Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-08-09
2003-09-16
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S041000, C435S468000, C435S419000, C800S295000, C800S298000
Reexamination Certificate
active
06620601
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for transforming plants, to transformed plants obtained by this method, to a process for preparing polyesters characterized by culturing or cultivating the transformed plants and subsequently collecting the polyesters from the cultured or cultivated plants.
BACKGROUND OF THE INVENTION
It is known that in eukaryotes the gene expression is controlled by promoter and terminator which exist every distinct gene and that poly A sequence is added to 3′-terminus of MRNA generated by transcription. Because the gene expression occurs within nucleus, any try to produce a plant with new trait using transformation methods for plants has been carried out predominantly by methods comprising introducing a gene into the nucleus chromosome.
However, genes of eukaryotes are monocistronic; that is, one gene is controlled by a single promoter. In prokaryotic cells which have the polycistronic expression mechanism where multiple genes are controlled by only one promoter, when a prokaryotic gene is transferred to the nucleus chromosome of a eukaryote then it is necessary to ligate a promoter to each gene. This procedure is thus complicated.
By the way, polyesters, such as poly-3-hydroxyalkanoic acids, that are biosynthesized by microorganisms have thermoplastic properties and are wide variety of biodegradable plastics from rigid ones to rubber-like ones having viscoelasticity.
Recently, the binary copolymer polyester P(3HB-co-3HH) of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HH) as well as its production methods have been studied and developed (e.g., see JP-A-5-93049 and JP-A-7-265065). The methods for the preparation of the copolymer P(3HB-co-3HH) as disclosed therein are based on its production from oleic acid or olive oil by fermentation using
Aeromonas caviae
isolated from soil. The copolymer, which is produced by fermentation process for the purpose of energy storage, becomes a material of flexible polymers because the crystallinity of the copolymer is decreased as the unit fraction of 3HH is increased. Hence, it has good thermostability and molding properties and can be processed into strong yarn and flexible films (Y. Doi, S. Kitamura, and H. Abe, Macromolecules 28, 4822-4823 (1995)). Additionally, because polyesters produced by microorganisms are biodegradable, they are useful in view of the protection of environment. Thus polyesters, which are produced and accumulated in microorganisms, may have wide variety of applications.
However, when polyesters are produced in microorganisms then certain installations including culture apparatus and medium would be needed. Further, when the installations are run, production costs are raised since the petroleum energy is consumed. Under these circumstances, there are demands for development of means of preparing polyesters, which have the above-mentioned properties, economically in large amounts without use of microbial culture means.
The object of the present invention is to provide a method for transformation of plants, transformed plants obtained by this method, and a process for preparing a polyester characterized by culturing or cultivating the transformed plant and subsequently collecting the polyester from the cultured or cultivated plant.
SUMMARY OF THE INVENTION
The present inventor studied intensively to solve the above-described problems. As a result, the inventor succeeded in obtaining a polyester by integrating an operon that has multiple genes including polyester synthase gene into the plastid of a plant to transform the plant in which polycistronic gene expression is possible even in eukarote like plant, culturing or cultivating the obtained transformed plant, and collecting the polyester from the cultured or cultivated plant, whereby the present invention was accomplished.
The present invention provides a method for the transformation of a plant wherein the method comprises ligating an operon to a vector, the operon containing a promoter and 2-100 genes of interest, and integrating the resulting recombinant vector into the plastid chromosome of a plant. The present invention also provides a transformed plant in which the said operon is integrated into the plastid chromosome. In the invention, for example, the said 2 genes of interest are a polyester synthase gene and a gene different from the polyester synthase gene; and the said 3 genes of interest are a polyester synthase gene, &bgr;-ketothiolase gene, and acetoacetyl-CoA reductase gene.
The present invention further provides a process for preparing a polyester wherein the process comprises integrating a recombinant vector to which an operon containing a promoter, a polyester synthase gene and 1-100 genes different from the polyester synthase gene is ligated, into a plastid chromosome of a plant to transform the plant, culturing or cultivating the obtained transformed plant, and collecting the polyester from the cultured or cultivated plant.
Example of the polyester synthase gene is a poly-3-hydroxybutyrate synthase gene. As the plant, exemplified are transformed plants belonging to any family selected from the group consisting of Solanaceae (e.g.
Nicotiana tabacum
), Gramineae, Malvaceae, Brassicaceae, Compositae, Pedaliaceae, Oleaceae, Myrtaceae, Rosaceae, Theaceae, Leguminosae, palmae, Sterculiaceae, and Rubiaceae.
Example of the polyester includes a copolymer of 3-hydroxyalkanoic acid represented by the following formula I:
wherein R represents a hydrogen atom or a C
1-4
alkyl group.
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Svab et al (1993) Proc. Natl. Aci. USA 90:913-917.*
Itaya (1999) J. Bacteriolgy 181:2045-1048.*
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Toshiaki Fukui et al., “Cloning and Analysis of the Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) Biosynthesis Genes ofAeromonas caviae.”, Journal of Bacteriology, vol. 179, No. 15, pp. 4821-4830, Aug. 1997, Polymer Chemistry Laboratory, Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako-shi, Saitama 351-01, Japan, XP-002113257.
Henry E. Valentin et al., “PHA production, from bacteria to plants.”, International Journal of Biological Macromolecules., vol. 25, No. 1-03, pp. 303-306, (1999), Monsanto Company, Agricultural Sector, 700 Chesterfield parkway North, St. Louis, Missouri 63198, XP-000892982.
Doi Yoshiharu
Nakashita Hideo
Yamaguchi Isamu
Yoshioka Keiko
Foley & Lardner
Katcheves Konstantina
Ketter James
Riken
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