JP2006223222A - Method for culturing cell, and gene, recombinant vector and transformant used for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for culturing a cell, inhibiting the increase of the amount of ROS (reactive oxygen species) in the cell and reducing the effect of oxidation stress against the cell. <P>SOLUTION: This method for culturing the cell is provided by introducing a gene consisting of a DNA shown in the following (a) or (b) into an objective cell and performing the culture. The gene can e.g. be introduced into the cell by a recombinant vector included in a vector. (a) A DNA consisting of a base sequence of at least one gene selected from a group consisting of yfiD, yggB and yggE. (b) A DNA consisting of a base sequence obtained by deleting, substituting or adding one or several bases in the base sequence (a) and encoding a protein having an activity of inhibiting the increase of the ROS. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for culturing various cells including microorganisms such as bacteria, and genes, recombinant vectors and transformants used therefor.

  Aerobic organisms preferentially utilize molecular oxygen as a final electron acceptor in respiration in order to effectively obtain energy for growth. For this reason, oxygen can be said to be an essential element for aerobic organisms.

However, oxygen is also involved in oxidative stress that damages cells. That is, a part of oxygen accepts electrons generated in various metabolic processes in the cell, and, for example, reactive oxygen species rich in reactivity such as superoxide anion (O ₂ ⁻ ), hydrogen peroxide, and hydroxy radicals. Converted to (ROS: reactive oxygen species). The converted ROS is known to cause cell degradation and cell destruction, which causes DNA destruction, enzyme inactivation, membrane lipid oxidation, etc., and this causes damage to cells (for example, non-patented) Reference 1).

Conventionally, with regard to aerobic cell culture, studies have been made on the operation theory and bioreactor design for effectively supplying oxygen to cells. Moreover, regarding oxygen conditions at the time of culture, since oxygen is an essential element, a high oxygen atmosphere is generally set (see, for example, Non-Patent Document 2). However, even if aerobic cells are cultured in a high oxygen atmosphere, the culture efficiency does not always improve, and for example, an extreme decrease in growth rate and cell yield has often been confirmed.
Finkel, T. and NJ Holbrook. 2000. Oxidants, oxidative stress and the biology of ageing. Nature. 408: 239-247. Hisaharu Taguchi, Microbial culture engineering (edited by Hisaharu Taguchi and Shiro Nagai), Chapter 5, Oxygen supply to fermenters, p153-199, 1985, Kyoritsu Publishing

  Therefore, the present inventors have found that such a decrease in the culture efficiency is caused by the occurrence of oxidative stress, that is, cells converted from oxygen, even though a sufficient amount of oxygen, which is essentially an essential element, is supplied. It was speculated that there was a cause for the occurrence of ROS. In the case of culturing absolute anaerobic cells or performing anaerobic fermentation, it is desired to culture under conditions with a very low amount of oxygen, particularly in the absence of oxygen. However, since the realization of such an environment setting becomes more difficult as the culture scale becomes larger, as described above, the inventors have also experienced cell growth caused by oxidative stress caused by ROS converted from oxygen. I guessed it might be affected.

  Accordingly, an object of the present invention is to provide a cell culture method that suppresses an increase in the amount of ROS in a cell and reduces the influence of oxidative stress on the cell.

In order to achieve the above-mentioned object, the cell culture method of the present invention, in order to reduce the influence of oxidative stress on the cells, prior to culturing the cells, the cells are subjected to DNA shown in (a) or (b) below. A step of introducing a gene comprising:
(A) DNA comprising a base sequence of at least one gene selected from the group consisting of yfiD, yggB and yggE
(B) DNA encoding a protein consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a), and having an activity of suppressing the increase of ROS

The gene of the present invention is a gene used in the cell culture method of the present invention, and is a gene consisting of DNA shown in the following (a) or (b).
(A) DNA comprising a base sequence of at least one gene selected from the group consisting of yfiD, yggB and yggE
(B) DNA encoding a protein comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a), and having an activity of suppressing an increase in ROS

  Furthermore, the recombinant vector of the present invention is a recombinant vector used in the cell culture method of the present invention, wherein the gene is incorporated into the vector, and the gene is the gene of the present invention. And The transformant of the present invention is a transformant in which a recombinant vector is introduced into a cell, and the recombinant vector is the recombinant vector of the present invention.

  According to the cell culture method of the present invention, the mechanism is unknown, but cells can be cultured while suppressing the influence of oxidative stress by introducing the gene of the present invention as described above into the cells in advance. In addition, although yfiD, yggB and yggE have not been reported for the function of each expressed protein, the present inventors have found for the first time that they have the function of avoiding the effects of oxidative stress by introduction into cells. That is. In addition, since the gene of the present invention is incorporated into the recombinant vector of the present invention, the recombinant vector is introduced into a target cell, and the transformant is used to obtain the culture method of the present invention. Can be performed easily.

In order to reduce the influence of oxidative stress on cells, the cell culture method of the present invention, as described above, prior to cell cultivation, the cells of the present invention, ie, the following (a) or ( a step of introducing a gene comprising DNA shown in b).
(A) DNA comprising a base sequence of at least one gene selected from the group consisting of yfiD, yggB and yggE
(B) DNA encoding a protein comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a), and having an activity of suppressing an increase in ROS

  As shown in (b), if the DNA can suppress an increase in intracellular ROS by introduction into a cell, one or several bases are deleted from the base sequence of (a), It may be a substituted or added base sequence. Here, the number of bases that can be deleted, substituted or added is, for example, from 1 to 6 by deletion / addition with respect to 40 bases, preferably 1 to 3, more preferably 1 to 2 and 1 to 4 by substitution, preferably 1 to 2 and more preferably 1.

  The DNA shown in (b) is not limited to the number of bases deleted, substituted, or added as described above. For example, the DNA hybridizes with the DNA of (a) under stringent conditions. And those having a homology with the DNA of (a) of 90% or more, preferably 95% or more, more preferably 97.5% or more. The hybridization can be performed by a conventionally known method. Examples of the stringent conditions include ± 10 ° C. with respect to the melting temperature (Tm value) of the DNA base sequence of (a). .

  For example, the base sequence of yfiD is represented by SEQ ID NO: 1, the base sequence of yggB is represented by SEQ ID NO: 3, for example, and the base sequence of yggE is represented by SEQ ID NO: 5, for example. In addition, deletion or substitution or addition of one or several bases is performed in the base sequence represented by these SEQ ID NOs as long as it is within the range showing the activity of suppressing the increase in ROS by introduction into cells. May be. The amino acid sequences of the proteins encoded by the base sequences represented by SEQ ID NOs: 1, 3, and 5 are represented by SEQ ID NOs: 2, 4, and 6, respectively.

Examples of the base sequence of yfiD, yggB or yggE include the base sequence of yfiD, yggB or yggE derived from Eschericia coli . The above-described base sequences of SEQ ID NOs: 1, 3, and 5 are sequences of yfiD, yggB, and yggE derived from E. coli K-12 W3110, respectively. Further, in the base sequence described in SEQ ID NO: 5, for example, the 5 ′ end may be “ATG” instead of “GTG”.

In the gene of the present invention, the base sequence of yfiD, yggB or yggE is not limited to the base sequence of E. coli , and may be, for example, the base sequence of yfiD, yggB or yggE derived from another organism. . The base sequences of yfiD, yggB and yggE are, for example, GenoBase (http://ecoli.aist-nara.ac.jp/GB5/search.jsp) and NCBI Entrez (http: //www.ncbi.nlm.nih .gov / Entrez / index.html) etc.

  A PCR amplification product obtained by PCR using a primer based on the known nucleotide sequence of yfiD, yggB or yggE can also be used as the gene of the present invention. PCR can be performed according to a conventional method.

The primer is preferably a base sequence complementary to a fragment containing a start codon or a stop codon in the base sequence of yfiD, yggB or yggE, for example. The length of the primer is not particularly limited, and is, for example, in the range of 10 to 40 mer, preferably 15 to 40 mer, and preferably 20 to 30 mer. Specific examples of primers for yfiD, yggB and yggE are shown below, but are not limited thereto.
(yfiD)
forward primer: SEQ ID NO: 7 5'-ATGATTACAGGTATC-3 '
reverse primer: SEQ ID NO: 8 5'-TTACAGGCTTTCAGT-3 '
(yggB)
forward primer: SEQ ID NO: 9 5'-ATGGAAGATTTGAAT-3 '
reverse primer: SEQ ID NO: 10 5'-TTACGCAGCTTTGTC-3 '
(yggE)
forward primer: SEQ ID NO: 11 5'-ATGAAGTTCAAAGTT-3 '
reverse primer: SEQ ID NO: 12 5'-TTATTGTGCTGCAGG-3 '

The template DNA for the PCR is not particularly limited, and for example, genomic DNAs such as various bacteria such as E. coli , microorganisms such as archaea, fungi and algae, cDNA and the like can be used. Specifically, examples of the template DNA for yfiD include genomic DNAs such as Shigella flexneri , Salmonella enterica , Salmonella typhimurium , Serratia liquefaciens , Vibrio vulnificus , Photobacterium profundum , and the template DNA for yggB includes Shigella flexneri , Salmonella. Examples include enterica , Salmonella typhimurium , and Pseudomonas aeruginosa , and examples of yggE template DNA include Shigella flexneri , Salmonella enterica , Salmonella typhimurium , Edwardsiella ictaluri , Agrobacterium tumefaciens , and Bacillus cereus . In addition, it is not restrict | limited to these.

In the culturing method of the present invention, cells to which this method can be applied are not particularly limited, and the effect can be obtained by the culturing method of the present invention as long as the cell is affected by oxidative stress. Therefore, for example, in addition to microorganisms such as bacteria such as Escherichia coli and Salmonella , archaea, and fungi, it can be applied to plant cells and animal cells. In the present invention, the “cell” to be cultured may be not only a unicellular organism or a multicellular organism, but also a culturable tissue, organ, or the like.

  In the present invention, the means for introducing a gene into a cell is not particularly limited, and a conventionally known method can be adopted. As an example, there is a method using the recombinant vector of the present invention in which the gene is incorporated into a vector. As described above, when the recombinant vector of the present invention is used, the gene of the present invention can be introduced into cells by a conventional method. Therefore, the present invention can be easily carried out by culturing the obtained transformant.

  Examples of the vector include a plasmid vector, a phage vector, and a virus vector, and these can be appropriately determined according to, for example, the type of cell into which the recombinant vector is introduced. In addition, the method for introducing the recombinant vector is not particularly limited, and a conventionally known method such as a calcium chloride treatment method or an electroporation method can be employed depending on the type of cell to be introduced.

  In the present invention, the culture conditions for the cells are not particularly limited, and various conditions such as aerobic conditions, anaerobic conditions, oxygen supply amount, temperature, and stirring can be determined as appropriate according to the type of cells. Among these, the culture method of the present invention is extremely useful under conditions where the influence of oxidative stress has conventionally occurred on cell growth. For example, in the conventional method, when excessive oxygen is supplied for the purpose of promoting the growth of aerobic bacteria or the like, the growth may be adversely inhibited by oxidative stress as described above. However, even under such conditions, if the culture method of the present invention is applied, the influence of oxidative stress is reduced, so that the supplied oxygen can be fully utilized and growth can be promoted. . In addition, for example, when anaerobic bacteria and the like are cultured anaerobically, the growth can be further stabilized by applying the present invention even under the same culture conditions as in the past. This is because the influence of oxygen (oxidative stress) can be further suppressed by introducing the gene of the present invention into cells in addition to anaerobic culture conditions.

The intracellular ROS content can be measured by a conventionally known method, and is not particularly limited, but can be measured by, for example, the trade name CM-H ₂ DCF-DA (Moleculer Probes).

  Reference examples are shown below.

(Reference Example 1)
1. Growth promotion of Escherichia coli IM303-I4 under oxidative stress (cells)
E. coli IM303-I4 was used as E. coli . This IM303-I4 is an isolated bacterium from E. coli IM303 provided by Professor Doto, Department of Biotechnology, Faculty of Engineering, Kansai University. Since E. coli IM303 lacks SOD (superoxide dismutase) for removing superoxide anion, which is ROS, even if ROS occurs in cells, it cannot usually be removed. It is said that growth is impaired.

(Culture medium)
An amino acid mixture was added to an M9 modified medium containing 8 g / L glucose and 5 mg / L thiamine. As the amino acid mixture, 0.25 g Met, 0.25 g Arg, 0.30 g Tyr, 0.60 g Lys, 0.80 g Ile, 0.40 g Phe, 1.5 g Val, 0.50 g Asp, 2.0 g Thr, 0.80 g Glu and 3.75 g Ser per liter (Hereinafter referred to as “2068 mixture”). This amino acid mixture is described as a synthetic medium for ATCC2068 in the catalog of American Type Culture Collection (ATCC).

(Culture conditions)
E. coli IM303-I4 was cultured under conditions that gave oxidative stress. Oxidative stress was applied by irradiating titanium oxide added to the medium with light and generating active oxygen from the excited titanium oxide. Specifically, 10 mL of the M9 modified medium to which TiO ₂ particles (trade name Degussa P25: manufactured by Nippon Aerosil Co., Ltd.) were added so as to be 10 mg / L was placed in an L-shaped test tube, and E. coli IM303-I4 was added thereto. And cultured with shaking under conditions of 37 ° C. and 45 strokes / min (spm). In order to excite titanium oxide, the L-shaped test tube was covered with a black screen during the culture, and a 20 W black light fluorescent lamp (trade name Type FL-20S BL- B: manufactured by Matsushita Electric Industrial Co., Ltd.) and irradiated with light at an incident light intensity I = 12.5 W / m ² . On the other hand, as a control, IM303-I4 was cultured in the same manner as described above using the above-mentioned M9 modified medium without addition of TiO ₂ particles and without light irradiation with a black light fluorescent lamp (normal culture conditions). . Then, the optical density (OD ₆₆₀ ) at a wavelength of 660 nm was measured for each culture solution, and the change over time was confirmed using this as the cell density. The results are shown in the growth curve graph of FIG. In FIG. 1A, the result of light irradiation in the presence of titanium oxide is represented by “◯: TiO ₂ and light”, and the control is represented by “●: -TiO ₂ and -light (normal condition)”. In addition, the maximum specific growth rate (μ _m), induction time. (Lag time: t _L) is, Zwietering, MH, et al. (Zwietering, MH, L. Jongenburger, FM Rombouts and K. van't Riet 1990 Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 56: 1875-1881.) was calculated by fitting the obtained growth curve data to the improved Gompertz equation.

(ROS content)
Moreover, the intracellular ROS content was measured about the cultured cell which is _OD660 = 0.2. The ROS content was measured by collecting cultured cells from the culture medium and using the product name CM-H ₂ DCF-DA (Moleculer Probes) according to the instructions for use. These results are shown in FIG.

As shown in Fig. 1 (A), when E. coli IM303-I4 is cultured in the presence of excited titanium oxide, it is approximately twice that of the control (in the absence of titanium oxide, non-light irradiation). showed maximum specific growth rate (mu _m). In addition, as shown in FIG. 1 (B), E. coli IM303-I4 was able to suppress an increase in ROS content when oxidative stress was applied, compared to a control cultured under normal conditions. Thus, E. coli IM303-I4 lacked SOD to remove superoxide anion and showed the result of reducing the ROS content despite the more severe culture conditions than the control. .

2. Analysis of gene expression of E. coli IM303-I4 under oxidative stress Based on the above results, subsequently, changes in gene expression in E. coli IM303-I4 by applying oxidative stress were confirmed.

E. coli IM303-I4 was cultured in the same manner as described above. The maximum specific growth rate of E. Coli IM303-I4 under titanium oxide present that is photoexcited (mu _m) is 0.4 h ^-1, the E. Coli IM303-I4 cultured in the absence of titanium oxide 2.56 times the maximum specific growth rate (μ _m = 0.16h ^-1). When the growth reached OD ₆₆₀ = 0.15, 18 mL of the culture solution was sampled and immediately mixed with an equal amount of cold ethanol containing 10% phenol. The cells were collected from this mixture by centrifugation (5 minutes, 4 ° C., 5,000 (g), washed with the phenol-containing cold ethanol solution, and then RNA was extracted from the collected cell pellet. Total RNA was extracted from all recovered cells using the trade name RNeasy Mini Kit and RNase-free DNase Set (manufactured by QIAGEN Inc.), and extracted RNAs were treated with phenol / chloroform and then ethanol. Purified RNAs were recovered again by precipitation, and the total concentration of purified RNAs was calculated from the difference in absorbance measured at wavelengths of 260 nm and 280 nm.

Labeled cDNAs use the product name RNA Fluorescence Labeling Core Kit (manufactured by Takara Bio Inc.) and the product name dUTP-Cy3 fluorescent nucleotide and the product name dUTP-Cy5 fluorescent nucleotide (manufactured by Amersham Biosciences Ltd.). According to the above, it was prepared from the aforementioned purified RNAs. DNA microarray analysis is a chip (trade name IntelliGene E. coli CHIP: manufactured by Takara Bio Inc.) on which 3,437 genes (cDNAs) corresponding to about 80% of open reading frames (ORFs) in E. coli K-12 are immobilized. ) And according to the instructions for use. The fluorescence intensity was determined by image analysis using a trade name GMS ^™ 418 Array Scanner (manufactured by Genetic MicroSystems Inc.) and a trade name ImaGene ^™ (manufactured by BioDiscovery Inc.). Then, the data of labeled cDNAs derived from cells cultured in the presence of oxidative stress and those derived from cells cultured in the absence of oxidative stress were compared. Specifically, the ratio of fluorescence intensity was determined for the value in the presence of oxidative stress with the value in the absence of oxidative stress as a reference. The gene whose ratio is 3 or more is a gene that is up-regulated under oxidative stress, and the gene that is less than 0.3 is a gene that is down-regulated under oxidative stress. It was judged that.

  As a result, fruB (313), fruK (939), pheA (1161), fruA (1692), yfiD (384), putA (3933), yghD (532), blc (534), spy (486), ymgD ( 330), pheT (2388), stpA (405), gltB (4545), gltD (1419), gpmI (1545), yqhC (957), yggG (885), nadB (1623), pheS (984), yggB ( 861), nagB (801), galP (1395), arp (2187), yggE (741), and osmB (219), the expression level was increased in the presence of oxidative stress. In addition, the number of bases of each gene is shown in parentheses. Among them, yfiD, yggB and yggE showed a large increase in the expression level, and particularly the yfiD gene showed a marked increase. From the above results, it can be said that these 25 genes, particularly yfiD, yggB and yggE, in which a marked increase in expression level was observed, are involved in the suppression of the increase in ROS content. The fact that these genes actually contribute to the suppression of the increase in the ROS content and the effect of suppressing the increase in the ROS can be obtained by introducing these genes into the cells will be described in the following examples.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

  Using recombinant plasmids into which yfiD, yggB and yggE were inserted, it was confirmed that the influence of oxidative stress during culture was avoided.

(Construction of recombinant plasmid)
yfiD, yggB and yggE were each amplified from the chromosomal DNA of the wild strain E. coli MM294 by PCR using the trade name KOD-Dash DNA polymerase (manufactured by Toyobo). The combinations of primers used in the PCR method are shown below.

(YfiD)
forward primer: SEQ ID NO: 13 5'-CCCAAGCTTGATGATTACAGGTATC-3 '
reverse primer: SEQ ID NO: 14 5'-CGGAATTCTTACAGGCTTTCAGT-3 '
(yggB)
forward primer: SEQ ID NO: 15 5'-CCCAAGCTTGATGGAAGATTTGAAT-3 '
reverse primer: SEQ ID NO: 16 5'-CGGAATTCTTACGCAGCTTTGTC-3 '
(yggE)
forward primer: SEQ ID NO: 17 5'-CCCAAGCTTGATGAAGTTCAAAGTT-3 '
reverse primer: SEQ ID NO: 18 5'-GCTCTAGATTATTGTGCTGCAGG-3 '

  Each of these PCR amplification primers has a base sequence serving as a restriction enzyme recognition site added to the 5 'end.

The cloning site of PUC19 was cleaved with a restriction enzyme, and the amplification products (yfiD, yggB, yggE) were ligated to the cleaved site using a trade name Ligation-Convenience Kit (manufactured by Nippon Gene Co., Ltd.). As restriction enzymes, HindIII and EcoRI were used for yfiD and yggB, and HindIII and XbaI were used for yggE. This recombinant plasmid was introduced into E. coli DH5a for transformation, and plasmid DNAs amplified in the cells were extracted according to a conventional method. The plasmid DNAs were purified using the trade name Wizard Plus SV Minipreps DNA Purification Systems (Promega Corp.). In the obtained recombinant plasmid, pUC19 in which yfiD is inserted is called “pYFD”, pUC19 in which yggB is inserted is called “pYGB”, and pUC19 in which yggE is inserted is called “pYGE”.

The recombinant plasmids (pYFD, pYGB, pYGE) were introduced into E. coli IM303-I4, and the resulting transformants were cultured to obtain an initial exponential growth period (OD ₆₆₀ = 0.2) and a medium exponential growth. The intracellular ROS content in the phase (OD ₆₆₀ = 0.5) was measured. In addition, for the culture, titanium oxide is not added to the M9 modified medium, 50 mg / L ampicillin and 10 μmol / L IPTG (isopropylthiogalactoside) are added, and the incident light intensity by the black light fluorescent lamp is increased. Except not having performed light irradiation, it carried out on the conditions similar to the said reference example 1. FIG. As a control, a transformant in which pUC19 was introduced into E. coli IM303-I4 was used, and culture and ROS content were measured in the same manner. The result is shown in FIG.

As shown in FIG. 2, each of the transformants into which the recombinant plasmids (pYFD, pYGB, pYGE) were introduced showed a lower ROS content than the control in both the initial and intermediate exponential growth periods. In particular, E. coli IM303-I4 with pYGE has an ROS level of about 46% of the control in the early exponential growth phase, and the control has an increase in ROS level in the intermediate exponential growth phase. Showed a further reduction in ROS content. Further, E. coli IM303-I4 having pYFD also showed a further decrease in ROS content during the medium-term exponential growth period.

A transformant of E. coli IM303-I4 introduced with pYGE was cultured, and its growth curve was confirmed.

In the same manner as in Example 1, E. coli IM303-I4 introduced with pYGE was cultured and its growth was confirmed. As a control, E. coli IM303-I4 introduced with pUC19 was also cultured in the same manner. These results are shown in FIG. The graph of FIG. 3 is a growth curve and shows the change in cell density (OD ₆₆₀ ) over time.

As shown in FIG. 3, E. Coli IM303-I4 introduced with pYGE, the average value of the maximum specific growth rate (mu _m) is 0.06 h ^-1, showed about 3 times the control.

A transformant of wild strain E. coli MM294 introduced with pYGE was cultured under oxidative stress, and its growth curve was confirmed.

In the same manner as in Example 1, an E. coli MM294 transformant into which pYGE was introduced was prepared. Then, the culture was carried out in the same manner as in Example 1 except that paraquat (PQ), which is an active oxygen generator, was added to the M9 modified medium supplemented with ampicillin and IPTG of Example 1 so as to be 10 μmol / L. The growth was confirmed. As a control, E. coli MM294 introduced with pUC19 was also cultured in the same manner. These results are shown in FIG. The graph of FIG. 4 is a growth curve and shows the change in cell density (OD ₆₆₀ ) over time.

As shown in FIG. 4, wild-type strain E was introduced pyge. Coli MM294, the average value of the maximum specific growth rate (mu _m) is 0.1 h ^-1, was about 5 times that of the control. As described above, by introducing a recombinant plasmid having yggE even in the wild strain, the ROS content was reduced, and the influence of oxidative stress was suppressed, so that growth could be recovered and enhanced.

Transformants of wild strain E. coli MM294 into which pYGE had been introduced were cultured under oxidative stress, and the ROS content of the cells in the early and middle exponential growth phases was confirmed.

In the same manner as in Example 3, a transformant of E. coli MM294 into which pYGE had been introduced was cultured in an M9 modified medium containing paraquat. Then, the ROS content of the cultured cells in the initial exponential growth period (OD ₆₆₀ = 0.2) and the middle exponential growth period (OD ₆₆₀ = 0.5) was measured. The result is shown in FIG.

As shown in the figure, the ROS content was about 54% of the control in the initial exponential growth period, and was about 34% of the control in the intermediate exponential growth period. Thus, E. coli MM294 introduced with pYGE showed a lower ROS content than the control in both the early and middle exponential growth periods, and the control increased the ROS content in the intermediate exponential growth phase. In contrast, E. coli MM294 introduced with pYGE showed a further decrease. From this result, it can be seen that the introduction of pYGE can not only suppress the increase in the ROS content but also reduce the content.

  The effect of pYGE on amino acid requirements changed by oxidative stress was confirmed.

(A) E. coli IM303-I4 transformant
introduced E. coli IM303-I4 transformants of pyge, OD ₆₆₀ was grown to 0.2 under the same conditions as in Example 1. 1 mL of the culture solution is centrifuged (4 ° C, 10,000 (g, 5 minutes) to recover the cultured cells, washed with physiological saline, and centrifuged again (4 ° C, 10,000 (g, 5 minutes). And then suspended in physiological saline (9 g / L NaCl aqueous solution), while M9 modified medium agar plate containing 11 amino acids (2068 mixture) and Lys, Met, Ile from 2068 mixture. M9 modified medium agar plates containing 7 types of amino acids except Thr and Thr were prepared, and the suspension of the cultured cells was ingested on each agar plate and incubated at 37 ° C. for 48 hours. The number of colonies was counted, and the number of colonies on each agar plate was substituted into the following formula to determine the degree of change in the amino acid requirement ratio (A _d ): As a first control, E. coli introduced with pUC19 Use IM303-I4 transformant as a second control. E. coli IM303-I4 into which rasmid was not introduced was used, and the amino acid requirement ratio was similarly calculated for these.

A _{d of} E. coli IM303-I4 = (N _a11 -N _a7 ) / N _a11
In the above formula, “N _a11 ” is the number of colonies in an 11 amino acid (2068 mixture) plate, and “N _a7 ” is the number of colonies in a 7 amino acid plate. These results are shown in FIG. Incidentally, as the A _d is large, as compared to amino acids 11 kinds of plates, poor growth at amino acid seven plates, can determine the type of request amino acids is large, as the A _d is small, the amino acid seven plates Therefore, it can be judged that the number of types of amino acids required is small.

(B) E. coli MM294 transformant
The E. coli MM294 transformant introduced with pYGE was cultured using paraquat-containing M9 modified medium in the same manner as in Example 3 until OD ₆₆₀ reached 0.2, and the culture was performed in the same manner as in (A) above. A body suspension was prepared. Other than using the A9 agar plate containing 11 amino acids (2068 mixture) and the M9 agar plate containing 20 amino acids (hereinafter referred to as “1904 mixture”) as the agar plate (A ) And the number of colonies was counted. Then, the number of colonies on each agar plate was substituted into the following formula to determine the degree of change in amino acid requirement ratio (A _d ). In addition, the E. coli MM294 transformant introduced with pUC19 was used as the first control, and E. coli MM294 without the plasmid was used as the second control. went. The “1904 mixture” contains 0.45 g / L of Leu per liter and 0.225 g / L of other amino acids (19 types), and is described as a synthetic medium for ATCC 1904 in the ATCC catalog.

A _{d of} E. coli MM294 = (N _a20 -N _a11 ) / N _a20
In the above formula, “N _a20 ” is the number of colonies in a plate with 20 types of amino acids, and “N _a11 ” is the number of colonies in a plate with 11 types of amino acids. These results are shown in FIG. Incidentally, as the A _d is large, as compared to amino acids 20 kinds of plates, poor growth at amino acid 11 kinds of plates, can determine the type of request amino acids is large, as the A _d is small, the amino acid 11 kinds of plate Therefore, it can be judged that the number of types of amino acids required is small.

As shown in FIG. 6 (A), E. Coli IM303-I4 but A _d values (first control) is about 73%, E. Coli IM303- I4 is A _d values with pYGE with pUC19 Was about 17% and about 1/4 of the control. Incidentally, A _d values of E not introduced plasmid. Coli IM303-I4 (second control) was 64%. Furthermore, as shown in FIG. 6 (B), A _d values of E. Coli MM294 with pYGE cultured under oxidative stress conditions with the addition of paraquat is 9.6%, E. Coli MM294 (first having pUC19 It was about 1/15 of 1 control), showing a remarkably low value. Incidentally, E not introduced plasmid. Coli MM294 A _d value (second control) was 40%. These results indicate that the introduction of pYGE could effectively suppress changes in amino acid requirements due to oxidative stress.

  As described above, according to the present invention, by introducing a gene as described above into a cell in advance, an increase in intracellular ROS content can be suppressed, and the influence of oxidative stress on growth can be reduced. Therefore, if the culture method of the present invention is used, for example, the culture efficiency can be improved even under the same culture conditions as before, and problems associated with an increase in the oxygen supply amount can be avoided. Therefore, it can be said that the culture method of the present invention is extremely useful for the production of various substances using a fermentation process, for example.

FIG. 1 (a) is a graph showing a growth curve of E. coli IM303-I4 in a reference example of the present invention, and FIG. 1 (b) shows the intracellular ROS content of E. coli IM303-I4. It is a graph to show. FIG. 2 is a graph showing the intracellular ROS content in the transformants prepared in the examples of the present invention. FIG. 3 is a graph showing the growth curve of E. coli IM303-I4 transformants in other examples of the present invention. FIG. 4 is a graph showing the growth curve of E. coli MM294 transformants in another example of the present invention. FIG. 4 is a graph showing the intracellular ROS content in E. coli MM294 transformants prepared in other examples of the present invention. Fig. 6 (a) is a graph showing the amino acid requirement ratio of the E. coli IM303-I4 transformant in another example of the present invention, and Fig. 6 (b) is the amino acid of the E. coli MM294 transformant. It is a graph which shows a request ratio.

Claims (26)

A method for culturing cells comprising:
In order to reduce the influence of oxidative stress on a cell, a method for culturing a cell comprising a step of introducing a gene comprising DNA shown in the following (a) or (b) into the cell prior to culturing the cell.
(A) DNA comprising a base sequence of at least one gene selected from the group consisting of yfiD, yggB and yggE
(B) a DNA encoding a protein having a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a) and having an activity of suppressing an increase in reactive oxygen species   The culture method according to claim 1, wherein the base sequence of yfiD is represented by SEQ ID NO: 1.   The culture method according to claim 1, wherein the base sequence of yggB is represented by SEQ ID NO: 3.   The culture method according to claim 1, wherein the base sequence of yggE is represented by SEQ ID NO: 5. The culture method according to claim 1, wherein yfiD, yggB and yggE are genes derived from Eschericia coli .   The culture method according to claim 1, wherein the gene is a PCR amplification product obtained by a PCR method using primers represented by SEQ ID NO: 7 and SEQ ID NO: 8.   The culture method according to claim 1, wherein the gene is a PCR amplification product obtained by a PCR method using primers represented by SEQ ID NO: 9 and SEQ ID NO: 10.   The culture method according to claim 1, wherein the gene is a PCR amplification product obtained by a PCR method using primers represented by SEQ ID NO: 11 and SEQ ID NO: 12. The culture method according to any one of claims 6 to 8, wherein the PCR amplification product is a product obtained by PCR using Eschericia coli genomic DNA as a template.   The culture method according to any one of claims 1 to 9, wherein the means for introducing a gene into a cell is a means for introducing a recombinant vector having the gene incorporated into a vector into the cell.   The culture method according to claim 10, wherein the vector is at least one selected from the group consisting of a plasmid vector, a phage vector, and a virus vector.   The culture method according to any one of claims 1 to 11, wherein the cells are cultured under aerobic conditions.   The culture | cultivation method as described in any one of Claims 1-12 which reduces the influence of oxidative stress by reducing the content of reactive oxygen species in a cell.   The culture method according to claim 1, wherein the cell is a microorganism. A gene used for the cell culture method according to any one of claims 1 to 14, which comprises a DNA shown in the following (a) or (b).
(A) DNA comprising a base sequence of at least one gene selected from the group consisting of yfiD, yggB and yggE
(B) a DNA encoding a protein having a base sequence in which one or several bases are deleted, substituted or added in the base sequence of (a) and having an activity of suppressing an increase in reactive oxygen species   The gene according to claim 15, wherein the base sequence of the yfiD gene is represented by SEQ ID NO: 1.   The gene according to claim 15, wherein the base sequence of the yggB gene is represented by SEQ ID NO: 3.   The gene of Claim 15 whose base sequence of a yggE gene is represented by sequence number 5. The gene according to claim 15, wherein yfiD, yggB and yggE are genes derived from Eschericia coli .   The gene according to claim 15, wherein the gene is a PCR amplification product obtained by a PCR method using primers represented by SEQ ID NO: 7 and SEQ ID NO: 8.   The gene according to claim 15, wherein the gene is a PCR amplification product obtained by a PCR method using primers represented by SEQ ID NO: 9 and SEQ ID NO: 10.   The gene according to claim 15, wherein the gene is a PCR amplification product obtained by a PCR method using primers represented by SEQ ID NO: 11 and SEQ ID NO: 12. The gene according to any one of claims 15 to 22, wherein the PCR amplification product is a product obtained by PCR using the genomic DNA of Eschericia coli as a template.   A recombinant vector used in the method for culturing cells according to any one of claims 1 to 14, wherein a gene is incorporated into the vector, and the gene is any one of claims 15 to 23. A recombinant vector characterized in that it is the gene described in 1.   A transformant having a recombinant vector introduced into a cell, wherein the recombinant vector is the recombinant vector according to claim 24.   The transformant according to claim 25, wherein the cell is a microorganism.

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