JP4806904B2 - Lactic acid production method - Google Patents

Description

  The present invention relates to a method for producing lactic acid.

  In recent years, attempts have been made to produce lactic acid such as L-lactic acid using genetically modified yeast. For example, a lactate dehydrogenase (LDH) gene is expressed under the control of a pyruvate decarboxylase (PDC) promoter or an alcohol dehydrogenase (ADH) promoter, which are strong promoters in yeasts such as Saccharomyces cerevisiae. (Patent Document 1, Non-Patent Document 1). However, it is important to establish lactic acid fermentation conditions peculiar to genetically modified lactic acid-producing yeast for more stable and efficient industrial lactic acid production by more effectively using the lactic acid-producing ability of yeast.

JP 2003-164295 A Enzyme and Microbial Technology 33 (2003) p. 38-46

  According to the present inventors, although the recombinant lactic acid-producing yeast is strongly promoted by using a strong promoter, the lactic acid yield in a certain period varies due to slight fluctuations in fermentation conditions. That is, it was found that there was a disadvantage that the lactic acid production rate fluctuated. If fermentation for reducing the fermentation rate to obtain a certain amount of yield is delayed, an uncertain factor in production control of the culture process is generated, which is not preferable. Moreover, even if fermentation rate is falling, if fermentation is complete | finished in fixed time, the sugar component which is a raw material will remain in a culture solution. Residual sugar content contributes to coloration of polylactic acid obtained by polymerizing the obtained lactic acid as well as reducing the lactic acid ratio in the lactic acid refining process.

  Therefore, one object of the present invention is to efficiently produce lactic acid in a method for producing lactic acid by culturing a genetically modified lactic acid-producing yeast. Another object of the present invention is to control fermentation characteristics such as fermentation period, fermentation rate, and yield in a method for producing lactic acid by culturing genetically modified lactic acid-producing yeast.

  The inventors of the present invention have examined the causes of changes in the lactic acid production rate and lactic acid yield of the genetically modified lactic acid-producing yeast and found that it greatly depends on changes in the oxygen transfer capacity coefficient (kLa). I found it. kLa is one of the fermentation conditions in fermentation production, but it is particularly sensitive to oxygen transfer capacity coefficient in genetically modified lactic acid producing yeast, and lactic acid production characteristics, i.e., due to slight differences in the low oxygen transfer capacity coefficient region, It was found that lactic acid production rate and lactic acid yield were different. Based on these findings, the present inventors have found that the lactic acid yield within the culture period and within a certain culture period can be easily controlled, and have completed the present invention. That is, according to the present invention, the following means are provided.

According to one aspect of the present invention, there is provided a method for producing lactic acid, the method comprising culturing a genetically modified lactic acid-producing yeast provided with a gene capable of expressing a gene encoding a protein having lactate dehydrogenase activity. In the culturing step, there is provided a lactic acid production method including a step of culturing in a range of oxygen transfer capacity coefficient (hr ⁻¹ ) of 6 or more and 36 or less. In this embodiment, it is also one preferred embodiment that the oxygen transfer capacity coefficient (hr ^-1) is cultured in a range of 16 or less, it is also preferable that the volumetric oxygen transfer coefficient (hr ^-1) is cultured in a range of 16 than It is one mode. Furthermore, in any one of these lactic acid production methods, it is preferable to change the oxygen transfer capacity coefficient (hr ⁻¹ ) in the culturing step.

Further, according to another embodiment of the present invention, there is provided a lactic acid production method comprising culturing a lactic acid-producing yeast that is capable of expressing a gene encoding a protein having lactate dehydrogenase activity. There is provided a method for producing lactic acid, wherein the oxygen transfer capacity coefficient (hr ⁻¹ ) is changed in the culturing step. In this embodiment, the culturing step includes (a) culturing the lactic acid-producing yeast in an oxygen transfer capacity coefficient (hr ⁻¹ ) in the range of 16 or less, and (b) lactic acid-producing yeast in an oxygen transfer capacity coefficient. It is preferable to include the step of culturing in a range where (hr ⁻¹ ) exceeds 16, and further, the step (b) is preferably performed after the step (a).

  Furthermore, according to another aspect of the present invention, there is provided a method for lactic acid production, which comprises culturing a lactic acid-producing yeast provided with a gene encoding a protein having lactate dehydrogenase activity under aeration and agitation. A method for producing lactic acid is provided.

  According to another aspect of the present invention, there is provided a method for producing lactic acid, wherein a lactic acid-producing yeast comprising a gene encoding a protein having lactate dehydrogenase activity is capable of being expressed. Below, a method for producing lactic acid is provided, which comprises a step of culturing in a range where the dissolved oxygen content of the culture solution is 1 mg / L or less. In this embodiment, it is a more preferable embodiment that the dissolved oxygen content of the culture solution in the culturing step is 0.5 mg / L or less.

  In any of the above lactic acid production methods, it is also a preferred aspect that the gene encoding the protein having lactate dehydrogenase activity is provided so as to be expressed under the control of a pyruvate decarboxylase gene promoter. Furthermore, it is a preferable aspect that the gene encoding the protein having lactate dehydrogenase activity is prepared so as to be expressed under the control of a pyruvate decarboxylase gene promoter on the yeast chromosome. Further, in the lactic acid-producing yeast, it is preferable that pyruvate decarboxylase and / or alcohol dehydrogenase activity is reduced, and the pyruvate decarboxylase gene on the yeast chromosome may be disrupted. This is a preferred embodiment. Furthermore, it is also a preferable aspect that the lactic acid-producing yeast is provided with two or more genes encoding a protein having the lactic acid dehydrogenase activity.

The lactic acid production method of the present invention includes a culture step of culturing a lactic acid-producing yeast that is capable of expressing a gene having lactate dehydrogenase activity. In the 1st form of this invention, in the said culture | cultivation process, it culture | cultivates in the range whose oxygen transfer capacity coefficient (hr < ^-1 >) is 6 or more and 36 or less. Preferred features for the lactic acid production rate, lactic acid yield, and lactic acid ratio (lactic acid ratio in the product) of the lactic acid-producing yeast when the oxygen transfer capacity coefficient (hereinafter simply referred to as kLa) (hr ⁻¹ ) is in the range of 6 to 36. Have For this reason, efficient lactic acid production becomes possible by cultivating lactic acid-producing yeast with kLa within this range. If kLa (hr ⁻¹ ) is less than 6, the lactic acid production rate tends to be slow, and if kLa (hr ⁻¹ ) exceeds 36, the lactic acid yield tends to decrease.

Moreover, in the 2nd form of this invention, kLa (hr < ^-1 >) is changed in the said culture | cultivation process, It is characterized by the above-mentioned. The lactic acid-producing yeast has a strong tendency to depend on kLa (hr ⁻¹ ) for its lactic acid production rate, and thus it is possible to realize lactic acid production in the intended culture period by adjusting kLa (hr ⁻¹ ). it can. Moreover, a high lactic acid yield and a lactic acid ratio can be obtained by suppressing the residual sugar content within a certain culture period.

  Furthermore, in the 3rd form of this invention, the said culture | cultivation process is equipped with the process of culturing the said lactic acid-producing yeast under aeration stirring. An unexpectedly good lactic acid yield and lactic acid ratio can be obtained by culturing under aeration and agitation which has not been used in this type of yeast or lactic acid bacteria, and the lactic acid production rate can be easily controlled.

  Furthermore, in the fourth aspect of the present invention, the culturing step comprises culturing the lactic acid-producing yeast in a range where the dissolved oxygen concentration of the culture solution is 1 mg / L or less under aeration and / or stirring of the culture solution. It is characterized by. By culturing under such oxygen conditions, an unexpectedly good lactic acid yield and lactic acid ratio can be obtained, and the lactic acid production rate can be easily controlled.

  Hereinafter, embodiments of the present invention will be described in detail.

(Gene lactic acid-producing yeast)
(yeast)
The lactic acid-producing transformed yeast of the present invention is a transformed yeast having a gene encoding a protein having an exogenous lactate dehydrogenase activity. Yeast for obtaining this lactic acid-producing yeast is a yeast that carries out alcoholic fermentation, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, Saccharomyces genus yeast, Pichia pastoris (Pichia pastoris), etc. Yeast can be mentioned. More preferred are yeasts including Saccharomyces genus such as Saccharomyces cerevisiae. Examples of preferred yeasts include Saccharomyces cerevisiae IFO2260 strain and YPH strain.

  In this lactic acid-producing yeast and lactic acid production method, lactic acid includes L-lactic acid, D-lactic acid, and DL-lactic acid, and any of these may be used.

(Reduction of pyruvate decarboxylase activity or alcohol dehydrogenase activity)
In the present lactic acid-producing yeast, it is preferable that pyruvate decarboxylase (PDC) activity and / or alcohol dehydrogenase (ADH) activity is reduced. These enzymes are enzymes of the yeast alcohol fermentation pathway, and are enzymes that are inherently present on the chromosome of yeast that performs alcohol fermentation. Pyruvate decarboxylase is an enzyme having a so-called autoregulation mechanism, such as pyruvate decarboxylase 1-6. Any one or two or more of these may be destroyed, but the activity of pyruvate decarboxylase 1 (PDC1), which is most highly active or expressed in large quantities, is preferably reduced. Is preferred. Moreover, although there are various subspecies (ADH1 to ADH7) in alcohol dehydrogenase, a large amount of alcohol dehydrogenase 1 (ADH1) is expressed. Therefore, it is preferable that the activity of alcohol dehydrogenase 1 is reduced.

  The fact that pyruvate decarboxylase activity and / or alcohol dehydrogenase activity is reduced means that the enzyme having a lower activity than the wild type is produced by artificial manipulation or screening, or the production of the enzyme The amount is preferably less than that of the wild type. As such an artificial operation for reducing the enzyme activity, it is preferable to destroy (knock out) the enzyme gene. Techniques for disrupting specific genes on chromosomes are well known to those skilled in the art.

(Lactate dehydrogenase)
This lactic acid-producing yeast holds a gene (LDH gene) encoding a protein having lactate dehydrogenase activity. The LDH gene is exogenous in yeast. As a lactate dehydrogenase (LDH), various homologues exist depending on the kind of organism or in vivo. The lactate dehydrogenase used in the present invention includes not only naturally derived LDH but also LDH synthesized chemically or genetically. As LDH, Preferably, Lactobacillus helveticus, Lactobacillus casei, Kluyveromyces thermotolerance, Torlas pora delbrukki, Schizosaccharomyces pombi, Rhizopus oryzae, It is derived from prokaryotes such as megaterium or eukaryotic microorganisms such as mold, more preferably derived from higher eukaryotes such as plants, animals and insects, and more preferably higher eukaryotes including mammals including cattle. It is derived from living things. For example, LDH derived from bovine (L-LDH). For example, a protein having the amino acid sequence shown in SEQ ID NO: 2 can be mentioned as LDH derived from bovine. Examples of the DNA encoding LDH include DNA consisting of the base sequence set forth in SEQ ID NO: 1. Furthermore, LDH in the present invention includes homologues of these LDHs. An LDH homolog is a protein having an LDH activity, which is an amino acid sequence having one or several amino acid substitutions, deletions, insertions and / or additions in the amino acid sequence of naturally occurring LDH, and a naturally occurring LDH It contains a protein having LDH and amino sequence homology of at least 70%, preferably 80% or more and having LDH activity. Therefore, DNA encoding any of these LDHs can be used as the LDH gene. The lactic acid-producing yeast holds at least one, preferably two or more LDH genes. More preferably, three or more are held. More preferably, four or more are held.

(PDC promoter)
The LDH gene is preferably provided so that it can be expressed under the control of a promoter having a strong promoter activity. For example, it is preferable that it is provided so that it can be expressed under the control of a pyruvate decarboxylase gene promoter (PDC promoter). Among these, it is preferable to use the PDC1 promoter which is a sufficiently strong promoter. More preferably, it is a PDC1 promoter of the genus Saccharomyces (preferably Seleviciae).

  For example, as the PDC1 promoter, in addition to DNA consisting of the base sequence set forth in SEQ ID NO: 3, DNA that hybridizes with the DNA under stringent conditions as long as it has an equivalent function, one or more in the base sequence DNA consisting of a sequence in which the bases are substituted, deleted, added and / or inserted, or homology with the DNA is 70% or more, more preferably 80% or more, still more preferably 90% or more, most preferably Can use DNA which is 95% or more. That is, the PDC1 promoter on the yeast chromosome may be replaced by homologous recombination or the like with DNA having these promoter activities that are not identical but have equivalent functions.

  In the present specification, the stringent condition means a hybridization condition in which the hybridization temperature is 37 ° C. in the presence of 50% formamide or a hybridization condition having the same stringency as this. According to conditions with higher stringency, DNA with higher homology can be isolated. Such hybridization conditions include, for example, hybridization conditions of about 42 ° C. in the presence of 50% formamide, and conditions of about 65 ° C. in the presence of 50% formamide. .

  Further, in this specification, with respect to the homology of nucleic acids such as DNA, the homology of the DNA base sequence can be determined by a gene analysis program BLAST or the like. The DNA base sequence homology is determined by the gene analysis program BLAST (http://blast.genome.ad.jp), FASTA (http://fasta.genome.ad.jp/SIT/FASTA.html), or the like. Can be determined.

(Other promoters)
The LDH gene can also be prepared so as to be expressed under the control of various other promoters including the promoter of the ADH gene (ADH promoter). As already mentioned, ADH is an enzyme immediately after PDC in the alcohol fermentation pathway. As the ADH promoter, it is preferable to use the ADH1 promoter which is a sufficiently strong promoter.

  In addition, the LDH gene includes yeast Saccharomyces cerevisiae hyperosmotic response 7 gene (HOR7 gene), glyceraldehyde 3-phosphate dehydrogenase 2 gene (TDH2 gene), and hexose transport protein 7 gene (HXT7 gene). , Heat shock protein 30 gene (HSP30 gene), thioredoxin peroxidase 1 gene (AHP1 gene), membrane protein 1 related gene (MRH1 gene) gene, phosphoglycerate kinase gene (PGK gene), glyceraldehyde 3-phosphate dehydrogenation It can also be introduced so that it can be expressed under the control of the promoters of enzyme 3 gene (TDH3 gene), galactose 1-phosphate kinase 1 gene (GAL1 gene) and transcription enhancer factor 1 gene (TEF1 gene).

  As long as these other promoters have the same function as each promoter on the yeast chromosome, DNA that hybridizes with the DNA of the promoter under stringent conditions, and one or more bases in the base sequence DNA comprising substitution, deletion, addition, and / or inserted sequence, or homology with ADH1 promoter is 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more The DNA can be used.

  The LDH gene may be retained in a plasmid or YAC maintained outside the yeast chromosome, but is preferably retained by being incorporated into the yeast chromosome.

  The LDH gene is preferably provided so that it can be expressed in the yeast chromosome under the control of the above various promoters. Therefore, expression cassettes containing the various promoters and LDH genes can be constructed and introduced into yeast chromosomes. In addition, the LDH gene can be incorporated so as to be able to be expressed under the control of the promoter while destroying the endogenous gene under the control of these various promoters on the yeast chromosome. In addition, the LDH gene operably linked under the control of these various promoters can be incorporated into the yeast chromosome so as to destroy the PDC gene and / or the ADH gene.

  Furthermore, in order to be prepared so that the LDH gene can be expressed under the control of the PDC promoter or the ADH promoter, the promoter can be controlled by these promoters at the original position of the yeast chromosome and the PDC gene is destroyed. Thus, it is preferably integrated into the yeast chromosome. In this way, alcohol fermentation can be suppressed by reducing PDC activity and ADH activity, and strong PDC promoter and ADH promoter activities that originally acted on PDC gene expression can be switched to LDH gene expression. The genes that are disrupted and whose promoters are utilized for the expression of the LDH gene are preferably the PDC1 gene and the ADH1 gene. The PDC1 gene controlled by the strongest PDC1 promoter is disrupted, and the LDH gene is expressed instead, thereby effectively reducing the PDC activity and expressing the LDH activity.

  The PDC1 gene is a gene having an autoregulation mechanism as already disclosed by the present inventors (Japanese Patent Laid-Open No. 2003-164295). The autoregulation mechanism is that multiple genes with the same function exist in the same organism, and at least one of them is normally expressed, but the rest is suppressed. It means a mechanism in which the remaining genes are expressed and continue their functions only when they stop functioning. Since such a mechanism is expressed, for example, even if the yeast PDC1 gene is disrupted, the physiological function of the yeast is maintained. By destroying the gene in which such an autoregulation mechanism exists, it is possible to maintain the survival and proliferation function of the organism itself, and as a result, the target product while maintaining the growth of the transformant holding the foreign DNA. Can be produced effectively.

  As is clear, the DNAs of the PDC promoter, the ADH promoter, and other promoters may be genomic DNA or chemically synthesized DNA.

  As described above, in the present lactic acid-producing yeast, it is possible to take various combinations of the above-described aspect of reducing the PDC activity and ADH activity and various types of LDH gene expression. A preferred lactic acid-producing yeast has a PDC (preferably PDC1) gene on the chromosome disrupted and an LDH gene that can be expressed under the control of the promoter of the disrupted gene. At the same time, the ADH (preferably ADH1) gene on the chromosome may be disrupted. In this embodiment, the LDH gene can be expressed under the control of the promoter of the disrupted ADH (preferably ADH1) gene. Such lactic acid-producing yeast preferably has 2 or more LDH genes on the chromosome, more preferably 4 or more, still more preferably 6 or more, and most preferably 8 or more.

  In order to obtain the lactic acid-producing yeast, it is preferable to transform the host yeast by disrupting the PDC gene and the ADH gene and introducing the LDH gene so that it can be expressed. In order to perform such genetic modification on yeast, a DNA construct for recombination is used. The DNA construct for recombination is not particularly limited, and may be a linear DNA fragment, a plasmid (DNA), a virus (DNA), a retrotransposon (DNA), an artificial chromosome (YAC), a foreign gene introduced form (extrachromosomal). Alternatively, it can be selected according to the intrachromosome etc. and can take the form of a vector.

  A DNA construct for disrupting the PDC gene or the ADH gene can be provided with a sequence for homologous recombination in order to introduce the gene into these gene sites and destroy the gene. The DNA sequence for homologous recombination is a DNA sequence that is homologous to the DNA sequence at or near the target site of the PDC gene and ADH gene to be destroyed. The DNA sequence for homologous recombination has one sequence that is homologous to at least one location in the vicinity of one target gene, or preferably, preferably a sequence that is homologous to at least two locations in the vicinity of the target gene. I have. For example, two DNA sequences for homologous recombination are made DNA sequences homologous to the upstream and downstream DNAs of the target gene on the chromosome, and the gene is destroyed between these DNA sequences for homologous recombination. The gene at the target site can be destroyed by introducing a DNA construct comprising DNA for the purpose into the yeast chromosome by homologous recombination.

  The selection of DNA for homologous recombination for realizing such integration on a chromosome is well known to those skilled in the art, and those skilled in the art can select appropriate DNA for homologous recombination as needed and perform homologous recombination. Recombinant DNA constructs can be constructed. For example, a DNA sequence homologous to at least part of the PDC promoter and at least part of the PDC gene or terminator can be used as the DNA sequence for homologous recombination. Similarly, a DNA sequence homologous to at least part of the ADH promoter and at least part of the ADH gene or terminator can be used as the DNA sequence for homologous recombination.

  The DNA construct that destroys the PDC gene can have the LDH gene in a form that can replace the PDC gene together with DNA for homologous recombination for targeting the PDC gene. According to such a DNA construct, the LDC gene can be introduced so as to be expressed under the control of the PDC promoter together with the destruction of the PDC gene.

  In addition to destroying the PDC gene, the LDH gene can be introduced under the control of the promoter of the disrupted PDC gene, but an equivalent promoter can be introduced instead of the PDC promoter on the chromosome by homologous recombination. it can. For example, the above-mentioned homologue of the PDC1 promoter.

  In addition, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selectable marker, and a ribosome binding sequence (SD sequence) are linked to the DNA construct to be used, if necessary, in addition to terminators such as CYC1 terminator and TDH3 terminator. be able to. The selectable marker is not particularly limited, and various known selectable marker genes such as drug resistance genes and auxotrophic genes can be used. For example, ampicillin resistance gene, kanamycin resistance G418 gene, hygromycin resistance gene, bleomycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene, chloramphenicol resistance gene, cyclohexamide resistance gene and the like can be used.

(Production method of lactic acid)
By culturing the present lactic acid-producing yeast in the presence of an appropriate carbon source, lactic acid which is an expression product of the LDH gene can be produced in the culture. According to this lactic acid production method, lactic acid can be obtained by carrying out the step of separating lactic acid from the culture system. In the present invention, the culture includes cultured cells or microbial cells, or crushed cells or cells in addition to the culture supernatant.

  In culturing the lactic acid-producing yeast of the present invention, a culture method and culture conditions can be selected according to the type of yeast. For example, a liquid culture method using a jar fermenter can be mentioned, and culture formats such as batch culture and semi-batch culture can be adopted. Moreover, as a medium for cultivating yeast, a natural medium, which contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by microorganisms and can efficiently culture transformants, Any of the synthetic media can be used. As a carbon source, carbohydrates such as glucose, fructose, sucrose, starch and cellulose, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used. As the nitrogen source, ammonium salt of inorganic acid or organic acid such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, etc. may be used. it can. Examples of inorganic substances that can be used include potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

  The culture temperature can be selected within the range in which the lactic acid-producing yeast to be used can grow, and can be, for example, about 20 ° C. to about 40 ° C. Moreover, it is preferable to maintain the pH of the medium at 2.0 to 6.0, and if necessary, neutralize the product lactic acid or the like, or remove the lactic acid continuously. It can also be done.

The culture period is preferably about 6 to 72 hours, more preferably about 48 hours, although it varies greatly depending on other culture conditions, in particular, kLa (hr ⁻¹ ) as described later.

  In this lactic acid production method, the lactic acid-producing yeast is preferably cultured under aeration and aerobic conditions rather than in a slightly aerobic state. In general, in lactic acid fermentation using lactic acid bacteria and ethanol fermentation using yeast, fermentation is performed in a microaerobic state without aeration. On the other hand, the present method is characterized by using oxygen conditions in the culture solution obtained by aeration and agitation which are not conventionally used in the fermentation production by yeast. That is, it is characterized in that the fermentation is performed under an aerobic oxygen condition rather than a microaerobic state. For lactic acid-producing yeast, efficient lactic acid fermentation is realized for the first time under such oxygen conditions. In particular, in a recombinant lactic acid-producing yeast, particularly a lactic acid-producing yeast in which alcohol fermentation is suppressed, such as a decrease in PDC activity, or a lactic acid-producing yeast provided with an LDH gene capable of being expressed under the control of a PDC gene promoter. Although the reason is not clear, practical lactic acid production characteristics can be obtained for the first time under such oxygen conditions. In this production method, as long as an aerobic state is obtained rather than a slightly aerobic state, in addition to performing both aeration and agitation, only one of aeration and agitation may be performed.

The oxygen condition to be obtained in the culturing step of the present method is not limited as long as the lactic acid-producing yeast to be used is capable of lactic acid fermentation or can promote lactic acid fermentation. For example, such an oxygen condition can be expressed as, for example, kLa (hr ⁻¹ ). kLa (hr ⁻¹ ) indicates the ability to transfer dissolved oxygen from the gas phase to the liquid phase per unit time during aeration and agitation culture, and can be expressed by the following formula (1). . In addition, kLa (hr ⁻¹ ) (hereinafter simply abbreviated as kLa) is a ratio of oxygen in the culture medium represented by oxygen supplied from the gas phase and oxygen consumed by microorganisms during aeration and agitation culture. It is used in the balance equation (2) (Biotechnical Experiment Manual, edited by Japanese Society for Biotechnology, Baifukan, p. 310 (1992)).

dC / dt = kLa × (C * −C) (1)
dC / dt = kLa × (C * −C) −Q02 × X (2)
Here, C: dissolved oxygen concentration DO (ppm) in the culture solution, C *: dissolved oxygen concentration DO (ppm) in equilibrium with the gas phase when oxygen is not consumed by microorganisms, X: bacterial cell concentration (g / L), Q02: specific respiration rate (mgO2 / (g · cell · h) kLa: oxygen transfer capacity coefficient (hr ⁻¹ ).

KLa in aeration and agitation culture equipment can be measured by sulfite oxidation method (batch method / continuous method), gas displacement method (Gassing out method (static method, dynamic method)), exhaust gas analysis method, etc. p311 etc.). Below, an example of the measurement of kLa by the gas substitution method (dynamic method) is shown. Place the water or medium to be used in the aeration and agitation culture apparatus, and replace the oxygen in these liquids with nitrogen gas or deoxygenate such as by adding sodium sulfite to a nearly saturated concentration. Reduce oxygen concentration. Next, a dissolved oxygen concentration electrode is inserted into the liquid, and the aeration rate, agitation rate, and temperature are set, and the rising process of the dissolved oxygen under a predetermined agitation agitation is measured. Here, since the following formula (3) is derived from the formula (1), kLa can be obtained by plotting the logarithm of C * -C against the aerated time.
In (C * -C) = − kLa × t (3)

  In the present invention, aeration and agitation conditions that combine several aeration conditions and agitation conditions are set, the relationship between these various aeration and agitation conditions and kLa is obtained in advance, and the aeration and agitation culture apparatus used is kLa. It is preferable to set in advance the aeration and stirring conditions such that is 6 or more and 36 or less. The kLa for various aeration and agitation conditions can be obtained by operating the aeration and agitation culture apparatus set for each aeration and agitation conditions according to the above-described method and measuring the rising process of the dissolved oxygen concentration and the equilibrium dissolved oxygen concentration. .

  When kLa is in the range of 6 or more and 36 or less, preferable culture characteristics can be obtained with respect to the lactic acid production rate, lactic acid yield and residual sugar content of the lactic acid-producing yeast, so that stable and efficient lactic acid production can be realized. Further, by carrying out the culturing step while changing within the range of kLa, the culturing can be completed while securing a preferable level of lactic acid yield and residual sugar content in a desired culturing period. In addition, lactic acid-producing yeast tends to have a low lactic acid production rate when kLa is low within this range, and tends to increase the lactic acid production rate when kLa is high. Thus, for example, when setting a relatively long culture period, a relatively low kLa can be set in this kLa range, and when setting a relatively short culture period, a relatively high kLa can be set. Can be set.

  For example, the final lactic acid yield can be increased by culturing at a kLa of 16 or less. It has been confirmed by the present inventors that when the kLa is 16 or less, a good final lactic acid yield can be obtained regardless of the LDH gene copy number. Moreover, such a tendency is more remarkable as kLa is smaller. Furthermore, the lactic acid ratio tends to increase as kLa decreases. On the other hand, lactic acid production rate can be improved by culturing with kLa exceeding 16. By increasing the lactic acid production rate, it is possible to control the culture process time. In order to improve the lactic acid production rate more remarkably, kLa is preferably 18 or more, kLa is more preferably 20 or more, and kLa is more preferably 22 or more.

  In the culturing step, kLa can be constant, but in the culturing step, kLa can also be changed. That is, a culture period with different kLa can be set in advance, or a different culture period of different kLa can be newly set according to the monitoring result of the lactic acid concentration and the sugar concentration in the culture process started by setting kLa constant. You can also make additional settings. Furthermore, different kLa culture processes may be set in advance, and also in such a culture process, the time of each culture process may be changed according to the monitoring result of the culture. By adjusting kLa in such a culture process, a preferable lactic acid yield and lactic acid ratio can be obtained within a convenient culture process time.

  For example, when the residual sugar content and / or lactic acid concentration at a predetermined time point is lower than the predetermined value in monitoring in a constant culture process of kLa, kLa is higher, preferably more than 16, more preferably 18 or more, more preferably 20 or more. More preferably, by changing to 22 or more, a preferable residual sugar content and / or lactic acid yield can be secured without greatly extending the culture process time.

  The culturing step may include a first culturing period in which kLa is 16 or less and a second culturing period in which kLa is greater than 16. When kLa is 16 or less, the lactic acid production rate of lactic acid-producing yeast is slower than that when kLa is in the range of more than 16, but the rate tends to be sustained, and the final lactic acid yield and lactic acid ratio are improved. Can be made. kLa is more preferably 14 or less, and still more preferably 11 or less. This is because when kLa is set to 14 or less and further 11 or less, such a tendency becomes remarkable. In addition, it is preferable that kLa in the first culture period is 6 or more because the lactic acid production rate does not become too slow. On the other hand, when kLa is in the range of more than 16, the final lactic acid yield tends to decrease, but the lactic acid production rate tends to be fast. Therefore, setting the second culture period shortens the culture period. can do. kLa is more preferably 18 or more, still more preferably 20 or more, and still more preferably 22 or more. In addition, if the upper limit in a 2nd culture period is 36 or less, a lactic acid yield will not be reduced significantly.

  Thus, by providing each with culture periods in different kLa regions, a desired culture period, a high lactic acid yield and lactic acid ratio, and a low residual sugar content can be obtained as appropriate. When combining the culture process in a different kLa area | region, it is preferable to implement the culture process of a lower kLa area | region, and to implement the culture | cultivation process of a higher area | region. By doing so, it is possible to easily balance both lactic acid yield and lactic acid production rate. For example, the second culture period is preferably performed after the first culture period described above. By performing different kLa culture periods in this order, favorable characteristics can be obtained with respect to the culture period, lactic acid yield, lactic acid ratio, residual sugar content, and the like, and it becomes easy to cope with changes in the culture process. In addition, when providing the culture process of a different kLa area | region, in addition to each culture process by 2 types of kLa, each culture process of 3 or more types of kLa can also be provided.

  The oxygen condition to be obtained in the main culture process can also be expressed as the dissolved oxygen amount of the culture solution in the culture process of lactic acid-producing yeast. In the lactic acid-producing yeast culture step, the amount of dissolved oxygen is preferably 1 mg / L or less. This is because the yield of lactic acid can be secured above a certain level when it is 1 mg / L or less. More preferably, it is 0.5 mg / L or less. It is because a high lactic acid yield is obtained as it is 0.5 mg / L or less. In addition, it is preferable that it is aerobic state rather than a microaerobic state also in such dissolved oxygen concentration. Such oxygen conditions may be obtained by aeration or agitation of the culture medium, and can also be obtained by combining both of them. It is preferable that the dissolved oxygen amount in the culture solution is not more than the above concentration throughout the entire culture process. Moreover, it is preferable to use the numerical value obtained from the measured value in the measurement position and measurement location which can represent the oxygen condition of a culture tank for the measurement of dissolved oxygen amount.

  In order to separate lactic acid or a salt thereof from the culture after completion of the culture, an ordinary lactic acid purification means can be used. For example, when produced in yeast, the cells can be disrupted by ultrasonic disruption treatment, grinding treatment, pressure disruption, etc., and the gene product can be separated from the cells by conventional methods. In this case, protease is added as necessary. Further, when lactic acid is produced outside the cells, the solid content is removed from the culture solution by filtration, centrifugation, or the like.

  For these crudely extracted fractions, lactic acid can be purified using various conventionally known purification separation methods. 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.

Although the specific example of this invention is described below, it is not the meaning which limits this invention to the following specific example, and can be implemented with a various aspect in the range which does not deviate from the summary of this invention. In the following examples, kLa means kLa (hr ⁻¹ ) unless otherwise specified.

(Production of lactic acid-producing transformed yeast)
Saccharomyces cerevisiae into which the LDH gene (derived from bovine L-LDH) was introduced was prepared using the pBTrp-PDC1-LDHKCB vector (described in JP-A No. 2003-259878) already constructed by the present inventors. This vector is a chromosomal transfer vector that targets the PDC1 gene on chromosome 12 of yeast, and has a DNA sequence encoding the PDC1 promoter on the chromosome and a homologous recombination site downstream of the PDC1 gene. (Fig. 1). If this vector is introduced into the PDC1 gene on the chromosome, the LDH gene will be expressed under the control of the PDC1 promoter. In addition, the gene introduction method to yeast is the method of Ito et al. (Ito, H., Y. Fukuda, K. Murata and A. Kimura, Transformation of intact yeast cells treated with alkali cations J. Bacteriol. Vol.153, p163 -168). That is, a strain lacking the ability to synthesize tryptophan of yeast IFO2260 strain (Saccharomyces cerevisiae strain registered in the Fermentation Research Institute), which is the host, is cultured in 10 ml YPD medium at 30 ° C. until the logarithmic growth phase. After washing with bacteria and TE buffer, and then adding 0.5 ml TE buffer and 0.5 ml 0.2 M lithium acetate and shaking culture at 30 ° C. for 1 hour, it is a chromosome transfer type vector The pBTrp-PDCl-LDHKCB vector was treated with restriction enzymes ApaI and SacI (both from Takara Shuzo) and added.

  After shaking this yeast suspension at 30 ° C. for 30 minutes, 150 μl of 70% polyethylene glycol 4000 (Wako Pure Chemical Industries, Ltd.) was added and stirred well. Furthermore, after shaking culture at 30 ° C. for 1 hour, heat shock was applied at 42 ° C. for 5 minutes to wash the cells, and the suspension in 200 μl of water was smeared on a tryptophan selective medium.

  The obtained colonies were streaked in a new tryptophan selection medium, and the selected strains that were confirmed to be stable were confirmed by PCR analysis for the presence or absence of gene transfer. Those having stable tryptophan synthesis ability and PCR amplification confirmed under these primers were judged as transformants (2 copies) into which the LDHKCB gene was appropriately introduced. As shown in FIG. 2, the 2-copy body is obtained by replacing the PDC1 gene of yeast chromosome 12 with the DNA of the chromosomal transfer vector.

  In this example, the chromosomal introduction vectors to the predetermined positions of the yeast chromosomes (Chromosome 7 and Chromosome 12) shown in FIG. 2 were further constructed according to the pBTrp-PDCl-LDHKCB vector, respectively. 2 copies were introduced and transformed to produce 6 copies, 8 copies and 10 copies.

(Fermentation test 1)
In various aeration and agitation conditions in an aeration and agitation type culture apparatus (TFL series, Chiyoda Kako Co., Ltd., jar capacity: 1 L), a fermentation test is performed by a batch method, and kLa for each aeration and agitation conditions is a dynamic method using nitrogen gas. Determined by The fermentation test was performed under the aeration stirring conditions shown in Table 1.

  The T167-10B strain (LDH8 copy) produced in Example 1 was cultured in a YPD medium 100 ml baffled Erlenmeyer flask with shaking at 80 rpm for 24 hours, and the cells were collected by centrifugation to obtain precultured cells. The pre-cultured cells have an initial cell concentration of OD600 = 1 to 3 in 500 mL of cane juice medium (121 ° C., heat sterilized at 30 minutes) diluted with distilled water to a sugar concentration of 15 wt%. The inoculation was carried out as described above, and the temperature was set to 32 ° C. and the cells were cultured under various aeration and stirring conditions shown in Table 1. During the cultivation, the pH was adjusted between 4.8 and 5.4 at any time using ammonia water as a neutralizing agent. The culture solution was collected at 24 hours, 36 hours and 48 hours after the start of the culture, and the lactic acid concentration was measured. The lactic acid concentration was measured using an enzyme sensor (Oji Scientific Instruments, Biosensor BF-4).

  After the fermentation test, kLa in each fermentation condition was measured by a dynamic method using a dissolved oxygen electrode. First, 500 mL of water was added under the same aeration and stirring conditions and temperature conditions used in the fermentation test, a dissolved oxygen electrode (T type made by METTLER TOLEDO) was inserted, and nitrogen gas was sufficiently blown into the water to minimize the electrode value. When the value was reached, zero calibration of the dissolved oxygen electrode was performed, the aeration gas was changed from nitrogen gas to air, the operation of the culture apparatus was started, and the change over time in the dissolved oxygen concentration was measured to obtain kLa. The obtained kLa is also shown in Table 1. FIG. 3 shows the results plotted with kLa as the horizontal axis and the lactic acid concentration as the vertical axis.

  As shown in FIG. 3, the lactic acid production rate increases when kLa is high, but the final lactic acid yield tends to decrease when kLa exceeds 36.0. In addition, when kLa was low, the initial lactic acid production rate was slow, and when kLa was smaller than 7.2, the tendency was strong. Furthermore, when kLa is 6.0 or more and 36.0 or less, a stable lactic acid yield is obtained and there is almost no residual sugar in the tested culture period, but the highest productivity is obtained when kLa is about 18.0. It was.

  From the above, it was found that the lactic acid-producing yeast exhibits preferable lactic acid production characteristics within a certain kLa range in a low kLa region of 40 or less. On the other hand, it was found that even within this kLa range, the lactic acid production rate greatly changed and greatly affected the lactic acid yield and residual sugar content within a certain culture period.

(Fermentation test 2)
Next, fermentation tests were performed under various aeration and stirring conditions that resulted in the same kLa, and the kLa dependency of lactic acid-producing yeast was confirmed. Table 2 shows the relationship between kLa and aeration stirring conditions. The fermentation test was performed in the same manner as the fermentation test 1 except that the various aeration stirring conditions shown in Table 2 were used. The results are shown in FIG.

  As shown in FIG. 4, the results of the fermentation test under various aeration stirring conditions with kLa of 18 showed almost the same lactic acid production characteristics regardless of the aeration stirring conditions. These results show that the fermentation characteristics of lactic acid-producing yeast do not depend on aeration or agitation, and oxygen is transferred from the gas phase to the liquid phase to generate dissolved oxygen in the unit time obtained according to the conditions set for these. He supported that it was dependent on ability.

(Fermentation test 3)
In this fermentation test, PDC1p-LDH2 copy bodies, 6 copy bodies and 10 copy bodies prepared in Example 1 were used as lactic acid-producing yeast, and various kLa (6, 11, 16) were used for these various lactic acid-producing yeasts. , 22 and 29) were set to conduct lactic acid fermentation, and the relationship between LDH copy number or kLa and fermentation characteristics was confirmed. The fermentation test uses these lactic acid-producing yeasts, except that fermentation was performed at a predetermined kLa up to 72 hours using a Kane juice medium diluted with distilled water so that the initial sugar concentration was 10 wt% as a fermentation medium. Was conducted in the same manner as in the fermentation test 1. In addition to the lactic acid concentration, the ethanol concentration and the glucose concentration were measured using an enzyme sensor (Oji Scientific Instruments, Biosensor BF-4). In addition, the relationship between kLa and aeration stirring conditions is as follows.

kLa Aeration rate (L / min) Stirring speed (rpm)
6 0.3 60
11 0.3 150
16 0.3 230
22 0.3 270
29 0.3 290

  Maximum lactic acid yield up to 48 hours of fermentation and ethanol amount, glucose amount and lactic acid ratio (%) at the maximum lactic acid yield (where lactic acid ratio (%) = lactic acid concentration / (lactic acid concentration + 2 × ethanol concentration) × 100) ) Is shown in Tables 3 to 5, and graphs of lactic acid production amounts up to 72 hours for various lactic acid-producing yeasts are shown in FIGS.

  As shown in Tables 3 to 5 and FIGS. 5 to 7, from the viewpoint of LDH copy number, the lactic acid production rate decreased as the copy number increased, but the lactic acid yield and lactic acid ratio tended to increase. . In addition, it was found that the sensitivity to kLa varies depending on the LDH copy number, and that the lactic acid production rate tends to vary greatly with kLa as the LDH copy number increases. That is, as the LDH copy number increases, the lactic acid production rate tends to increase when kLa is large, and the lactic acid production rate tends to decrease when kLa is small. On the other hand, from the viewpoint of kLa, it was found that the smaller the kLa, the higher the final lactic acid yield and the lactic acid ratio. From the above, it was found that lactic acid-producing yeast is affected by kLa in terms of lactic acid production rate, lactic acid yield, and lactic acid ratio, and these effects are greater in LDH multicopy bodies having a high lactic acid yield. Therefore, control of kLa is effective for obtaining desired lactic acid production characteristics in lactic acid-producing yeast, and especially for LDH multi-copy, process management such as fermentation time can be easily performed by control of kLa. all right.

(Fermentation test 4)
In this fermentation test, the effect of changing kLa in the culturing process on the lactic acid production characteristics was confirmed. Fermentation was started under the aeration and stirring conditions (aeration rate: 0.3 L / min, agitation speed: 150 rpm) with a kLa of 10.8, and after 6 hours and 12 hours, kLa was 18.0 (aeration rate: 0.0. 3 L / min, stirring speed: 240 rpm) The fermentation test was conducted in the same manner as in the fermentation test 1 except that the culture was performed for a maximum of 55 hours and a medium in which cane juice had an initial sugar concentration of 15 wt% was used. The results are shown in FIG.

  As shown in FIG. 7, the lactic acid production rate was clearly increased after switching kLa to 18.0, and the fermentation time until consuming the sugar content could be accelerated by 5 to 10 hours. That is, it was found that even when kLa was switched during the culturing process, the lactic acid production characteristics of the lactic acid-producing yeast corresponding to kLa after the switching were expressed. From this, by adjusting the lactic acid production rate by changing kLa during the culturing process, it is possible to adjust the culturing period of the lactic acid producing process using lactic acid producing yeast while ensuring a lactic acid yield and lactic acid ratio above a certain level. all right. The above supports the fact that the lactic acid production rate is highly dependent on kLa, and at the same time, the control of kLa or the change of kLa in the culture process adjusts the lactic acid production rate and consumes sugar. It supports that it is effective in controlling the fermentation end time.

(Fermentation test 5)
In the present fermentation test, using the PDC1p-LDH10 copy produced in Example 1 as a lactic acid-producing yeast, two types of kLa (16 and 29) were set for the lactic acid-producing yeast, and lactic acid fermentation was performed. In the middle, the dissolved oxygen concentration was measured using a dissolved oxygen electrode (T type manufactured by METTLER TOLEDO). The fermentation test was performed in the same manner as the fermentation test 1 except that the fermentation was performed at a predetermined kLa. The measurement results for the lactic acid concentration and dissolved oxygen concentration of the fermentation broth under each kLa condition are shown in FIG. 9 and FIG.

  As shown in FIGS. 9 and 10, the dissolved oxygen concentration was 1 ppm or less throughout the fermentation test, and was 0.5 ppm or less throughout the fermentation test under the condition of kLa = 16, which is excellent in lactic acid production.

The figure which shows the structure of pBTrp-PDCl-LDHKCB vector. The figure which shows the LDH gene introduction | transduction site | part on the chromosome in 2 copy body, 6 copy body, 8 copy body, and 10 copy body which are the LDH gene transfer yeast produced in Example 1. FIG. The graph which plotted kLa on the horizontal axis and the lactic acid concentration on the vertical axis. kLa (18) is a graph showing changes in the amount of lactic acid produced under constant aeration stirring conditions. The graph which shows the change of the lactic-acid production amount under different kLa about LDH2 copy body. The graph which shows the change of the lactic acid production amount under different kLa about LDH6 copy body. The graph which shows the change of the lactic acid production amount under different kLa about LDH10 copy body. The graph which shows the change of the lactic-acid production amount at the time of changing kLa during a culture | cultivation process. The figure which shows the change of the dissolved oxygen concentration in the fermentation test 5. FIG. The figure which shows the change of the lactic acid density | concentration in the fermentation test 5. FIG.

Claims (10)

A method for producing lactic acid,
A culture step of culturing a lactic acid-producing yeast comprising a gene encoding a protein having lactate dehydrogenase activity so that the gene can be expressed under the control of a pyruvate decarboxylase gene promoter on the yeast chromosome ,
With
The said culture | cultivation process is a lactic acid production method provided with the process of culture | cultivating in the range whose oxygen transfer capacity coefficient (hr < ^-1 >) is 6 or more and 36 or less. The lactic acid production method according to claim 1, wherein the culturing step includes culturing in a range where an oxygen transfer capacity coefficient (hr ⁻¹ ) is 16 or less. The lactic acid production method according to claim 1, wherein the culturing step includes culturing in a range where an oxygen transfer capacity coefficient (hr ⁻¹ ) exceeds 16. The lactic acid production method according to any one of claims 1 to 3, wherein an oxygen transfer capacity coefficient (hr ^-1 ) is changed in the culturing step. The culture step includes
(A) culturing the genetically modified lactic acid-producing yeast in an oxygen transfer capacity coefficient (hr ⁻¹ ) of 16 or less;
(B) culturing the genetically modified lactic acid-producing yeast in an oxygen transfer capacity coefficient (hr ⁻¹ ) exceeding 16;
The lactic acid production method of Claim 4 containing this.   The lactic acid production method according to claim 5, wherein the step (b) is performed after the step (a).   The said culture | cultivation process is a process of supplying oxygen under aeration and / or stirring of the culture solution of the said lactic acid examination yeast, and culturing in the range whose dissolved oxygen amount of this culture solution is 1 mg / L or less. The method for producing lactic acid according to any one of 6.   The method for producing lactic acid according to claim 7, wherein the amount of dissolved oxygen in the culture solution in the culturing step is 0.5 ppm or less.   The lactic acid-producing yeast according to any one of claims 1 to 8, wherein the lactic acid-producing yeast has reduced pyruvate decarboxylase and / or alcohol dehydrogenase activity.   The lactic acid-producing yeast according to any one of claims 1 to 9, wherein the lactic acid-producing yeast comprises two or more genes encoding a protein having the lactic acid dehydrogenase activity.

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