Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
2000-03-15
2002-12-10
Whisenant, Ethan C. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C800S278000, C536S023100
Reexamination Certificate
active
06492134
ABSTRACT:
The present invention relates to method for producing polyhydroxyalkanoates in recombinant organisms.
BACKGROUND OF THE INVENTION
Plastic materials have become an integral part of contemporary life because they possess many desirable properties, including durability and resistance to degradation. Over the past 10-20 years, their widespread use have been increasingly regarded as a source of environmental and waste management problems. Industrial societies are now more aware of the impact of discarded plastic on the environment, and of their deleterious effect on wildlife and the aesthetic qualities of cities and forests. Problems associated with the disposal of waste and reduction in the availability of landfills have also focused attention on plastics, which accumulate in the environment at a rate of 25 million tonnes per year (Lee, 1996). These problems have created much interest in the development and production of biodegradable plastics. Biodegradable polymers are composed of material which can be degraded either by non-enzymatic hydrolysis or by the action of enzymes secreted by microorganisms. Estimates of the current global market for these biodegradable plastics range up to 1.3 billion kg per year (Lindsay, 1992).
Among the various biodegradable plastics available, there is a growing interest in the group of polyhydroxyalkanoates (PHAs). These are natural polymers produced by a variety of bacteria and they are 100% biodegradable. By changing the carbon source and bacterial strains used in the fermentation processes, PHA-biopolymers having a wide variety of mechanical properties have been produced. Their physical characteristics range from hard crystalline to elastic, depending on the composition of monomer units (Anderson & Dawes, 1990). The majority of PHAs are composed of R-(−)-3-hydroxyalkanoic acid monomers ranging from 3 to 14 carbons in length (C3-C14). The simplest member of the family, P(3HB) (C4), is highly crystalline, relatively stiff, and becomes brittle over a period of days upon storage under ambient conditions (Barham et al., 1984; De Koning et al., 1992; Doi, 1990; Holmes, 1988). Therefore, attempts have been made to decrease the brittleness of P(3HB) either by incorporating comonomers such as P(3HV), by blending with other polymers or blending with chemically synthesized atactic P(3HB) (Holmes, 1988; Kumagai & Doi, 1992 a, 1992 b, 1992 c; Pearce & Marchessault, 1994).
The P(3HB-co-3HV) copolymer, developed by ZENECA under the tradename BIOPOL™, has improved mechanical properties compared to P(3HB). As the fraction of P(3HV) (C5) increases, the polymer becomes tougher, more flexible and have an higher elongation to break (Doi et al., 1990). The medium-chain-length (MCL) PHAs are semicrystalline elastomers with a low melting point, low tensile strength, and high elongation to break. They thus have physico-chemical characteristics that make them more appealing than homogeneous P(3HB); they can even be used as a biodegradable rubber after cross linking by electron-beam irradiation (De Koning et al., 1994; Gagnon et al., 1992; Gross et al., 1989; Preusting et al., 1990).
PHAs have been shown to occur in over 90 genera of Gram-positive and Gram-negative bacteria species (Steinbüchel, 1991). Over 40 different PHAs have been characterized, with some polymers containing unsaturated bonds or various functional groups (Steinbüchel, 1991). Bacteria synthetise and accumulate PHAs as carbon and energy storage materials or as a sink for redundant reducing power under the condition of limiting nutrients in the presence of excess carbon sources (Byrom, 1994; Doi, 1990; Steinbüchel & Valentin, 1995). When the supply of the limiting nutrient is restored, the PHAs are degraded by intracellular depolymerases and subsequently metabolized as carbon and energy source (Byrom, 1990; Doi, 1990). The monomer 3HAs released from degradation of these microbial polyesters are all in the R-(−)-configuration due to the stereo specificity of biosynthetic enzymes (Anderson & Dawes, 1990). The molecular weights of polymers are in the range of 2×10
5
to 3×10
6
Daltons, depending on the microorganism and growth condition (Byrom, 1994). PHAs accumulate in the cells as discrete granules, the number per cell and size of which can vary among the different species; 8 to 13 granules per cell of 0.2 to 0.5 &mgr;m diameter have been observed in
Alcaligenes eutrophus
(Byrom, 1994).
PHAs can be subdivided in two groups depending on the number of carbon atoms in the monomer units: short-chain-length-(SCL) PHAs, which contain 3-5 carbon atoms, and medium-chain-length-(MCL) PHAs, which contain 6-14 carbon atoms (Anderson & Dawes, 1990). This is mainly due to the substrate specificity of the PHA synthases that can only accept 3HA monomers of a certain range of carbon lengths (Anderson & Dawes, 1990). The PHA synthase of
Alcaligenes eutrophus
can polymerize C3-C5 monomers, but not C6 or higher. On the other hand, the PHA synthase of
Pseudomonas oleovorans
only accepts C6-C14 monomers. Of particular interest is the capacity of some PHA synthase to polymerize 3-hydroxy-, 4-hydroxy- and 5-hydroxy-alkanoates (Steinbüchel & Schlegel, 1991). Even though most of the PHA synthases examined to date are specific for the synthesis of either SCL- or MCL-PHAs, at least six cases were recently reported in which the bacteria were able to synthesize copolymer consisting of SCL and MCL units (Lee, 1996).
P(3HB) is the most widespread and thoroughly characterized PHA, and most of the knowledge has been obtained from
Alcaligenes eutrophus
(Steinbüchel, 1991). In this bacterium, P(3HB) is synthesized from acetyl-CoA by the sequential action of three enzymes (FIG.
1
).The first one, 3-ketothiolase, catalyses the reversible condensation of two acetyl-CoA moieties to form acetoacetyl-CoA. Acetoacetyl-CoA reductase subsequently reduces acetoacetyl-CoA to R-(−)-3-hydroxybutyryl-CoA, which is then polymerized by the action of PHA synthase to form P(3HB). A number of PHAs with different C3 to C5 monomers have been produced in
A. eutrophus
, the nature and proportion of these monomers being influenced by the type and relative quantity of the carbon sources supplied to the growth media (Steinbüchel & Valentin, 1995).
Pseudomonas oleovorans
and most pseudomonades belonging to the ribosomal rRNA homology group I synthesize MCL-PHAs from various MCL-alkanes, alkanols, or alkanoates (Steinbüchel & Valentin, 1995). The composition of PHA produced is related to the substrate used for growth, with the polymer being mostly composed of monomers which are 2n carbons shorter than the substrates used. It was suggested that the acyl-CoA derived from alkanoic acids enter the &bgr;-oxidation pathway and R-(−)-3hydroxyacyl-CoA intermediates used by the PHA synthase are generated either through reduction of 3-ketoacyl-CoA by a ketoacyl-CoA reductase, conversion of S-(+)-3hydroxyacyl-CoA normally produced by the pathway to the R-(−)-isomer by an epimerase, or the direct hydration of enoyl-CoA by an enoyl-CoA hydratase (Poirier et al., 1995).
Most pseudomonades belonging to rRNA homology group I, except
P. oleovorans
, also synthesize MCL-PHAs when grown on substrates non related to fatty acids and alkanoates, such as gluconate, lactate, glycerol, and hexoses (Anderson & Dawes, 1990; Huijberts et al., 1994; Timm & Steinbüchel, 1990). These substrates must be first converted into acetyl-CoA to be used for the PHAs biosynthesis. This suggests that, in theory, microorganisms, plants and even animals, must be able to synthesize PHA following the transfection of a limited number of genes. In these bacteria, three main pathways have been proposed for the synthesis of PHA precursors (Huijberts et al., 1992, 1994).
(i) A detailed analysis of the composition of PHA produced by
P. putida
grown on glucose have shown that the monomers are structurally identical to the acyl-moieties of the 3-hydroxyacyl-ACP intermediates of the novo fatty acid biosynthesis. Since it has not been shown that PHA synthase
Aquin Stéphanie
Vezina Louis-P.
Nixon & Peabody LLP
Universite Laval
Whisenant Ethan C.
LandOfFree
Method for producing polyhydroxyalkanoates in recombinant... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for producing polyhydroxyalkanoates in recombinant..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for producing polyhydroxyalkanoates in recombinant... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2962941