JPS626690A - Method for converting organism - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method of bioconverting a precursor to a desired product by means of mitotically arrested cells. Cell division is caused by X-rays,
It is stopped by gamma radiation or other radiation used to essentially stop cell division. The irradiation field is allt
a) Choose one that arrests division, but does not essentially impair the biotransformation ability of the cells. This is contrary to expectations in the art, and it was generally accepted that if cell division was stopped by electromagnetic radiation, this would result in a substantial reduction or cessation of the cell's ability to biotransform. It also happens.

A wide variety of cells can be used, including plant cells, bacteria, yeast cells, and mammalian cells. The cells can be used in suspension, or they can be embedded in a suitable matrix or attached to a suitable support. It can also be embedded in polymer beads. It has been established that the process of the invention is suitable for industrial conversion. Arrested cells can be used for long periods of time in batch or continuous conversion methods.

BACKGROUND OF THE INVENTION The production of organic compounds by living cells is as old as human history. It is explained by the fermentation of sugar to ethanol and the baking of bread.

More specifically, in vitro cultured plants 1IIIri
are widely used in the production of useful organic compounds such as flavors, medicines and perfumes, for example, Starbucks E.G., Devel, Hicro, 4.192-198.
1963: Kurz et al., Advanced Applied Microbiology, 209-24011979).

Production of such compounds in cultured cells can be accomplished by two main techniques. Adding specific precursors to cultured cells and bioconverting them into useful substances without supplying them (Reinhard et al., Advanced Biochemical Engineering,
16.50-83.1980: Chabur P.D., Applied Biochemical Biotechnology, 1982). There have been several obstacles to the industrial use of plant cells and other cells using the above techniques. For example: (1) Freely suspended cells are inferior to immobilized enzymes in advanced biotechnology (Proprius et al., Advanced Applied Microbiology 11
．． 1-26.1982), +21 Normally dividing cultured cells require a constant supply of expensive organic compounds, among other medium components, to support the metabolic activity involved in cell division, thus reducing production costs. increase

(3) Dividing cells cultured in vitro are treated with polymer (
When immobilized with alginate beads, etc., the cells are gradually released from the immobilization, resulting in a mixture of immobilized cells and freely suspended cells. Such mixtures interfere with the use of the cells for the production of organic compounds.

A method that retains the ability of cultured cells to produce and/or convert specific organic compounds, but does not impair cell division, must overcome the above difficulties. Adding metabolic inhibitors to arrest cell division and/or starve certain media components is undesirable for several reasons, such as adding toxic components to the system and causing metabolic changes.

Cell division arrest due to irradiation, such as X-rays, gamma rays, other electromagnetic radiation, or particulate irradiation, is a well-established phenomenon in plant, animal, and bacterial cells (Lee D. E., Effects of Irradiation on Living Cells). (2nd edition), Cambridge University Press, 1955).

Irradiation-induced mitotic cells of animals, plants, and microorganisms have not been previously used for industrial biotechnology in the bioconversion and production of organic compounds. The present invention provides a simple and generally applicable method for arresting cell division while retaining the ability of the cell to bioconvert and produce specific organic compounds.

SUMMARY OF THE INVENTION In accordance with the present invention, a method is provided for converting compounds on an industrial scale by means of cytostatic II cells.

In order to arrest cell division without impairing the biotransformation capacity of the cells, it is advantageous to treat the cells with X-rays or gamma radiation.

Contrary to what was previously accepted in the art, cessation of cell division by such means does not necessarily reduce the biotransformation potential of cells so treated.

The present invention is applicable to a wide variety of cells, including plants, yeast, bacteria, and vertebrate cells. There are various types of irradiation used to stop this cell division, such as electric vA.
t1i radiation and particulate radiation. X-rays and gamma rays of various wavelengths and intensities are selected. The invention is described with reference to plant cells, yeast, bacteria and mammalian cells. It is emphasized that this is merely illustrative and is applicable to a wide range of other cells.

Cells derived from Mensa sp., Nicotiana sp., other plant explants or other organisms by means conventional in the art (Reinert et al., Plant Cells, Tissue and Organ Culture, p. 803, Springelverlag, Berlin; 1
977) in a liquid medium such as 85 (Gambleg et al., Experimental Cell Research 50.151.1
968) or similar nutrient medium appropriate for the given cell type. The cells thus cultured are maintained and propagated in shaking culture or by other means suitable to provide growth conditions for these plant cells. Cells were harvested using standard methods detailed in the literature (e.g. Reinert and Baja, supra, 1
977; Chabourg ibid., 19B2) and periodically diluted with nutrient medium.

Biotransformation of certain precursors into corresponding products for use in the production of specific biochemicals (e.g., Starbucks, ibid., 1963) or by commonly used methods. I! (Reinhard and Alferman, supra, 1980), the cells are transferred to production or conversion medium, respectively. The latter contains precursors for biotransformation. Mentha suspended freely
To convert (-) menthone to (+) neomenthol by line 193, 20-80 η/1 menthone was added to the conversion medium consisting of Gamborg B5 components and the cells were shaken at a temperature of 23-27°C. maintained as a culture (Aviv et al., Planta mesoica, ±2.236-243.
1981). Bioconversion techniques can similarly be applied to other cellular and plant sources (Rainbird and Alferman, supra, 1980).
) or can be applied to those derived from living organisms for the direct production of biological chemicals. According to the method of the invention, the cells are irradiated (before being used for biotransformation or production) with a dose that actually arrests further cell division. Each dose for Mentha and Nicotiana cells is 5 Q rad, but other doses are applicable for specific cell cultures.

Gamma irradiation is cobalt 601G, 8.150A.

A cobalt-60 source such as an Atomic Energy Canada device is used. X-ray irradiation is performed using an X-ray generating X-ray device. The former source is recommended as it is less time consuming.

Following irradiation, the cells are washed once with a suitable medium and maintained in shaking culture for an extended period of time (usually 18-24 hours) before being washed again and then produced by conventional methods (e.g. Chabourg, supra, 1982). exposure to biotransformation precursors for

Irradiated IB cells are cultured during bioconversion or production processes with freely suspended cells or embedded and immobilized cells by established methods (e.g., Proprius and Mosbach, supra, 1982; Garn et al. , Planta Mezoica, Lord 2.9-13.1983). Although batch methods or shake culture techniques are commonly practiced, the method of the present invention can be applied to cells in column reactors or continuous flow reactors.

In batch culture techniques, the medium can be separated from the cells after culture;
Cells are washed and maintained in nutrient medium for at least 6 hours.

Additional manufacturing cycles can then be applied and this operation repeated several times with or without marginal degradation of bioconversion and/or with the productivity of the irradiated cells. In single, multiple or continuous cultures, the liquid medium is separated from the cells by means conventional in the art (usually centrifugation). The products are extracted from the liquid medium.

The following specific example demonstrates that mitotically arrested cells retain their biochemical transformation potential. The prone position is for illustrative purposes only and is not limiting.

Example 1 The following experiment was performed to test the ability of cytostatic menta cells to convert (-) menthone to (+) neomenthol. Mentaline 193 cells were maintained in a shaking culture in modified B5 medium (Aviv et al., supra, 198]) and transferred weekly to fresh medium. These are stock cultures. All steps up to product extraction were performed under aseptic conditions. For gamma irradiation, cells were harvested from a stock fluid, washed once in B5, resuspended in 85 to 50% back volume density and transferred to a 500 d Erlenmeyer flask. This flask is cobalt 60
．． G, 8.150A Atomic Energy Canada Equipment (3kr
ad/min) to 5 Q rad gamma rays,
It was then resuspended in B5 medium.

To evaluate the mitotic effect of irradiated cell samples of 11d were incubated with solidified (1,0% agar) B5 medium containing 9c.
m plastic vetri dishes.

In parallel, non-irradiated cells were plated in the same way (non-irradiated control). This plate was cultured at 25±2°C (
in the dark) or in a cool place (2-4°C as an uncultured control).
). After 12 days, plate 1IIIll! The growth of was evaluated. Each of the four treatment groups (culture with irradiation, culture without irradiation, irradiation with cold storage, cold storage without irradiation) consists of four plates. Among the four treatments, only the non-irradiated cultured cells divided and overflowed onto the surface of the plate (Figure 1), indicating the effect of cell division due to gamma irradiation.

To assess the transforming potential of mitotically arrested Mensa cells, each irradiated cell obtained by the method described above was transferred to a 250d Erlenmeyer flask and 100 ml of B5 liquid medium at 30% filling volume density. (total volume) and 100 μ
/at! Methanolic menthone solution 20j! added.

In parallel (control), non-irradiated cells were similarly suspended in menthone. The suspension was kept on a rotary shaker (120 rpm) at 25 ± 2°C, and samples were taken periodically to determine the monoterpene composition by standard gas-liquid chromatography methods (Aviv et al., supra, 1981; Garn et al., supra, 1983). Tested on things. The rate of conversion of menthone to neomenthol shown in Figure 2 clearly demonstrates that gamma-irradiated menta cells (If cell division has ceased as shown in Figure 1) maintain their conversion potential. Illuminated.

Example 2 The following experiment was performed to test whether biotransformation capacity was maintained in immobilized, mitotically arrested cells.

The same Mensa cell stock as in Example 1 was used. Conversion of menthone to neomenthol served to demonstrate bioconversion potential. Free suspension a with or without gamma irradiation
The transformation system with M was as in Example 1, but 5Rg of menthone was added to each flask. Although the immobilization method was slightly modified, it followed the criteria of Garn et al. (1983). That is, a stock of Mensa line 193 was cultured to 30% filling capacity. The cells were then filtered through a [Nalji J 0.45 um filter, washed once with 85 medium on the same filter, and
5 g of filter dried cells were used for immobilization with polymer. 1.5 g of linear polyacrylamide hydrazide (PAAH) was suspended in 50% water resistance, heat sterilized and heated at 20°C.
It was cooled to Cold sterile PAAH eggplant suspension was mixed with filter-dried cells on ice and stoichiometric values (approximately 4d, 5% solution) of glyoxal were added with vigorous stirring to prepare PAAH.
The AH was cross-linked and the cells were embedded. Leave the gel to solidify (15 minutes) and cut into cubes of approximately 0.3 cm2.
It was further solidified. Gel in standard syringe (without needle)
Spread it to make beads, wash it once with 85 medium,
250Il+J! Suspended in a total volume of 100 ae in Erlenmeyer. Finally, 5 μm of menthone (from the ethanol solution) was added and the flask was shaken at 25°C for 1 mm (1
Cultured at >2 Orpm. The same operation was performed on the gamma-irradiated cells obtained in Example 1.

Each of the 4 treatments; (1) Jiaoyu gA/free suspension; (2) Teru9
A/Free suspension; (3) Heat irradiation/PAAH embedding; (4) Irradiation/PAAH embedding - were performed twice. Samples were taken periodically and tested for monoterpene composition as in Example 1.

The results are shown in Figure 3. Multiple results showed the same result (3rd
Results for gamma-irradiated/free suspension cells in the figure are shown separately for each of the two flasks). Embedded or freely suspended gamma-irradiated cells maintained their biotransformation potential, complementing and extending the results of Example 1.

Example 3 To test whether mitotically arrested cells can survive repeated bioconversions, the following experiment was performed. Cell suspensions were made and gamma-irradiated as in Example 1. Four flasks of cell suspension were cultured once with menthone for 18 hours.

Two flasks were then taken and tested for monoterpene composition (1 day total extraction), and the cells in the other two flasks were separated from the medium by centrifugation.

Media was analyzed for monoterpenes (+1 day media extraction) and cells were resuspended in B5 media for 30 hours.

Menthone (5η/flask) was then added to the suspended cells,
Cells were cultured again for 18 hours. The cells should be isolated from the culture medium as described above and tested for monoterpene composition.
3 times medium extraction). Cells were resuspended in 85 medium for 30 hours. Menthone (5 μ/flask) was added to the resuspended cells;
Two flasks were incubated in triplicate for 18 hours. The medium was separated and the monoterpene composition of the medium was analyzed as before (+5
day culture medium extraction).

Additionally, two flasks containing gamma-irradiated cells were
It was kept on a rotary shaker for 6 days at °C. 5η menthone was then added and the flasks were incubated for 18 hours. The contents of the flask were analyzed for noterben composition as in Example 1.
6 days total extraction). Gas-liquid chromatography analysis is shown in FIG. The results show that gamma-irradiated cells are capable of multiple bioconversions and that such cells can be maintained in shaking culture for 6 days without compromising their bioconversion potential.

Example 4 To test whether the retention of bioconversion capacity by mitotically arrested cells is restricted to neomenthol conversion of mentha cells and menthone, or whether the bioconversion capacity of mitotic cells is a general phenomenon, converting geraniol. Bioconversion to products followed gamma-irradiated Mensa and Nicotiana cells.

Erlenmeyer flask (250
d) was prepared as in Example 1. A flask containing 100 m of gamma-irradiated Nicotiana sylvestris line Sl-1 cell suspension was also prepared. Furthermore, Mensa Line 193 and F. Silvestris Line S
Flasks containing H gamma-irradiated and PAAH-embedded cells were also prepared.

Each of the four gamma-irradiated cell suspensions: (
1) Freely suspended Mentha [21P A A H-Embedded Mentha (3) Freely suspended Nicotiana (41P
AAH-embedded Nicotiana was processed as follows.

10 #I9 geraniol/flask was added and the flask was incubated on a rotary shaker at 25° C. for 6 hours (Mensa cells) or 13 hours (Cochia ± cells). The medium was removed and analyzed for monoterpenes (as in Example 3). Cells are 250ai! 1001+ in flask! i! Resuspend in B5 medium and shake as above for 18 hours (Mentha) or 9 hours (Nicotiana). 10#Ig geraniol was then added, a second transformation was performed as above, and the medium was analyzed for monoterpenes as above. This method was repeated once (Mensa) or twice (Nicotiana) and subjected to a total of three or four consecutive biotransformations with Mensa or Nicotiana cells. Gas-liquid chromatographs are shown in Figures 5 and 6 for gamma-irradiated Mensa and Nicotiana cells, respectively.

Mensa and Nicotiana gamma-irradiated cells were able to perform multiple bioconversions of geraniol with little or no loss of conversion capacity. Only free-suspending/gamma-irradiated Mensa cells lost some of this conversion capacity after three rounds of biotransformation. This conversion power is due to the gamma irradiation of Mensa and Nicotiana.
P, AA) Well retained by the embedded cells.

Example 5 In order to test whether the retention of biotransformation capacity is maintained by microorganisms whose cell division has been arrested by gamma irradiation, this method was applied to Saccharomyces relevisiae. 6911 cells of strain Y-567 were exposed to gamma rays (40
0 krad) to induce 99.5% mitotic arrest, and then dried per d of 10% glucose-containing medium [IWi
It was suspended at a density of 4. Non-irradiated yeast cells were suspended as above and served as a control. The suspension was maintained at 30°C as a shaking culture. Samples were taken periodically and analyzed for glucose and ethanol during the culture. The mitosis-arrested yeast cells maintained the ability to ferment glucose to ethanol (Fig. 7), indicating that the initial fermentation was faster than the control (non-irradiated) yeast cells.

Example 6 To test whether retention of bioconversion potential was maintained by bacteria whose cell division was arrested by gamma irradiation, it was tested with Mycobacterium sp. NRRL3805. This mycobacterium system saturates the Δ1 bond of 1,4-androstadiene-3,17-dione (ADD). 50 ■ fresh type A (8,4 ~ dry heavy) bacteria were (1) not irradiated (control), (21
Irradiation was performed with 50 krad gamma rays. This bacteria is 0.05
M phosphate buffer, pH 7.0 and 0.1 mL
Suspended with ADD. This suspension at 30℃
The cells were cultured in a shaker (130r11m). The product (AD) was sampled for reverse phase 1'' PLC analysis.

The results obtained, expressed in terms of the product ulyl, are as follows.

No irradiation 5.95 11.57 16.2B
20.86 25.73 gamma light tJA5.92
10.86 17.26 22.94 24.57 The calculated slopes for non-irradiated and gamma-irradiated bacteria are 5.09 and 5.15, respectively, and 100% and 101. Means a total efficiency of 2%. Therefore, bacteria whose cell division was arrested by gamma irradiation fully retained their sdelloid conversion ability.

Example 7 To test the ability of radiation-induced cytostatic mammalian cells to convert specific metabolites, the macrophage-like murine system P388
D1 cells were plated at approximately 4 x 105 cells/plate. After 4 days of growth, a group of plates was exposed to gamma light 11FJ.
(15 krad) L, cell division arrested. The white plate group (control) was not irradiated. The medium was changed on each plate, and arachidone'fa (3 uM)
added. After 4 hours, the medium was removed from each plate, and the concentration of staglandin-E211 in the medium was measured by radioimmunoassay. In the gamma-irradiated plate and the non-irradiated (control) plate, the prostaglandin concentration was 1250+)! It was found that II/d±150. This indicates that arrest of cell division by irradiation did not reduce this biotransformation in mammalian cell lines.

[Brief explanation of the drawing]

Figure 1: Effect of gamma irradiation on cell division of Mensa cells (
50 krad). Non-irradiation (control)
Cells and irradiated cells were plated on solid nutrient medium and stored in the cold (no culture) or cultured at 25°C for 12 days. Figure 2: Non-irradiated and gamma-irradiated Mensa line 1
93 shows the bioconversion of menthone to neomenthol by 93 cells. Figure 3 Free suspension and PAAH with or without gamma irradiation
Bioconversion of menthone by embedded Mensa cells is shown. Figure 4: Multiple biotransformations of mentone by gamma-irradiated Mensa cells. FIG. 5 shows multiple biotransformations of geraniol by gamma-irradiated Mensa cells. FIG. 6 shows multiple biotransformations of geraniol by Nicodiana cells irradiated with two gamma rays. Figure 7 = all cell division 4 Q Q krad gamma
The dotted line shows the conversion of glucose to ethanol by S. cerevisiae with or without irradiation (black dotted line) or not (control).

Claims (6)

[Claims] (1) In the method of bioconverting a precursor into a target compound, the method described above is characterized in that the precursor is brought into contact with a cell division-arrested cell that retains bioconversion ability. (2) The method according to claim 1, which uses cells that have been irradiated with electromagnetic radiation or particle radiation to undergo cell division arrest without impairing their bioconversion ability. (3) The method according to claim 1 or 2, wherein the cells are embedded in a polymer matrix or bonded to a solid substance. (4) The method according to any one of claims 1 to 3, wherein the cells are selected from plants, yeast, bacteria, and vertebrates. (5) A method according to any one of claims 1 to 4, wherein a particular precursor is converted into a compound that is a flavor, a pharmaceutically active compound or a perfume. (6) The method according to any one of claims 1 to 5, wherein the cells are irradiated in suspension, embedded in a polymer matrix or bound to a solid support.