GB2175312A - Bioconversion - Google Patents

Abstract

Process for the bioconversion of specific chemicals by means of division arrested cells. Such cells can be provided in suspension, entrapped in a suitable matrix or attached to a certain substrate. Cell-division arrest is attained by controlled irradiation which is suited to substantially arrest cell division, yet which does not impair to any appreciable extent the bioconversion capability of such cells. The cells used can be plant cells, yeasts, bacteria or any other cell type suitable for the desired bioconversion process.

Description

SPECIFICATION Bioconversion The invention relates to a process for the bioconversion of certain precursor chemicals to desired products by means of division-arrested cells. Cell division is arrested by irradiation of such cells by means of X-rnys, gamma-rays or other radiation adapted to essentially arrest cell division. The dosage of radiation is chosen in such a manner that cell division is arrested, yet the capability of the cells to effect bioconversions is essentially not impaired. This is contrary to the expectations of the art, as is was generally accepted that when cell division-arrest is obtained by an electromagnetic radiation, this results also in a substantial decrease, or even cessation of ability of the cells to bring about bioconversions.

There may be used a wide variety of cells: plant cells, bacteria, yeast cells, mammalian cells etc. The cells can be used in suspension or they may be entrapped in a suitable matrix or attached to a suitable support.

They can be entrapped in polymer beads. It has been established that the process of the invention is suitable for industrial scope conversions. The division arrested cells can be used over prolonged periods of time in either batch processes or in continuous conversion processes.

The production of organic compounds by living cells is as old as human history. It is exemplified by fermentation of sugar to ethanol and the baking of bread. More specifically, in vitro cultured plant cells were used abundantly for the production of valuable organic compounds such as flavours, pharmaceuticals and scents (e.g. Staba, E.J. Devel. Micro 4, 192-198(1963); Kurz et al., Adv. Appl. Microbiol. 209-240 (1979)).

The production of such compounds by cultured cells can be performed by either of two main technologies: without feeding of a specific precursor and with the addition, to the cultured cells, of specific precursors which are biotransformed into valuable products (Reinhard et al., Adv. Biochem. Eng. 16, 50-83(1980); Shargool, P.D., Appl. Biochem. Biotech. 7(1982)). The industrial use of plant-cells and other cells by either of the above technologies was hampered by several difficulties; among these are the following: (1) Freely-suspended cells are inferior to immobilized cells in advanced biotechnologies (Brodelius et al., Adv. Appl.Micro. 28,1-26(1982); (2) Normally-dividing cultured cells require, among other medium components, a constant supply of expensive organic compounds to support the metabolic activity involved in cell-division, thus increasing production costs.

(3) When dividing in vitro cultured cells were immobilized in polymers (such as alginate beads), the cells tended to be released gradually from the entrapment, thus establishing a mixture of immobilized and freely-suspended cells. Such a mixture hampers efficient biotechnological application of these cells for organic-compound production.

A process which will retain the capacity of cultured cells to produce and/or transform specific organic compound, but which impairs cell-division, should overcome the above mentioned difficulties. Arresting cell division by the addition of metabolic inhibitors and/or causing starvation for certain medium components is undesirable for several reasons (inclusion of toxic compounds into the system, causing gross metabolic changes, etc.).

Cell division arrest by irradation such as X-rays, gamma-rays and other electromagnetic and particulate irradiations, is a well established phenomenon in plant, animal and microbial cells (Lea, D. E., Action of Radiation on Living Cells (second Edition), University Press, Cambridge, (1955)). No previous use has been made of irradiation-induced division-arrested cells of plants, animals or microorganisms as regards the industrial biotechnology of biotransformation and production of organic compounds. This invention furnishes a simple and generally applicable process to arrest cell division while retaining the capacity of such cells for biotransformation and production of specific organic compounds.

According to the invention there is provided a process for the industrial scale conversion of chemicals by means of cell-division arrested cells.

The cells are advantageously treated by X-ray or gamma-radiation so as to arrest cell division, while not impairing their capability to effect bioconversions.

Contrary to what has been accepted hitherto in the art, arrest of cell division by such means does not necessarily curtail substantially the bioconversion capabilities of such treated cells.

The invention is applicable to a wide variety of cells, such as plant cells, yeast cells, bacterial cells, vertebrate cells, etc. The irradiation used to arrest cell division of such cells can be of a variety of types, such as electromagnetic radiation, particulate irradiation, etc. The irradiations of choice are gamma-rays, and X-rays of varying wave lengths and intensity. In the following, the invention is illustrated with reference to plant cells, yeast, bacteria and mammalian cells. It is stressed that this is by way of illustration only, and that the invention is applicable to a wide scope of other cells.

Cells, derived by the common means of the art (Reinert et al. Plant Cell, Tissue and Organ Culture, p. 803, SpringerVerlag, Berlin, (1977)) from a plant-organ plant of Mentha sp., Nicotiana sp., other plants or other organisms are suspended in liquid medium such as B5 (Gamburg et al., Exp. Cell Res. 50, (1968)) or any similar nutrient medium suitable for the given cell type. The thus cultured cells are maintained and propagated in shake cultures or by any other means suitable to furnish growth conditions of these plant cells. The cells are periodically diluted by nutrient medium, by standard procedures which are amply documented in the pertinent literature (e.g. Reinert and Bajaj, ibid. (1977); Shargool, ibid. (1982).For utilization in the production of specific biochemicals (e.g. Staba, ibid. (1963)) orforthe biotransformation of a specific precursor to a specific product (Reinhard and Alfermann, ibid. (1980)) by the commonly used procedure, the cells are transferred into a production - or transformation medium respectively. The latter contains the precursor for the biotransformation. For the transformation of (-) menthone to (+) neomenthol by freely suspended Mentha line 193, 20 to 80 mg/liter of menthone are added to the transformation medium composed of Gamborg's B5 components and the cells are maintained as shake culture at temperatures ranging between 23 and 27"C (Aviv et al., Planta Medica, 42, 236-243 (1981)).The biotransformation techniques can be similarly applied to other cells, whether of plant origin (Reinhard and Alfermann, ibid.

(1980)) or from other organisms as well as for the direct production of biochemicals. According to the process of this invention, the cells are exposed (before use for biotransformation or production) to irradiation, at a dose which virtually arrests further cell-division. The respective dose for Mentha and Nicotiana cells is 50 Krad but other doses may apply for specific cell-cultures. The gamma-irradiation is furnished by a cobalt60 source such as a Cobalt 60, G.B. 1 50A, Atomic Energy of Canada machine and X-ray irradiation is furnished by an X-ray emitting Roentgen machine. The former source is recommended because it is much less time consuming.Following irradiation, the cells are washed once in the appropriate culture medium and maintained as shake culture for a number of hours (usually, 18 to 24 hours) before being washed again and then exposed to the precursor for biotransformation for production according to conventional techniques (e.g. Shargool, ibid. (1982)). The irradiated cells are then incubated during the biotransformation or production process either as freely suspended cells or with cells entrapped and immobilized by established procedures (e.g. Brodelius and Mosbach, ibid. (1982); Galun petal., Planta Medica 49, 9-13 (1983)). A batch or shake-culture technology is commonly used but the process of this invention can also be applied to cells in column reactors or continuous flow reactors.

In the batch-culture technology, the medium can be separated from the cells after incubation and the cells are washed and maintained for 6 or more hours in nutrient medium; thereafter an additional production cycle is applied and this procedure can be repeated several times with or without marginal-deterioration of the biotransformation and/or production capability of the irradiated cells. In either the single, multiple or continuous incubations, the liquid medium is separated from the cells by one of the common means of the art (usually by centrifugation) and the product is extracted from the liquid medium. The invention will now be illustrated by Examples with reference to the accompanying drawings in which Figure 1 shows the effect of gamma irradiation (50krad) on the cell division of Mentha cells.Non-irradiated (contr.) and irradiated cells were plated over solidified nutrient medium and either cold-stored (no-culture) or cultured at 25 C for 12 days.

Figure 2 shows kinetics of menthone to neomenthol biotransformation by non-irradiated and gammairradiated Mentha Line 193 cells.

Figure 3 shows menthone biotransformation by freely suspended and PAAH-entrapped Mentha cells which were either gamma-irradiated or non-irradiated.

Figure 4 shows multiple menthone biotransformation by gamma-irradiated Mentha cells.

Figure 5 shows multiple geraniol biotransformation by gamma-irradiated Mentha cells.

Figure 6 shows multiple geraniol biotransformation by gamma-irradiated Nicotiana cells.

Figure 7 shows kinetics of glucose-to-ethanol conversion by Saccaromyces cerevisiae cells in which cell division was arrested by 400 krad gamma-radiation (solid line) or which were non arrested (control) - broken line.

The following specific examples demonstrate that division-arrested cells retain their ability to bring about biochemical conversions. The following examples are of an illustrative, non-restrictive nature.

Example 1: To test the capability of cell-division arrested Mentha cells to transform (-) mentone to (s) neomenthol, the following experiment was performed. Mentha Line 193 cells were maintained in shake culture on modified B6 medium (Aviv et al., ibid. (1981)) and transferred weekly to fresh medium. These were the stock cultures. All operations up to extraction of products were performed under axenic conditions.For gamma-ray irradiation, the cells were harvested from stock culture, washed once in B6, resuspended in B6 to a 50% packed-volume density and transferred to 500 ml Erlenmeyer flasks. The flasks were exposed to 50 Krad gamma rays from a Cobalt 60, G.B. 1 50A Atomic Energy Canada machine (at a rate of 3 Krad per minute) and then resuspended in B6 medium. To evaluate the division-arrest effect of the irradiation, 1 ml samples of irradiated cells were plated in 9 cm plastic petri dishes over solidified (1.0% agar) B5 medium. In parallel, non-irradiated cells were similarly plated (non-irradiated control).The plates were then either incubated (in the dark) at 25 + 2"C or transferred to the cold (2-4"C) as non-cultured controls. After 12 days, the growth of the plated cells was evaluated. Each of the four groups of treatments (irradiated and cultured, non-irradiated and cultured, irradiated and cold-stored, non-irradiated and cold-stored) consisted of four plates. Of the four treatments only the non-irradiated and cultured cells divided and filled the surface of the plates (Figure 1), indicating the efficiency of cell division arrest by the gamma irradiation.

For the evaluation of the transformation capacity of division-arrested Mentha cellos, the respective irradiated cells, obtained by the process detailed above, were transferred to 250 ml Erlenmeyerflasks and suspended in 100 ml (total volume) of B6 liquid medium, at a 30% packed volume density and 201 of a 100 mg/ml ethanolic menthone solution was added. In parallel (control), non-irradiated cells were suspended with menthone in the same manner. The suspensions were then maintained on a rotation shaker (120 rpm) at 25 * 2"C and samples were withdrawn periodically and tested for monoterpene composition by the standard gas-liquid chromatography procedure (Aviv et al., ibid. (1981), Galun et al., ibid. (1983)).The kinetics of methone to neomenthol conversion presented in Figure 2, clearly demonstrated that gamma-irradiated Mentha cells (in which cell division was arrested as demonstrated in Figure 1) maintained their conversion capacity.

Example 2: To test whether or not the biotransformation capacity is maintained in division-arrested cells which are immobilized, the following experiment was performed.

The same stock of Mentha cells as in Example 1 was used. The conversion of menthone to neomenthol served to demonstrate biotransformation capability. The transformation system by freely suspended cells, either gamma-irradiated or non-irradiated was as in Example 1, but 5 mg menthone were added to each flask. The immobilization procedure was according to the protocol presented by Galun et al. ibid. (1983) with some modifications, as follows: A stock of Mentha Line 193 was cultured up to 30% packed volume. The cells were then filtered over a "Nalgi" 0.45 um filter, washed once with B6 medium over the same filter and 15 g of filter-dry cells served for immobilization with the polymer.One and one half g of a linear polyacrylamide hydrazide (PAAH) were suspended in 50 ml water, heat-sterilized and cooled to 20"C. The cooled sterile PAAH suspension was then mixed, over ice, with the filter-dried cells and a stoichiometric amount (ca. 4 ml, of a solution 5%) of glyoxal was added during vigorous mixing, to cross-link the PAAH and to cause cell-entrapment. The gel was left for hardening (15 minutes), cut into cubes of ca 0.3cm2 and left for further hardening. The gel was then extruded through a standard syringe (without the needle) to produce beads, which were washed once in B5 medium and then suspended in a total volume of 100 ml in a 250 ml Erlenmeyer.Finally 5 mg menthone (from an ethanolic solution) were added and the flasks were incubated on a rotary shaker (120 rpm) at 25"C. The same procedure was followed with gamma-irradiated cells obtained as in Example 1.

Each of the four treatments; (1) non-irradiated and freely suspended; (2) irradiated and freely suspended; (3) non-irradiated and PAAH entrapped; (4) irradiated and PAAH entrapped -was performed in duplicate.

Samples were withdrawn periodically and tested for monoterpene composition as in Example 1. The results are presented in Figure 3. Duplicates gave similar results (see gamma-irradiated and freely suspended cells in Figure 3 for which the results were presented separately for each of the two flasks, rather than as means, in the other three treatments). Gamma-irradiated cells, whether entrapped or freely suspended, obviously maintained their biotransformation capability, complementing and extending the results of Example 1.

Example 3: To test whether or not division-arrested cells can undergo repeated biotransformation, the following experiment was performed. Cell suspensions were produced and gamma-irradiated as in Example 1. Cell suspensions in 4 flasks were incubated once with menthone for 18 hours. Then 2 flasks were harvested and tested for monoterpene composition (1 day total extraction) and the cells of the two other flasks were separated by centrifugation from the media. The media were analysed for monoterpenes (+ 1 day medium extract) and the cells were resuspended in B6 medium for 30 hours. Menthone (5 mg per flask) was then added to the suspended cells and the cells were incubated again for 18 hours. The cells were then separated from the medium as above and tested for monoterpene composition (+ 3 days medium extract).The cells were resuspended in B5 medium for 30 hours. Menthone (5 mg per flask) was then added to the resuspended cells and the two flasks were incubated a third time for 18 hours. The medium was then separated and the monoterpene composition of the medium was anlaysed as before (+ 5 days medium extract).

In addition, 2 flasks containing gamma irradiated cells were maintained on a rotational shaker, at 250C for 6 days. Then 5 mg menthone were added and the flasks were incubated for 18 hours. The content of the flasks was then analyzed for monoterpene composition as in Example 1 (+ 6 days total extract). The gas liquid chromatographs of the analyses are presented in Figure 4. The results show that the gamma-irradiated cells were capable of multiple biotransformation and that such cells can be maintained as shake cultures for 6 days without impairment of their biotransformation capability.

Example 4: To test whether or not the retention of biotransformation capability by division-arrested cells is confined to Mentha cells and menthone to neomenthol conversion, or the biotransformation capability of divisionarrested cell is a general phenomenon - the biotransformation of geraniol to conversion products was followed with gamma-irradiated Mentha and Nicotiana cells.

Erlenmyerflasks (250 ml) with 100 m I g ml gamma-irradiated cell-suspensions of Mentha Line 193 were prepared as in Example 1. Such flasks were also prepared with 100 ml of a gamma irradiated Nicotiana sylvestris Line SH cell suspension. In addition, such flasks were prepared with gamma-irradiated and PAAH-entrapped cells of Mentha Line 193 and of N. sylvestris Line SH.

Each of the 4 gamma-irradiated cell-suspensions: (1) freely suspended Mentha (2) PAAH-entrapped Mentha (3) freely suspended Nicotiana (4) PAAH-entrapped Nicotiana were then treated in the following manner. Ten mg geraniol, per flask, were added and the flasks were incubated for 6 hours (Mentha cells) or for 13 hours (Nicotiana) cells on a rotary shaker at 25do. The medium was removed and analyzed for monoterpenes (as in Example 3) and the cells were resuspended in 100 ml B5 medium, in 250 ml flasks and shaken as above for 18 hours (Mentha) or 9 hours (Nicotiana). Then, 10 mg geraniol were added, a second transformation was performed as above and the medium was analysed for monoterpenes, as above. The process was repeated once (Mentha) or twice {Nicotiana), furnishing in total 3 or 4 consecutive biotransformations with Mentha or Nicotiana cells.Gas-liquid chromatographs are presented in Figures 5 and 6 for gamma-irradiated Mentha and Nicotiana cells, respectively. Gamma-irradiated cells of Mentha and Nicotiana were capable of performing multiple biotransformations of geraniol with little or no loss of conversion capability. Only freely suspended and gamma-irradiated Mentha cells lost some of this conversion capability at the third round of biotransformation. This conversion capability was fully retained by gamma-irradiated and PAAH-entrapped cells of both Mentha and Nicotiana.

Example 5: To test whether or not the retention of biotransformation capability is maintained also by microorganisms in which cell-division was arrested by gamma-irradiation the process was applied to yeast (Saccharomyces cerevisiae). Yeast cells of strain Y-567 were exposed to a gamma-ray dose (400 Krad) which causes 99.5% division-arrest and then suspended at a density of 4 mg dried cells per ml medium containing 10% glucose.

Non-irradiated yeast cells were suspended as above to serve as control. The suspensions were maintained as shake-cultures at 30"C. Aliquots were removed periodically to analyze glucose and ethanol concentrations during incubation. The division-arrested yeast cells retained their glucose-to-ethanol fermentation capability (Figure 7) showing an initial faster fermentation than control (non-irradiated) yeast cells.

Example 6: To test whether or not the retention of biotransformation capability is maintained also by bacteria in which cell-division was arrested by gamma-i rradiation, we tested Mycobacterium sp NRRL 3805. This Mycobacterium line saturates the A 1 bond of 1, 4-androstadiene 3, 1 7-dione (ADD). 50 mg fresh weight (8.4 mg dry weight) of bacteria were (1) not irradiated (control), (2) irradiated with 500 Krad gamma-rays. The bacteria were suspended in 0.05 M phosphate buffer, pH 7.0 and 0.1 mU of ADD. The suspensions were incubated on a shaker (130 rpm) at 30"C. Samples were withdrawn for reverse-phase HPLC analysis of the product (AD).

The following results expressed in uM of product, were obtained.

Time 1h 2h 3h 4h 5h Non irradiated: 5.95 11.57 16.28 20.86 25.73 Gamma-irradiated: 5.92 10.86 17.26 22.94 24.57 The calculated slope for the non-irradiated and gamma-irradiated bacteria was 5.09 and 5.15 respectively, meaning a total relative efficiency of 100% and 101.2% for non-irradiated and irradiated bacteria, respectively. Hence the bacteria in which cell division was arrested by gamma irradiated fully retained their steroid transformation capacity.

Example 7: To test the ability of irradiation-induced cell-division arrested mammalian-cells to perform bioconversion of a specific metabolite, cells of macrophage-like murine-line P388DI were plated at about 4 x 105 cells/plate.

After 4 days of growth one group of plates was gamma-irradiated (15 Krad) to arrest cell-division. Another group of plates (control) was not irradiated. The medium was changed in each of the plates and arachidonic acid (3 uM) was added. After 4 h the medium was removed from each plate and the prostaglandin-E2 concentration in the medium was determined by radioimmunoassay. In both gamma-irradiated and the non irradiated (control) plates, the prostaglandin concentration was found to be 1250 pg/ml t 150, indicating that the irradiation imposed cell-division arrest did not reduce this biotransformation in a mammalian cell-line.

Claims (13)

1. A bioconversion process for converting a precursor to a desired product which comprises contacting the precursor with cell-division arrested cells, which retain their capacity to effect bioconversion.

2. A process according to claim 1, in which the cells have been irradiated by electromagnetic or particle radiation such that cell-division is arrested without impairing the bioconversion capability of the cells.

3. A process according to claim 1 or claim 2, in which the cells are entrapped in a polymer matrix or are attached to a solid substrate.

4. A process according to any one of claims 1 to 3, in which the cells are plant cells, yeast cells, bacterial cells or vertebrate cells.

5. A process according to any one of claims 1 to 4, in which a precursor chemical is converted to a compound which is a flavour, a pharmaceutically active compound or a scent.

6. A process according to any of claims 1 to 5, comprising irradiating cells which are in suspension, which are entrapped in a polymer matrix or which are adhering to a solid support.

7. A bioconversion process for the conversion of a chemical precursorto a chemical compound, by means of cell-division arrested cells, substantially as herein before described with reference to the Examples.

8. A chemical product obtained by the bioconversion of a precursor according to a process as claimed in any of claims 1 to 7.

9. Cell-division arrested cells having the ability to effect bioconversion.

10. A process for producing cells according to claim 9 comprising irradiating cells with electromagnetic or particle radiation.

11. A process according to claim 10 comprising irradiating cells which are in suspension, which are entrapped in a polymer matrix or which are adhering to a solid support.

12. Cells according to claim 9 substantially as hereinbefore described with reference to any one the Examples.

13. A process according to claim 10 substantially as hereinbefore described with reference to any one of the Examples.