Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
2000-10-10
2004-08-10
Fox, David T. (Department: 1638)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C800S298000, C800S281000, C800S306000
Reexamination Certificate
active
06773917
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to genetically engineered plants. More particularly, the present invention relates to the optimization of substrate pools to facilitate the biosynthetic production of commercially useful polyhydroxyalkanoates (PHAs) in plants.
The present invention especially relates to the production of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), designated P(3HB-co-3HV) copolymer, and derivatives thereof.
2. Description of Related Art
Polyhydroxyalkanoates
Polyhydroxyalkanoates are 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. Due to these properties, PHAs 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, 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
Alcaligenes eutrophus
(Byrom, 1987). The current market price is $6-7/lb, and the annual production is 1,000 tons. By best estimates, this price is likely to be reduced only about 2-fold via fermentation (Poirier et al., 1995). Competitive synthetic plastics such as polypropylene and polyethylene cost about 35-45¢/lb (Layman, 1994). The annual global demand for polyethylene alone is about 37 million metric tons (Poirier et al., 1995). It is therefore likely that the cost of producing P(3HB-co-3HV) by microbial fermentation will restrict its use to low-volume specialty applications.
Nakamura et al. (1992) reported using threonine (20 g/L) as the sole carbon source for the production of P(3HB-co-3HV) copolymer in
A. eutrophus
. These workers initially suggested that the copolymer might form via the degradation of threonine by threonine deaminase, with conversion of the resultant &agr;-ketobutyrate (=2-oxobutyrate) to propionyl-CoA. However, they ultimately concluded that threonine was utilized directly, without breaking carbon-carbon bonds, to form valeryl-CoA as the 3HV precursor. The nature of this chemical conversion was not described, but since the breaking of carbon-carbon bonds was not postulated to occur, the pathway could not involve threonine deaminase in conjunction with an &agr;-ketoacid decarboxylating step to form propionate or propionyl-CoA. In the experiments of Nakamura et al., the PHA polymer content was very low (<6% of dry cell weight). This result, in conjunction with the expense of feeding bacteria threonine, makes their approach impractical for the commercial production of P(3HB-co3HV) copolymer.
Yoon et al. (1995) have shown that growth of Alcaligenes sp. SH-69 on a medium supplemented with threonine, isoleucine, or valine resulted in significant increases in the 3HV fraction of the P(3HB-co-3HV) copolymer. In addition to these amino acids, glucose (3% wt/vol) was also added to the growth media. In contrast to the results obtained by Nakamura et al. (1992), growth of
A. eutrophus
under the conditions described by Yoon et al. (1995) did not result in the production of P(3HB-co-3HV) copolymer when the medium was supplemented with threonine as the sole carbon source. From their results, Yoon et al. (1995) implied that the synthetic pathway for the 3HV component in P(3HB-co-3HV) copolymer is likely the same as that described in WO 91/18995 and Steinbüchel and Pieper (1992). This postulated synthetic pathway involves the degradation of isoleucine to propionyl-CoA (FIG.
3
).
The PHB Biosynthetic Pathway
Polyhydroxybutyrate (PHB) was first discovered in 1926 as a constituent of the bacterium
Bacillus megaterium
(Lemoigne, 1926). Since then, PHAs such as PHB have been found in more than 90 different genera of gram-negative and gram-positive bacteria (Steinbüchel, 1991). These microorganisms produce PHAs using R-&bgr;-hydroxyacyl-CoAs as the direct metabolic substrate for a PHA synthase, and produce polymers of R-(3)-hydroxyalkanoates having chain lengths ranging from C3-C14 (Steinbüchel and Valentin, 1995).
To date, the best understood biochemical pathway for PHB production is that found in the bacterium
Alcaligenes eutrophus
(Dawes and Senior, 1973; Slater et al., 1988; Schubert et al., 1988; Peoples and Sinskey, 1989a and 1989b). This pathway, which is also utilized by other microorganisms, is summarized in FIG.
1
. In this organism, an operon encoding three gene products, i.e., PHB synthase, &bgr;-ketothiolase, and acetoacetyl-CoA reductase, encoded by the phbC, phbA, and phbB genes, respectively, are required to produce the PHA homopolymer R-polyhydroxy-butyrate (PHB).
As further shown in
FIG. 1
, acetyl-CoA is the starting substrate employed in the biosynthetic pathway. This metabolite is naturally available for PHB production in the cytoplasm and plastids of plants.
Poirier et al. (1992) demonstrated that a multi-enzyme pathway can be introduced into plants to produce polyhydroxybutyrate (PHB). In that work, the genes encoding the
Alcaligenes eutrophus
acetoacetyl-CoA reductase (phbB) and PHB synthase (phbC) genes were introduced into
Arabidopsis thaliana
, where the enzymes were expressed cytoplasmically. A 3-ketothiolase is already expressed in the cytoplasm of Arabidopsis. Although PHB was produced in the plants which expressed the three enzymes, the yield was low and the plants were stunted and had reduced seed production.
Nawrath et al. (1994) provided a solution to these problems. There, the genes for the three bacterial PHB enzymes (phbC, phbA, and phbB) were modified to comprise a pea chloroplast targeting peptide (=“transit peptide”), which targeted the enzymes to the chloroplast. Arabidopsis plants which produced these three enzymes in the chloroplast accumulated large amounts of PHB. There was also no apparent affect of these transgenes, or of the PHB accumulation, on the growth and development of the transgenic plants.
The P(3HB-co-3HV) Copolymer Biosynthetic Pathway
As noted above, P(3HB-co-3HV) random copolymer, commercially known as Biopol™, is produced by fermentation employing
A. eutrophus
. A proposed biosynthetic pathway for P(3HB-co-3HV) copolymer production is shown in FIG.
2
. Production of this polymer in plants has been reported (oral presentation by Mitsky et al., 1997).
Since the production of PHB in chloroplasts apparently does not affect plant growth and development as does production of PHB in the cytoplasm (Nawrath et al., 1992), the chloroplast is the preferred site of P(3HB-co-3HV) biosynthesis. The successful production of P(3HB-co-3HV) copolymer in plants thus requires the presence of three PHA biosynthetic enzymes as well as the substrates required for the copolymer biosynthesis (FIG.
2
), preferably in the plastids. For the 3HB component of the polymer, the substrate naturally exists in chloroplasts in sufficient concentration in the form of acetyl-CoA (Nawrath et al., 1994). However, this is not true for the 3HV component of the polymer, where the starting substrate is propionyl-CoA.
FIG. 3
is an overview of enzyme pathways which are related to the provision of these substrates. The engineering of plants to generate sufficient chloroplast pools of propionyl-CoA, along with the proper PHA biosynthetic enzymes (i.e., a &bgr;-ketothiolase, a &bgr;-ketoacyl-CoA reductase, and a PHA synthase), makes it possible to produce copolyesters of poly(3HB-co-3HV) in these organisms.
Methods for optimization of PHB and P(3HB-co-3HV) production in various crop plants are disclosed in Gruys et al. (1998). A major focus in that invention is the optimization of the substrate pools fo
Johnston Mark L.
Luethy Michael H.
Miernyk Jan A.
Mooney Brian P.
Randall Douglas D.
Fox David T.
Kallis Russell
Senniger Powers Leavitt & Roedel
The Curators of the University of Missouri
LandOfFree
Use of DNA encoding plastid pyruvate dehydrogenase and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of DNA encoding plastid pyruvate dehydrogenase and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of DNA encoding plastid pyruvate dehydrogenase and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3354244