic acid medium having a lactic acid concentration of 5% is shown in Table 3 and FIG. Show.

From the results shown in Table 3 and FIG.
(1) As a result of culturing the pol3 mutant strain in 3% lactic acid medium, the cell density increased and many viable cells were observed.
(2) As a result of transplanting the pol3 mutant grown in culture in 3% lactic acid medium to 4% lactic acid medium, an increase in cell density and viable cells were observed.
(3) As a result of transplanting the pol3 mutant grown in culture in 4% lactic acid medium to 5% lactic acid medium, an increase in cell density was observed on the 4th day from the start of the culture.
(4) As a result of trypan blue staining, many viable cells were observed in the culture with 5% lactic acid medium (FIG. 3).
(5) The pol3 mutant strain grown by culturing in 5% lactic acid medium was transplanted again into 5% lactic acid medium, and subculture was repeated in the same medium. As a result, there was a tendency for the cell growth rate to increase as the number of passages increased (Table 3, FIG. 4).

  From the above results, it was shown that resistance to lactic acid at a concentration of 5% can be imparted to the pol3 mutant by using the yeast production method as described above.

Reference Example 3 Culture Characteristics of 5% Lactic Acid-Resistant Strain In Reference Example 2, the culture characteristics of the pol3 mutant strain that acquired resistance to lactic acid at a concentration of 5% (hereinafter referred to as 5% lactic acid resistant strain) were examined. The experimental method is shown below.

-Yeast strain-The same wild strain as in Reference Example 1 and the 5% lactic acid resistant strain obtained in Reference Example 2 were used.

-Experimental method-
The 5% lactic acid resistant strain obtained in Reference Example 2 was subcultured in a 200 mL baffled Erlenmeyer flask containing 70 mL of 5% lactic acid medium. This culture was performed at 30 ° C. and 200 rpm, and about 1/10 of the cells were passaged with respect to the whole culture solution.

(1) A colony of a wild 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 of the wild strain was centrifuged at 5,000 × 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) The 5% lactic acid resistant strain subcultured in the 5% lactic acid medium and the wild strain cultured as described above were collected in a 50 mL centrifuge tube, and centrifuged at 1,000 × g for 5 minutes to collect the cells.
(4) The cells collected above 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, each was diluted with a liquid medium without lactic acid so that the cell densities of the wild strain and the 5% lactic acid resistant strain were equal.

(6) The cell dilution prepared so that the final cell density is about 2.0 × 10 ⁷ cells / mL is collected in a 5 mL centrifuge tube, centrifuged at 1,000 × g for 5 minutes, and the cells are removed. It was collected. Next, the recovered cells were suspended in a 200 mL baffled Erlenmeyer flask containing 70 mL of lactic acid medium with 5%, 6% and 7% lactic acid concentrations, and cultured with shaking at 30 ° C. and 200 rpm. The wild strain was cultured only in a 5% lactic acid medium. These lactic acid media were used after being filtered with a 0.45 μm sterilizing filter (ADVANTEC).
(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 three times for each measurement, and the average value and standard deviation were calculated.

-Experimental results-
As described above, changes in cell density when wild strains and 5% lactic acid resistant strains were cultured in lactic acid medium at various concentrations are shown in Table 4 and FIGS. 5 and 6 below.

From the results shown in Table 4 and FIGS. 5 and 6, the following facts were recognized.
(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) It was found that the 5% lactic acid resistant strain grew rapidly under culture conditions containing lactic acid at concentrations of 5% and 6%, 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).

  From the above results, it was found that the 5% lactic acid resistant strain obtained by acclimatizing the pol3 mutant strain to lactic acid has acquired excellent resistance to 5% and 6% lactic acid.

Reference Example 4 Preparation of 7% Lactic Acid Resistant Strain Resistant to 7% concentration of lactic acid using the 5% lactic acid resistant strain shown in Reference Example 2 and Reference Example 3 using the method for producing yeast having lactic acid resistance of the present invention The experiment which gives is performed.

-Experimental method-
The 5% lactic acid resistant strain (pol3 mutant) obtained in Reference Example 2 and Reference Example 3 was subcultured at 30 ° C. and 200 rpm using a 200 mL baffled Erlenmeyer flask containing 70 mL of 5% lactic acid medium. went. Passage to a new medium was performed every 2 to 3 days, and 1/10 amount of cells were collected from the whole culture and transplanted.

(1) A 1/5 volume of the whole culture was collected from the subculture of a 5% lactic acid resistant strain, and centrifuged at 1,000 × g for 5 minutes to collect cells. The recovered cells were suspended in a 200 mL baffled Erlenmeyer flask containing 70 mL of 7% lactic acid medium having a lactic acid concentration of 7%, and cultured overnight at 30 ° C. and 200 rpm (first passage).
(2) One-fifth of the whole culture solution after the above culture was collected and centrifuged at 1,000 × 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 (second passage).
(3) A 1/10 volume of the whole culture solution after the culture was collected and centrifuged at 1,000 × 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 (the third passage).
(4) A 1/10 volume of the whole culture solution after the above culture was collected and centrifuged at 1,000 × 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 (passage 4th time).
(5) The fourth subculture strain and a part of the wild strain previously cultured in the liquid medium were transplanted into 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 concentration.

-Experimental results-
As described above, changes in cell density when a 5% lactic acid resistant strain is subcultured in a 7% lactic acid medium are shown in Table 5 and FIG.

From the results shown in Table 5 and FIG.
(1) When cultured for 5 days in the second subculture, about 6.7 times the proliferation was observed as compared to immediately after the subculture (Table 5, FIG. 7).
(2) When the cells were cultured for 5 days at the third subculture, about 66.9 times the proliferation was observed as compared to immediately after the subculture (Table 5, FIG. 7).
(3) When cultured for 4 days in the 4th subculture, the growth was about 310 times that immediately after the subculture (Table 5, 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 lactate resistant strain (FIG. 8).

  The lactic acid resistant strain obtained in the example was named BYD5-LA7 (can grow in YPD medium containing 7% lactic acid).

Reference Example 5 Yeast Production Using Yeast Production Method with Lactic Acid Resistance- Yeast Strain-
In Reference Example 5, Saccharomyces cerevisiae YPH499 strain was used as a wild strain. The YPH499 strain is a known wild strain, and is introduced as a catalog number NBRC10505, for example, in a sales agency operated by NITE.
-Medium-YPDA Broth (YPD Broth (Difco), 20 mg / L Adenine sulfate (Wako Pure Chemical Industries)) was used as a liquid medium. As the solid medium, YPDA Broth (YPD Broth (Difco), 20 mg / L Adenine sulfate (Wako Pure Chemical Industries), 20 g / L Bacto Agar (Becton Dickinson)) was used. L-lactic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was used as lactic acid. Also, as a 5-FOA solid medium, (20 g / L D-Glucose (manufactured by Wako Pure Chemical Industries, Ltd.), 6.7 g / L Yeast Nitrogen Base). w / o Amino acids (manufactured by Beckton Dickinson), 0.79 g / L Complete supplement mixture (manufactured by FORMIUMIUM, 40 mg / L Adenine sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), 1 g / Lor oc Kogyo Co., Ltd.), 20 g / L Bacto Agar (Becton Dickinson)).

-Experimental method-
(1) Saccharomyces cerevisiae YPH499 strain was transformed with the mutant DNA polymerase δ expression plasmid YEplac195 pol3-01. FIG. 9 shows a construction diagram of the above mutant DNA polymerase δ expression plasmid. The mutant DNA polymerase δ expression plasmid YElac195 pol3-01 is a plasmid constructed by incorporating a mutant DNA polymerase δ gene into the multicloning site of YElac195, a vector plasmid for introducing genetic information into yeast cells. . Similar plasmids that can incorporate the mutant DNA polymerase δ gene include the YEp series and YRp series, which are high-copy plasmids that replicate independently of chromosomes in yeast cells, and the low-value that replicate independently of chromosomes in yeast cells. Examples thereof include the YCp series which is a copy plasmid and the YIp series which is a plasmid integrated into a chromosome in yeast cells.
(2) The colony of the transformed strain was scraped off, inoculated into a test tube containing 4 mL of a liquid medium, and cultured with shaking at 30 ° C. and 180 rpm overnight.
(3) A part of the culture solution was seeded on a solid medium containing 3.5% lactic acid and cultured at 30 ° C. for 5 days, and several colonies that had grown were caught.
(4) The colony fished in (3) was inoculated in a solid medium containing lactic acid at a concentration of 3.5% and cultured at 30 ° C. overnight, and the grown colonies were fished.
(5) The colony fished in (4) was inoculated in a liquid medium, cultured at 30 ° C. for 6 hours, and all cells were again collected from the culture solution by centrifugation.
(6) The cells collected in (5) were inoculated into a liquid medium containing 4.0% lactic acid and cultured overnight at 30 ° C.
(7) A part of the culture solution was seeded on a solid medium containing 4.0% lactic acid and cultured at 30 ° C. for 4 days, and the grown colonies were fished.
(8) The colony fished in (7) was inoculated in a liquid medium, cultured at 30 ° C. for 8 hours, and all cells were again collected from the culture solution by centrifugation.
(9) The cells collected in (8) were inoculated into a liquid medium containing 5% lactic acid and cultured at 30 ° C. for 3 nights.
(10) YEplac195 pol3-01 is removed from cells in 5% culture solution, 5% culture solution is seeded on 5-FOA solid medium, cultured at 30 ° C. for 3 nights, and plasmids are retained for the colonies that have grown. The absence was confirmed by PCR.
(11) The strain in which YElac195 pol3-01 is eliminated in (10) is cultured in a liquid medium containing 4.5% lactic acid at 30 ° C. for 2 nights. By culturing until about 5 to 10 times, a yeast strain having acquired resistance to 4.5% lactic acid concentration was isolated.

  The lactic acid resistant strains obtained in Reference Example 5 were named HRSC207, HRSC208, HRSC210, and HRSC214, respectively.

Reference Example 6 Yeast Production Using Yeast Production Method with Lactic Acid Resistance- Yeast Strain-
The same strain as in Reference Example 5 was used.
-Medium-
The same medium as in Reference Example 5 was used.

-Experimental method-
(1) Saccharomyces cerevisiae YPH499 strain was transformed with the mutant DNA polymerase δ expression plasmid YEplac195 pol3-01.
(2) The colony of the transformed strain was scraped off, inoculated into a test tube containing 4 mL of a liquid medium, and cultured with shaking at 30 ° C. and 180 rpm overnight.
(3) A part of the culture solution was spread on a solid medium containing lactic acid having a concentration of 5.0%, cultured at 30 ° C. for 2 days, and several colonies that had grown were caught.
(4) The colony fished in (3) was inoculated into a culture solution containing lactic acid at a concentration of 5.5% and cultured at 30 ° C. overnight.
(5) All the cells were again collected from the culture solution by centrifugation.
(6) In order to drop YEplac195 pol3-01 from the cells in the 5.5% culture solution, the 5.5% culture solution was seeded on 5-FOA solid medium and cultured at 30 ° C. for 3 nights. It was confirmed by PCR that the plasmid was not retained.
(7) The strain in which YEplac195 pol3-01 has been confirmed to drop in (6) is cultured in a liquid medium containing 4.5% lactic acid at 30 ° C. for 2 nights. By culturing until about 5 to 10 times, a yeast strain having acquired resistance to 4.5% lactic acid concentration was isolated.

  The lactic acid resistant strain obtained in Reference Example 6 was named TBSCR5 and TBSCR8.

Reference Example 7 Culture characteristics of 4.5% lactic acid resistant strain 1
In Reference Examples 5 and 6, the culture characteristics of mutant strains that acquired resistance to lactic acid at a concentration of 4.5% (hereinafter referred to as 4.5% lactic acid resistant strains) were examined. The experimental method is shown below.

-Yeast strain-The same wild strain (YPH499 strain) as in Reference Example 5 and 4.5% lactic acid resistant strains (HRSC207, HRSC208, HRSC210, HRSC214, TBSCR5 and TBSCR8) obtained in Reference Examples 5 and 6 were used. . -Medium-YPDA Broth (YPD Broth (Difco), 20 mg / L Adenine sulfate (Wako Pure Chemical Industries)) was used as a liquid medium.

-Experimental method-
(1) Wild strains and 4.5% lactic acid resistant strains were inoculated into test tubes containing 4 mL of liquid medium, and cultured overnight at 30 ° C. and 180 rpm. Cells were collected from the culture broth by centrifugation, washed 3 times with sterilized water, allowed to stand for 2 hours, diluted, and turbidity (OD660) was measured.
(2) Inoculate 0.05 mL each of a cell culture solution diluted to OD660 = 1.0 in 3 mL of a liquid medium containing 4.5% lactic acid, and after culturing at 30 ° C. and 180 rpm for 36 hours, Each OD660 value was measured.

-Experimental results-
As a result of the above experiment, as shown in FIGS. 10 and 11, the 4.5% lactic acid resistant strain showed remarkable growth compared to the wild strain. The TBSCR5 strain had low lactic acid resistance, but exhibited high aggregation properties.
Based on the above results, the 4.5% lactic acid resistant strain obtained by acclimatizing the mutant DNA polymerase δ-expressing strain to lactic acid exhibits remarkable resistance in the presence of 4.5% lactic acid and proliferates cells. It has been shown.

Reference Example 8 Culture characteristics of 4.5% lactic acid resistant strain 2
In Reference Examples 5 and 6, the culture characteristics of mutant strains that acquired resistance to a lactic acid concentration of 4.5% (hereinafter referred to as 4.5% lactic acid resistant strains) were examined.
(1) Wild strains and 4.5% lactic acid resistant strains were obtained from YPDA medium (10 g / L Bacto Yeast extract (Difco), 20 g / L Bacto trypton (Difco), 20 g / L Glucose, 40 mg / L Adenine. sulfate (manufactured by Sigma)) was inoculated into each test tube containing 10 mL each, and cultured overnight at 30 ° C. and 120 rpm.
(2) 0.1 mL of the culture solution was added to 10 mL of YPDA liquid medium containing 4% lactic acid, and cultured with shaking at 30 ° C. and 120 rpm.
(3) The OD600 of the culture was measured 14 hours, 22 hours, 38 hours, 45 hours, and 70 hours after the start of the culture. As a result, as shown in FIG. 12, it was shown that the 4.5% lactic acid resistant strain obtained in Reference Examples 5 and 6 proliferated significantly in the presence of 4% lactic acid compared to the wild strain. It was.

Reference Example 9 Cloning of Lactate Synthase (Lactate Dehydrogenase, L-ldh) Gene Phagemid was prepared from a skeletal muscle-derived cDNA library of bovine (Bos taurus) (manufactured by STRATAGENE) according to the attached protocol. did. The lactate synthase gene was cloned by PCR using KOD Plus DNA polymerase using the obtained phagemid as a template and the oligonucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 as a primer set. Each PCR amplified fragment was purified, and the end was phosphorylated with T4 Polynucleotide Kinase (Takara Shuzo Co., Ltd.), and then ligated to pUC118 vector (cleaved with restriction enzyme Hinc II and the cleavage surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver. 2 (Takara Shuzo) was used. Escherichia coli DH5α was transformed with the ligation plasmid product, and the plasmid DNA was recovered to clone the bovine-derived lactic acid synthase (L-ldh-b) gene (SEQ ID NO: 3). The cloned L-ldh-b gene was ligated under the control of a GAPDH (glyceraldehyde-3′-phosphate dehydrogenase) promoter to construct an L-ldh-b expression plasmid pLDH-b.

Lactic acid production test with 4.5% lactic acid resistant strains In 4.5% lactic acid resistant strains, which were produced in Reference Example 5 and confirmed to grow better than Reference YPH499 strain in Reference Examples 7 and 8 in the presence of lactic acid. Among them, HRSC207 strain, HRSC208 strain, TBSCR8 strain and parent strain YPH499 strain were transformed with pLDH-b. FIG. 13 shows a construction diagram of pLDH-b. The transformation was carried out by the lithium acetate method using a YASTMAKER Yeast Transformation System (manufactured by CLONTECH). For details, the attached protocol was followed.
The HRSC207, HRSC208, TBSCR8, and YPH499 strains used as hosts are strains lacking the ability to synthesize uracil, and transformants are selected on a uracil-free medium by the action of the URA3 gene on pLDH-b. be able to. Using the prepared HRSC207 / pLDH-b strain, HRSC208 / pLDH-b strain, TBSCR8 / pLDH-b strain, and YPH499 / pLDH-b strain, a lactic acid fermentation test was performed as follows.
(1) 5 mL of SC2-Ura medium (100 g / L glucose, 6.7 g / L Yeast Nitrogen Base w / o Amino acids (manufactured by Difco), 3.84 g / L Drop-out supplement with uracil (manufactured by Sigma) ), And cultured with shaking at a temperature of 30 ° C. for 24 hours.
(2) After inoculating 0.5 mL of the culture solution into a 200 mL Erlenmeyer flask containing 50 mL of SC2-Ura medium, the culture was shaken at a temperature of 30 ° C. for 65 hours, and 1 mL was sampled in time. To prevent glucose from being depleted during the culture, 10 mL of 20% glucose solution and 5 mL of 50% glucose solution were added after sampling after 40 hours and 50 hours, respectively, and cultured with shaking for 65 hours.
(3) The cells were removed from the sampled culture solution by centrifugation, and the glucose concentration and lactic acid concentration in the supernatant were measured. The glucose concentration was measured using Test Wako GLU-II (manufactured by Wako Pure Chemical Industries, Ltd.), and the lactic acid concentration was measured using HPLC under the following conditions.
Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
-Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
・ Detection method: Electric conductivity ・ Column oven temperature: 45 ℃

  As a result of the experiment, as shown in FIG. 14, compared with the YPH499 / pLDH-b strain, the HRSC207 / pLDH-b strain and the HRSC208 / pLDH-b strain were 5%, and the TBSCR8 / pLDH-b strain was 12%, respectively. It was shown that the productivity of lactic acid was improved, and it was clarified that a strain producing higher lactic acid could be constructed by creating a strain with improved lactic acid tolerance.

FIG. 1 is a photograph showing changes in the culture medium due to the addition of lactic acid (photo left: YPD liquid medium without lactic acid, photo right: YPD liquid medium with 3% concentration of lactic acid). FIG. 2 is a graph comparing the cell growth of the wild strain and the pol3 mutant by addition of lactic acid. FIG. 3 is a photograph of a 5% lactic acid resistant strain on the second day of culture observed with trypan blue staining. The cells shown in black in the photograph are dead cells stained blue with trypan blue, and the unstained white cells are viable cells. FIG. 4 is a graph showing changes in cell proliferation of the pol3 mutant in 5% lactic acid culture. FIG. 5 is a graph comparing cell growth of a wild type strain and a 5% lactate resistant strain in 5% lactic acid culture. FIG. 6 is a graph showing the effect of lactic acid addition on cell growth of wild strains and 5% lactic acid resistant strains. FIG. 7 is a graph showing changes in cell density when a 5% lactic acid resistant strain is subcultured in a 7% lactic acid medium. FIG. 8 is a photograph of a wild strain and a lactic acid resistant strain on day 1 cultured in 7% lactic acid medium stained with trypan blue. The cells shown in black in the photograph are dead cells stained blue with trypan blue, and the unstained white cells are viable cells. FIG. 9 is a construction diagram of the plasmid YEplac195 pol3-01 used in the present invention. FIG. 10 is a graph showing a growth comparison of 4.5% lactic acid resistant strains (HRSC207, HRSC208, HRSC210, HRSC214) in Reference Example 7 under a lactic acid concentration of 4.5%. FIG. 11 is a graph showing a growth comparison of 4.5% lactic acid resistant strains (TBSCR5, TBSCR8) in Reference Example 7 under a 4.5% lactic acid concentration. FIG. 12 is a graph showing growth comparison of the 4.5% lactic acid resistant strain in Reference Example 8 under a 4% lactic acid concentration. FIG. 13 is a construction diagram of plasmid pLDH-b used in the present invention. FIG. 14 is a graph comparing lactic acid production using a 4.5% lactic acid resistant strain and a wild strain in Example 1.

Sequence number 1: Primer Sequence number 2: Primer

Claims (13)

  After culturing a yeast mutant strain in which the proofreading function of the DNA polymerase is controlled under a lactic acid concentration of at least 1% (v / v) or more, the yeast obtained by isolating viable cells is transformed into a lactic acid synthase gene. A method for producing yeast having the ability to synthesize lactic acid.   The method for producing yeast according to claim 1, wherein the mutant strain of yeast is a strain into which a gene encoding mutant DNA polymerase δ has been introduced.   The method for producing yeast according to claim 1, wherein the mutant strain of yeast is a mutant strain of budding yeast obtained by modifying the amino acid sequence of Pol3. After culturing under the specified lactic acid concentration, the process of isolating viable cells is repeated,
The predetermined lactic acid concentration is 1% (v / v) or more and 7% (v / v) or less,
As the culture and isolation steps are repeated, the lactic acid concentration is increased.
The method for producing a yeast according to claim 1. A step of isolating viable cells after culturing a mutant strain of yeast in which a lactic acid synthase gene has been introduced and the proofreading function of DNA polymerase is controlled at a lactic acid concentration of at least 1% (v / v). Including,
A method for producing yeast having the ability to synthesize lactic acid.   The step of isolating viable cells after culturing under a lactic acid concentration of at least 1% (v / v) or more using a mutant strain of yeast in which the DNA polymerase proofreading function is controlled, v) A yeast having the ability to synthesize lactic acid, which is obtained by introducing a lactic acid synthase gene after obtaining a yeast having lactic acid resistance by repeating while increasing it within a range of not less than 7% (v / v). The yeast according to claim 6, wherein the yeast mutant is a strain into which a gene encoding mutant DNA polymerase δ has been introduced. A process of isolating viable cells after culturing under a lactic acid concentration of at least 1% (v / v) or more using a yeast mutant strain into which a lactic acid synthase gene has been introduced and the proofreading function of DNA polymerase is controlled. Yeast having the ability to synthesize lactic acid, obtained by repeating while increasing the lactic acid concentration within the range of 1% (v / v) to 7% (v / v). The yeast according to claim 8, wherein the mutant strain of yeast is a strain into which a gene encoding mutant DNA polymerase δ has been introduced.   The yeast according to claim 6 or 8, wherein the yeast belongs to the genus Saccharomyces.   The yeast according to claim 6 or 8, wherein the yeast is Saccharomyces cerevisiae.   Lactic acid synthase gene was introduced into yeast deposited under the accession number “NITE AP-319”, “NITE AP-320”, or “NITE AP-321” to the Patent Microorganism Depositary, National Institute of Technology and Evaluation Yeast having the ability to synthesize lactic acid.   A yeast obtained by the yeast production method according to any one of claims 1 to 5, or the yeast according to any one of claims 6 to 12 is cultured in the presence of a carbohydrate to produce lactic acid. And producing the lactic acid by collecting the lactic acid from the culture.

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