Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
1999-12-14
2001-12-04
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S252100, C435S173100, C435S173800, C210S635000
Reexamination Certificate
active
06326203
ABSTRACT:
SUMMARY OF THE INVENTION
(1) Field of the Invention
The present invention relates to a process for growing cells, particularly to produce chemicals either intracellularly or extracellularly, in a magnetofluidized bed bioreactor apparatus using aphrons in the culture medium to provide oxygen or other necessary gases to the cells. The apparatus preferably uses a magnetic valve to allow sequential removal of a portion of bioparticles from a column after a pre-selected residence time in the bioreactor.
(2) Description of Related Art
Many secondary metabolites of living cells (procaryotes and eucaryotes) have value as pharmaceuticals, enzymatic catalysts, food colors, flavors and fragrances. This can include recombinant DNA containing cells which produce metabolites.
Plant pharmaceuticals include for instance: taxol, genistein, diadzein, codeine, morphine, quinine, shikonin, ajmalacine and serpentine. Food product examples are anthocyanins, saffron, vanilla, and a wide variety of other fruit and vegetable flavors and texture modifying agents.
Bacteria are known to naturally produce a wide range of industrially important products. Examples include alcohols, such as ethanol; organic acids, such as lactic acid; and amino acids, such as phenylalanine (Crueger, W. and A. Crueger, Biotechnology: A text book of Industrial Microbiology 1-275 (1984)). The range of products can be widely expanded through the use of recombinant-DNA techniques. A rapidly increasing spectrum of high-value pharmaceuticals can be produced by bacteria by transferring DNA coding for those products from eucaryotic cells into bacteria (A. Prokop, R. Bajpai, and C. Ho, Recombinant DNA Technology and Applications, McGraw Hill, N.Y. (1991)). For instance, human insulin is now commercially produced by recombinant
E. coli
in large-scale fermentations.
Production of biochemicals from higher plants is attractive where compounds do not naturally occur in microbial or mammalian systems, or where compounds are produced by polygenic (multiple gene) processes not amenable to gene transfer (Vieth, W., Gene expression with plant cells. In Bioprocess engineering: kinetics, mass transport, reactors and gene expression. John Wiley & Sons: N.Y. 265-324 (1994)). The exploitation of plant production of biochemicals has been largely limited, however, to whole plant systems. While at least 30 compounds are known to accumulate in plant cell culture at concentrations equal or greater than in whole plants, literature reports of commercial production are limited to shikonin from
Lithospermum erythrorhizon
and taxol and other products from Taxus (Phyton Gesellschaft für Biotechnik mbH) (Dörnenburg, H., et al., Enzyme and Microbial Technology 17:674-684 (1995)). Plant cell culture offers several potential advantages over harvest and extraction of whole plants. Continuous production, rather than seasonal, is possible in a bioreactor where conditions can be controlled for optimal product formation. Plant cell culture can provide a high quality, uniform product while reducing labor costs (Shuler, M., et al., Bioreactor considerations for producing flavors and pigments from plant tissue culture. In Biotechnology and food process engineering; Scwartzberg, H., Rao, M., Eds; Marcel Dekker: N.Y. pp. 45-65 (1990
3
)) and eliminating the effects of weather and disease.
Industrial application of plant cell culture has been limited by a number of drawbacks including slow growth rates, low product yields, intracellular storage of product, and poorly understood metabolic regulation. Perhaps the largest roadblock to industrial application of plant cell culture is low productivity per volume (Ten Hoopen, H., et al., Possibilities, problems and pitfalls of large-scale plant cell cultures. In Progress in plant cellular and molecular biology; Nijkamp, H.; Van der Plas, L.; Van Aartrijk, J. Eds; Kluwer Academic publishers: Boston, 673-681 (1990)). Production of shikonin is an example where this has been overcome however. High-yield cell lines obtained by repeated selection had a shikonin content 25 times that of the original strain (Sahai, O., et al., Biotechnology Progress 1:1-9 (1985)). In addition to isolation of high yield cell lines, genetic manipulation may enable dramatic increases in product yields. Progress in understanding of biochemical pathways and genetic engineering of plants has been reported for several systems (Dörnenburg, H., et al., Enzyme and Microbial Technology 17:674-684 (1995); Christou, P., Euphytica 74:165-185 (1994); Vieth, W., Gene expression with plant cells. In Bioprocess engineering: kinetics, mass transport, reactors and gene expression. John Wiley & Sons: N.Y. 265-324 (1994); Jende-Strid, B., Hereditas 119:187-204 (1993); Yun, D., Proc. Natl. Acad. Sci. USA 89:11799-11803 (1992); Waugh, R., et al., Plant Genetic Engineering, 1-37 (1991); Mol, J., et al., Use of genetic engineering to improve yields in cell cultures, e.g. (anti)sense DNA technology. In Progress in plant cellular and molecular biology; Nijkamp, H.; Van der Plas, L.; Van Aartrijk, J. Eds; Kluwer Academic publishers: Boston 712-716 (1990); Uchimiya, H., et al., Journal of Biotechnology 12:1-20 (1989)). Application of this knowledge base is likely to create new opportunities for production of plant cell biochemicals. As metabolic barriers to commercially interesting yields are overcome, other barriers to plant cell culture should simultaneously be addressed such as bioreactor design. Experiments need to be performed at an early stage in the type of reactor to be used at large scale, as growth and production rates may change completely with reactor type (Ten Hoopen, H. et al., Possibilities, problems, and pitfalls of large-scale plant cell cultures. In Progress in plant cellular and molecular biology; Nijkamp, H.; Van der Plas, L.; Van Aartrijk, J. Eds; Kluwer Academic publishers: Boston 673-681 (1990)).
Several reactor schemes are available for plant cell cultures, including stirred tanks, bubble columns, air-lift reactors, hollow-fiber membranes, liquid-dispersed trickle and incline reactors, and mist bioreactors for hairy root culture (Gulik, W., Biotechnology progress 10:335-339 (1994); Reinhard, E., et al., Biotechnology and Bioengineering 34:502-508 (1989); Panda, A., et al., Enzyme Microb. Technol. 11:386-397 (1989); McKelvey, S. et al., Biotechnol. Prog. 9:317-322 (1993)). However, each of these reactors has limitations, such as cell death from shear, insufficient mixing at high cell densities, settling of cells, and difficulty in scale-up (Panda, A., et al., Enzyme Microb. Technol. 11:386-397 (1989)). Furthermore, many of these reactors are poorly suited for a continuous operation in which the product is accumulated intracellularly.
Immobilization offers several advantages for using plant cells. Cell densities may reach 110 g/L in alginate beads, while suspension culture is limited to about 30 g/L (Sahai, O., et al., Biotechnology Progress 1:1-9 (1985)). The close proximity of immobilized cells allows intercellular communication and transport that can be important for differentiation and secondary-metabolite production (Shuler et al., 1990). Attachment and plugging caused by free cells are eliminated by immobilization (Sahai, O., Biotechnology Progress 1:1-9 (1985)).
Fluidized bed bioreactors are well suited for immobilized cell systems. Low shear forces and pressure drops prevent damage to fragile cells. Excellent contact between the gas, liquid, and solid phases eliminates the need for mechanical mixing.
An important disadvantage of conventional immobilized cell systems, however, is that plant secondary metabolites are typically stored intracellularly (Brodelius, P., Transport and accumulation of secondary metabolites. In Progress in plant cellular and molecular biology; Nijkamp, H.; Van der Plas, L.; Van Aartrijk, J. Eds; Kluwer Academic Publishers: Boston, 567-576 (1990)). Soybean isoflavonoids, as an example, exist predominantly as glucoside-malonylated conjugates (Graham, T., et al., Mol. Plant-Microbe Interact. 3:157-166 (1990); Kudou, S., et al., Ag
Ames Tyler T.
Worden Robert Mark
Afremova Vera
Board of Trustees operating Michigan State University
McLeod Ian C.
Saucier Sandra E.
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