Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-03-03
2002-09-10
Horlick, Kenneth R. (Department: 1656)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S287000, C800S298000, C435S006120, C435S183000, C435S320100, C435S419000
Reexamination Certificate
active
06448473
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the construction and use of multigene expression vectors useful to enhance production of materials by multienzyme pathways. In particular, the construction and use of multigene vectors encoding proteins in the polyhydroxyalkanoate biosynthetic pathway is disclosed.
BACKGROUND OF THE INVENTION
Metabolic engineering is a process by which the normal metabolism of an organism is altered to change the concentration of normal metabolites, or to create novel metabolites. This process often involves introduction or alteration of numerous enzymatic steps, and thus often requires introduction of multiple genes. An efficient system for introducing and expressing multiple genes is therefore desirable. In prokaryotes such as
Escherichia coli,
introduction of multiple genes is relatively straightforward in that operons can be constructed to express multiple open reading frames, or multiple complete genes can be expressed from a single plasmid. However, introduction of pathways into plants is more difficult due in part to the complexity of plant genes, the difficulty of constructing vectors harboring multiple genes for expression in plants, and the difficulty of introducing large vectors intact into plants.
Polyhydroxyalkanoates are bacterial polyesters that accumulate in a wide variety of bacteria. These polymers have properties ranging from stiff and brittle plastics to rubber-like materials, and are biodegradable. Because of these properties, polyhydroxyalkanoates are an attractive source of non-polluting plastics and elastomers.
Currently, there are approximately a dozen biodegradable plastics in commercial use that possess properties suitable for producing a number of specialty and commodity products (Lindsay,
Modern Plastics
2: 62, 1992). One such biodegradable plastic in the polyhydroxyalkanoate (PHA) family that is commercially important is Biopol™, a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). This bioplastic is used to produce biodegradable molded material (e.g., bottles), films, coatings, and in drug release applications. Biopol™ is produced via a fermentation process employing the bacterium
Ralstonia eutropha
(Byrom, D.
Trends Biotechnol.
5: 246-250, 1987). (
R. eutropha
was formerly designated
Alcaligenes eutrophus
[Yabuuchi et al.,
Microbiol. Immunol.
39:897-904, 1995]). The current market price is $6-7/lb, and the annual production is 1,000 tons. By best estimates, this price can be reduced only about 2-fold via fermentation (Poirier, Y. et al.,
Bio/Technology
13: 142, 1995). Competitive synthetic plastics such as polypropylene and polyethylene cost about 35-45¢/lb (Layman,
Chem. & Eng News, p.
10 (Oct. 31, 1994). The annual global demand for polyethylene alone is about 37 million metric tons (Poirier, Y. et al.,
Int. J. Biol. Macromol.
17: 7-12, 1995). It is therefore likely that the cost of producing P(31HB-co-3HV) by microbial fermentation will restrict its use to low-volume specialty applications.
Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids (Anderson. A. J. and Dawes, E. A.
Microbiol. Rev.
54: 450-472, 1990; Steinbüchel, A. in
Novel Biomaterials from Biological Sources,
ed. Byrom, D. (MacMillan, New York), pp. 123-213, 1991); Poirier, Y., Nawrath, C. & Somerville, C.
Bio/Technology
13: 143-150, 1995). Polyhydroxybutyrate (PHB) is the most well-characterized PHA. High molecular weight PHB is found as intracellular inclusions in a wide variety of bacteria (Steinbüchel, A. in
Novel Biomaterials from Biological Sources,
ed. Byrom, D.: (MacMillan, New York), pp. 123-213, 1991). In
Ralstonia eutropha,
PHB typically accumulates to 80% dry weight with inclusions being typically 0.2-1 &mgr;m in diameter. Small quantity of PHB oligomers of approximately 150 monomer units are also found associated with membranes of bacteria and eukaryotes, where they form channels permeable to calcium (Reusch, R. N.,
Can. J. Microbiol.
41 (Suppl. 1): 50-54, 1995). High molecular weight polyhydroxyalkanoates have the properties of thermoplastics and elastomers. Numerous bacteria and fungi can hydrolyze polyhydroxyalkanoates to monomers and oligomers, which are metabolized as a carbon source. Polyhydroxyalkanoates have accordingly attracted attention as a potential source of renewable arid biodegradable plastics and elastomers. PHB is a highly crystalline polymer with rather poor physical properties, being relatively stiff and brittle (de Koning, G.,
Can. J. Microbiol.
41 (Suppl. 1): 303-309, 1995). In contrast, PHA copolymers containing monomer units ranging from 3 to 5 carbons for short-chain-length PHA (SCL-PHA), or 6 to 1,4 carbons for medium-chain-length PHA (MCL-PHA), are less crystalline and more flexible polymers (de Koning, G.,
Can. J. Microbiol.
41 (Suppl. 1): 303-309, 1995).
PHB has been produced in the plant
Arabidopsis thaliana
expressing the
R. eutropha
PHB biosynthetic enzymes (Poirier, Y. et al.,
Science
256: 520-523, 1992; Nawrath, C., et al.,
Proc. Natl. Acad. Sci. U.S.A.
91: 12760-12764, 1994). In plants expressing the. PHB pathway in the plastids, leaves accumulated up to 14% PHB per gram dry weight (Nawrath, C., et al.,
Proc. Natl. Acad Sci. U.S.A.
91: 12760-12764, 1994). High-level synthesis of PHB in plants opened the possibility of utilizing agricultural crops as a suitable system for the production of polyhydroxyalkanoates on a large scale and at low cost (Poirier, Y. et al.,
Bio/Technology
13: 143-150, 1995; Poirier, Y. et al.,
FEMS Microbiol. Rev.
103: 237-246, 1992; Nawrath, C., et al.
Molecular Breeding
1: 105-22, 1995). PHB was also shown to be synthesized in insect cells expressing a mutant fatty acid synthase (Williams, M. D., et al.,
Appl. Environ. Microbiol.
62: 2540-2546, 1996), and in yeast expressing the
R. eutropha
PHB synthase (Leaf, T. A., et al.
Microbiol.
142: 1169-1180, 1996).
A number of pseudomonads, including
Pseudomonas putida
and
Pseudomonas aeruginosa,
accumulate MCL-PHAs when cells are grown on alkanoic acids (Anderson, A. J. & Dawes, E. A.
Microbiol. Rev.
54: 450-472, 1990; Steinbüchel, A. in
Novel Biomaterials from Biological Sources,
ed. Byrom, D. (MacMillan, New York), pp. 123-213, 1991; Poirier, Y., Nawrath, C. & Somerville, C.
Bio/Technology
13: 143-150, 1995). The nature of the PHA produced is related to the substrate used for growth and is typically composed of monomers which are 2n carbons shorter than the substrate. These studies indicate that MCL-PHAs are synthesized by the PHA synthase from 3-hydroxyacyl-CoA intermediates generated by the &bgr;-oxidation of alkanoic acids (Huijberts, G. N. M., et al.
Appl. Environ. Microbiol.
58: 536-544, 1992; Huijberts, G. N. M., et al.,
J. Bacteriol.
176: 1661-1666, 1994).
Chen et al. (
Nature Biotech.,
16: 1060-1064, 1998; reviewed by Gelvin, S. B.,
Nature Biotech.,
16: 1009-1010, 1998) describes the cobombardment of embryogenic rice tissues with a mixture of 14 different pUC based plasmids. Integration of multiple transgenes was observed to occur at one or two genetic loci.
Creating a transgenic host cell or plant that produces multiple enzymes within a biosynthetic pathway is often a daunting task. Individual vectors must be created for each enzyme. Transformation of the host cell or plant is typically accomplished by one of three general methods: serial transformation, parallel transformation followed by crossing, or batch transformation. Each method has serious practical drawbacks.
Serial transformation involves transforming a host cell or plant with the first vector, selecting and characterizing the transformed cell or plant, transforming with the second vector, and so on. This process can become quite laborious and time consuming.
Parallel transformation followed by crossing involves separately transforming cells with each of the individual vectors, and subsequently mating or crossbreeding the transformed cells or plants to obtain a final cell or plant which contains all of the in
Hao Ming
Houmiel Kathryn L.
Mitsky Timothy A.
Reiser Steven E.
Slater Steven C.
Bond Gary M.
Horlick Kenneth R.
Howrey Simon Arnold & White , LLP
Monsanto Technology LLC
Strzelecka Teresa
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