Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2001-05-15
2004-01-27
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S287000, C435S069100, C435S069700, C435S468000, C536S023600
Reexamination Certificate
active
06683231
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of plant gene expression and molecular biology and microbiology. More specifically, a method is presented for the production of p-hydroxybenzoic acid (pHBA) in green plants which relies on the expression of a unique expression cassette comprising a gene encoding chorismate pyruvate lyase operably linked to a specific chloroplast targeting sequence.
BACKGROUND OF THE INVENTION
p-Hydroxybenzoic acid (pHBA) is the major monomeric component (~65% by weight) of Zenite™, a Liquid Crystal Polymer (LCP). LCP's have superior properties over conventional resins such as high strength/stiffness, low melt viscosity, excellent environmental resistance, property retention at elevated temperatures, and low gas permeability. However, current synthetic methods for the synthesis of pHBA (Kolbe-Schmitt reaction (Kolbe and Lautemann,
Ann.
113:125 (1869)), are prohibitively expensive, and an inexpensive route to LCP monomers would open up many new applications for their use in the automotive, electrical, and other industries. Biological production offers one potential, less expensive route to pHBA production.
pHBA has been produced in microbial systems. For example, JP 06078780 teaches pHBA preparation by culturing benzoic acid in the presence of microorganisms (preferably Aspergillus) that oxidize benzoic acid to pHBA. Additionally, strains of Enterobacter with the ability to convert p-cresol to pHBA have been isolated from soil (JP 05328981). Further, JP 05336980 and JP 05336979 disclose isolated strains of
Pseudomonas putida
with the ability to produce pHBA from p-cresol. Similarly, commonly owned WO 9856920 teaches a method for the production of pHBA from toluene using a
Pseudomonas mendocina
mutant lacking the ability to express para-hydroxybenzoate hydroxylase (pHBH). Finally, U.S. Pat. No. 6,030,819 teaches the production of pHBA in genetically engineered
E. coli
expressing the chorismate pyruvate lyase (CPL) gene.
In spite of these successes the ability to produce commercially useful quantities of pHBA in microbial platforms is hampered by the use of toxic starting materials and limited biomass. A method for pHBA production that overcomes these problems is needed.
Coincidentally, pHBA is naturally occurring in nearly all plants, animals, and, microorganisms, albeit in miniscule quantities. In many bacteria, the generation of pHBA occurs by way of chorismate, an important branchpoint intermediate in the synthesis of numerous aromatic compounds, including phenylalanine, tyrosine, p-aminobenzoic acid, and ubiquinone. In
E. coli
, chorismate itself undergoes five different enzymatic reactions to yield five different products, and the enzyme that is ultimately responsible for the synthesis of pHBA is chorismate pyruvate lyase, which is known as CPL. The latter is the product of the
E. coil
ubiC gene, which was independently cloned by two different groups (Siebert et al.,
FEBS Lett
307:347-350 (1992); Nichlols et al.,
J. Bacteriol
174:5309-5316 (1992)). The enzyme is a 19 kDa monomeric protein with no known co-factors or energy requirements. Through elimination of the C
3
enolpyruvyl side chain of its sole substrate, CPL catalyzes the direct conversion of 1 mol of chorismate to 1 mol of pyruvate and 1 mol of pHBA. Recombinant CPL has been overexpressed in
E. coli
, purified to homogeneity, and partially characterized both biochemically and kinetically (Siebert et al.,
Microbiology
140:897-904; Nichlols et al.,
J. Bacteriol
174:5309-5316 (1992)). In addition a detailed mechanism for the CPL enzyme reaction has also been proposed (Walsh et al.,
Chem Rev.
90:1105-1129).
In plants pHBA has been found in carrot tissue (Schnitzler et al.,
Planta,
188, 594, (1992)), in a variety of grasses and crop plants (Lydon et al., (
J. Agric. Food. Chem.,
36, 813, (1988), in the lignin of poplar trees (Terashima et al., Phytochemistry, 14, 1991, (1972); and in a number of other plant tissues (Billek et al.,
Oesterr. Chem.,
67, 401, (1966). The fact that plants possess all of the necessary enzymatic machinery to synthesize pHBA suggests that they may be a useful platform for the production of this monomer. For example, as a renewable resource a plant platform would require far less energy and material consumption than either petrochemical or microbial methods. Similarly, a plant platform represents a far greater available biomass for monomer production than a microbial system. Finally, the natural presence of pHBA in plants suggests that host toxicity as a result of overproduction of the compound might not be a problem. Nevertheless, in spite of the obvious benefits of using plants as a means to produce pHBA, high level production of the monomer has been elusive.
One difficulty to be overcome lies in the metabolic fate of chorismate in plant tissues. Indeed, the production of pHBA from chorismate is vastly more complicated in higher plants than microbes, since the former lack an enzyme that is functionally equivalent to CPL. For example, the biosynthetic pathway leading to pHBA in
Lithospermum erythrorhizon
is thought to consist of up to 10 successive reactions (Loscher and Heide,
Plant Physiol.
106:271-279 (1992)), presumably all catalyzed by different enzymes. Moreover, most of the enzymes that catalyze these reactions have not been identified, nor have their genes been cloned. Even less information is available on how pHBA is synthesized in other plant species. To further complicate matters, those enzymes that are known to participate in plant pHBA production span two different pathways, that are differentially regulated and located in different cellular compartments. Thus, chorismate is an intermediate of the shikimate pathway which is largely confined to chloroplasts and other types of plastids (Siebert et al.,
Plant Physiol.
112:811-819 (1996)) Sommer et al.,
Plant Cell Physiol.
39(11):1240-1244 (1998)), while all of the intermediates downstream from phenylalanine belong to the phenylpropanoid pathway which takes place in both the cytosol and endoplasmic reticulum.
Despite the lack of understanding of how plants normally synthesize pHBA and the enzymes that are involved in this process, transgenic plants that accumulate significantly higher levels of pHBA than wildtype plants have been described. For example, Kazufumi Yazaki, (
Baiosaiensu to Indasutori
(1998), 56(9), 621-622) discusses the introduction of the CPL encoding gene into tobacco for the production of pHBA in amounts sufficient to confer insect resistance. Similarly, Siebert et al., (
Plant Physiol.
112:811-819 (1996)) have demonstrated that tobacco plants (
Nicotiana tabacum
) transformed with a constitutively expressed chloroplast-targeted version of
E. coli
CPL (referred to as “TP-UbiC”) have elevated levels of pHBA that are at least three orders of magnitude greater than wildtype plants (WO 96/00788 granting as DE 4423022). Interestingly, the genetically modified tobacco plants contained only trace amounts of free pHBA. Instead, virtually all of the compound (~98%) was converted to two glucose conjugates, a phenolic glucoside and an ester glucoside, that were present in a ratio of about 3:1 (Siebert et al.,
Plant Physiol
112:811-819 (1996); Li et al.,
Plant Cell Physiol.
38(7):844-850 (1997)). Both glucose conjugates were 1-&bgr;-D-glucosides, with a single glucose residue covalently attached to the hydroxyl or carboxyl group of pHBA. The best transgenic plant that was identified in this study had a total pHBA glucoside content of 0.52% of dry weight, when leaf tissue was analyzed. Correcting for the associated glucose residue, the actual amount of pHBA that was produced in the transgenic tobacco plants was only about half of this value.
In more recent studies, the same artificial fusion protein was expressed in transformed tobacco cell cultures using both a constitutive promoter (Sommer et al.,
Plant Cell Physiol.
39(11):1240-1244 (1998)) and an inducible promoter (Sommer et al.,
Plant Cell Reports
17:891-896 (1998)). While the accum
Meyer Knut
Van Dyk Drew E.
Viitanen Paul V.
Braum Stuart F
Bui Phuong T.
E. I. du Pont de Nemours and Company
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