Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1995-09-29
2001-01-16
McElwain, Elizabeth F. (Department: 1649)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S306000, C800S312000, C800S317300, C800S322000
Reexamination Certificate
active
06175061
ABSTRACT:
SUMMARY OF THE INVENTION
An object of the present invention is to provide materials and a method for the efficient production of polyhydroxyalkanoate.
According to the present invention there is provided a plant adapted for the production of polyhydroxyalkanoate comprising a recombinant genome of an oil-producing plant, which genome contains genes encoding enzymes necessary for catalyzing the production of polyhydroxy-alkanoate together with gene regulatory sequences directing expression of the said genes to target plant call components.
These regulatory sequences include promoter sequences directing expression of the biosynthetic pathway specifically to the developing seed, and transit peptide sequences targeting the enzymes to appropriate subcellular compartments.
The genes encoding the enzyme or enzymes necessary for the catalysis of polyhydroxyalkanoate production may be isolated from a micro-organism, such as
Alcaligenes eutrophus
, which is known to produce polyhydroxybutyrate and other polyhydroxy-alkanoates.
It is preferable, for reasons which will later be explained, that the plant be of a species which produces substantial quantities of oil, rather than starch. such plant species are well known and are simply referred to as “oil-seed” crops and include, oilseed rape, canola, soya and sunflower. Methods for the genetic transformation of many oil crops are known; for example, transformation by
Agrobacterium tumefaciens
methods are suitable for most. Such methods are well-described in the literature and well-known and extensively practised in the art.
The biosynthesis of polyhydroxybutyrate from the substrate, acetyl-CoA involves three enzyme-catalysed steps, illustrated in
FIG. 1
herewith.
The three enzymes involved are &bgr;-ketothiolase, NADP linked acetoacetyl-CoA reductase, and polyhydroxybutyrate synthase, the genes for which have been cloned from
Alcaligenes eutrophus
(Schubert et al, 1988, J Bacteriol, 170). When cloned into
Escherichia coli
the three genes are known to facilitate production of polyhydroxyalkanoate up to 30% of the cell weight.
Genes specifying the production of alkanoates higher than the butyrate are known to exist in bacteria. Isolation of the appropriate genes allows expression of these higher polyhydroxy-alkanoates. For example, genes specifying production of the polyhydroxy-octanoate and the —decanoate exist in the bacterial species
Pseudomonas oleovorans
and
Pseudomonas eruginosa
. Howeverr genes for analogous polymers are widespread in bacterial species.
All the microorganisms required for performance of this invention are publicly available from public culture collections.
An important preferred feature of this invention is the use of an oilseed plant for expression of the polyhydroxyalkanoate. The reason behind our selection of oil-producing crops is that such plants naturally produce large amounts of acetyl-CoA substrate (under aerobic conditions) in the developing seed, which is normally used in fatty acid synthesis. Diversion of this substrate into polyhydroxyalkanoate production will reduce the amount of oil stored by the seed but will have minimal influence on other aspects of the cell's metabolism. It is therefore possible to produce commercial viable quantities of polyhydroxy-alkanoate such as polyhydroxybutyrate in an oilseed.
It has been previously suggested that
Alcaligenes eutrophus
genes could be expressed in a starch crop but this has certain problems. In order to optimise polyhydroxyalkanoate production in such a crop, it would probably be necessary to down-regulate starch synthesis. However, even if this down-regulation were to be effected it would not guarantee an increased rate of acetyl-CoA production. Moreover, even if this increased production were actually achieved, it is possible that the acetyl-COA would be rapidly utilised by respiration in the starch crop.
For expression in higher plants the bacterial (for example
Alcaligene eutrophus
) genes require suitable promoter and terminator sequences. various promoters/torminators are available for use. For constitutive expression the cauliflower mosaic virus CaMV35S promoter and nos terminator may be used. It is however preferred to target synthesis of polyhydroxyalkanoate only to the developing oil storage organ of the oilseed such as the embryo of oilseed rape. The promoter of the rape seed storage protein, napin, could be used to obtain embryo specific expression of polyhydroxyalkanoate genes. Expression of the polyhydroxyalkanoate genes during the precise period when lipid is being made will ensure effective omrpetition by the polyhydroxyalkanoate enzymes for available acetyl-CoA. The promoters of fatty acid synthesis genes whose expressions are switched on at this time are thus most appropriate candidates to be used as polyhydroxyalkanoate gene promoters. Examples of such promoters are those of seed specific isoforms of rape acyl carrier protein (ACP) or &bgr;-ketoacyl ACP reductase.
In inserting the polyhydroxyalkanoate genes into eukaryotic cells, consideration has to be given to the most appropriate subcellular compartment in which to locate the enzymes. Two factors are important: the site of production of the acetyl-CoA substrate, and the available space for storage of the polyhydroxyalkanoate polymer.
The acetyl-CoA required for fatty acid synthesis in, for example, developing rapeseed embryo is produced by two routes. The first, direct, route involves the activity of a pyruvate dehydrogenase enzyme located in the plastid. The second route involves the initial production of acetyl-CoA by mitochondrial pyruvate dehydrogenase, lysis to free acetate, and diffusion of the acetate into the plastid where it is re-esterified to CoA by acetyl-CoA synthase. Rapeseed also produces acetyl-CoA in the cytosol, though at a lower rate than in the plastid, via the activity of a cytosolic citrate lyase enzyme.
Considering substrate supply, the bacterial (for example,
Alcaligenes
) &bgr;-ketothiolase enzyme may function in the mitochondrion, using acetyl-CoA produced in excess of the requirements of respiration, or in the cytosol. The regulatory sequences of the invention may thus direct expression of the &bgr;-ketothiolase gene to the mitochondrion or to the cytosol. It is however preferred to target this enzyme to the plastids, where highest rates of acetyl-CoA generation occur.
The mitochandrion lacks sufficient space for storage of the polyhydroxyalkanoate polymer. Significant storage space exists in the plaitids, at least in rape embryo. Highest storage space exists in the cytosol, the compartment normally occupied by the oil bodies.
It is not known whether the acetcacetyl-CoA or hydroxybutyryl-CoA pathway intermediates can be transported from plastid to cytosol. Certainly they would not be able to traverse the plastid envelope membrane as CoA esters. Export would require that the acetoacetate or hydroxybutyrate groups are recognised by the transport systems involved in export of fatty acids from plastids. These have been suggested to involve: lysis of the CoA ester, export of the free acid, and resynthesis of the CoA ester in the cytosol; or transfer of the acyl groups to carnitine, and export of acyl carnitine. if acetoacetyl groups may be exported from the plastid by one of these mechanisms then it would be possible to target &bgr;-ketothiolase to the plastid, to utilise acetyl-CoA destined for lipid synthesis, and target acetoacetyl-CoA reductase and polyhydroxybutyrate synthase to the cytosol to achieve polymer synthesis in thie more spacious compartment. If neither acetoacetate nor hydroxybutyrate groups may be exported from the plastid, polyhydroxyalkanoate synthesis will require that all three pathway enzymes are targeted to this organelle so that they are expressed in the same cell compartment.
To target the three bacterial (such as
Alcaligenes eutrophus
) enzymes for polyhydroxyalkanoate synthesis to the plant plastid requires the use of specific targeting regulatory elements called transit peptides. Possible sources of plastid stroma targ
Bright Simon William Jonathan
Byrom David
Fentem Philip Anthony
Beusen Jon
Howrey Simon Arnold & Durkee
McElwain Elizabeth F.
Monsanto Company
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