Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2001-06-29
2003-11-04
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S041000, C435S193000, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06642036
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding plant sinapoylglucose:malate sinapoyltransferase (SMT) and its use in the conjugation of small molecules for materials.
BACKGROUND OF THE INVENTION
Recent advances in genetic engineering have enabled the development of new biological platforms for the production of molecules, heretofore only synthesized by chemical routes. Although advances in fermentation technology have resulted in the use of microorganisms for the production of pharmaceutically useful proteins (antibiotics, enzymes etc.), the possibility of using green plants for the manufacture of high volume materials is becoming increasingly more attractive.
There are two obvious advantages of using green plants to produce large amounts of compounds that are traditionally synthetically manufactured. First, plants are a renewable energy resource. The photosynthetic ability of green plants means that the only raw materials that are required to produce carbon-based compounds in plants are CO
2
, water and soil nutrients. Second, in contrast to microbial fermentation, green plants represent a huge biomass that can easily accommodate the large amounts of chemicals that are required for high-volume, low-cost applications. The use of plants as production platforms for materials is complicated only in that they comprise a vastly more differentiated and complex genetic and biochemical systems as compared with microbes. Thus, production of molecules and materials from plants will be greatly enhanced if the materials to be produced are native, at least in some amounts to the plant.
Two classes of materials that are native to plants are aromatic acids and aromatic esters. In particular, p-hydroxybenzoic acid (pHBA) and esters of pHBA can readily be found. Both of these materials find use in various polymers useful in paints and other coatings. In addition, pHBA is the key monomer in Liquid Crystal Polymers (LCPs) which contain approximately 67% pHBA. Esters of pHBA can be used as backbone modifiers in other condensation polymers, i.e., polyesters, and are also used to make parabens preservatives.
It is known that aromatic acids, aromatic esters and pHBA are endogenous to plants as well as other organisms. In most 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 also known as CPL. The latter is the product of the
E. coli
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)). In higher plants the biosynthetic pathway leading to pHBA in
Lithospermum erythrorhizon
is thought to consist of up to ten successive reactions (L{haeck over (s)}scher and Heide,
Plant Physiol.
106:271-279 (1992)), presumably all catalyzed by different enzymes.
Recently it has been shown that levels of pHBA production in plants may be enhanced through genetic manipulation. Several recent publications (Severin et al.,
Planta Medica
, (1993) Vol. 59, No. 7, pp. A590-A591; Siebert et al.,
Plant Physiol.
112:811-819 (1996); WO 9600788), including Applicants own work (U.S. Ser. No. 09855,341) 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. However, it should be noted that these studies indicated that virtually all of the pHBA was converted to its two glucose conjugates, a phenolic glucoside and an ester glucoside. The conversion of the glucoside to a useful product will require a chemical step and represents an obstacle for the production of free pHBA or other aromatic acids. Therefore, a method of further processing the pHBA glucosides is needed.
There are no reports of endogenous plant transconjugation reactions that involve the transfer of benzoic acids from glucose esters to organic acids. However, there are reports of the processing of esters of hydroxycinnamic acids such as sinapic acid to malate conjugates as a function of secondary metabolism in cotyledon and leaf tissues of cruciferous plant species. Sinapic acid is generated from phenylalanine through the action of phenylalanine ammonia lyase (PAL) cinammate-4-hydroxylase, coumarate-3-hydroxylase, caffeic acid o-methyltransferase and ferulate-5-hydroxylase. Sinapoyl glucose is synthesized from sinapic acid and uridinediphosphate glucose (UDPG) through the action of UDPG sinapoyltransferase (SGT). Sinapoyl glucose is subsequently translocated to the vacuole. Sinapoyl glucose is a 1-O-glucose ester that has a free energy of hydrolysis (Mock and Strack,
Phytochemistry
32:575-579 (1993)). This linkage provides the necessary free energy for the transacylation reaction catalyzed by sinapoylglucose:malate sinapoyltransferase (SMT) (Strack,
Planta
155:31-36 (1982)), which generates sinapoyl malate in the expanding cotyledons (Sharma and Strack,
Planta
163:563-568 (1985)). It is instructive to note that sinapoyl malate accumulated in the vacuole in these plants, although little is known about how vacuolar transport might be effected (Sharma and Strack (1985), supra). During seed maturation, sinapic acid is converted to sinapoyl choline by the combined actions of SGT and sinapoylglucose:choline sinapoyltransferase (SCT) (Strack et al.,
Z Naturforsch
38c:21-27 (1983)). Recently SMT has been partially characterized (Graewe et al.,
Planta
187(2):236-41 (1992)). However, despite the detailed biochemical understanding of these enzymes, none of the genes involved had been cloned, and relatively little is known about their regulation. Additionally, it is unclear how or if this enzymatic system may be adapted to the processing of benzoic acid glucosides and related molecules.
The problem to be solved therefore is to design a system for the production of benzoic acid derivatives and particularly pHBA derivatives in plants. Applicants have solved the stated problem by the discovery that sinapoylglucose:malate sinapoyltransferase (SMT) has the ability to convert glucosides of p-hydroxybenzoic acid to its corresponding malate conjugate where the malate product is localized in the plant vacuole. This further processing of the native p-hydroxybenzoic acid glucoside advances the art of materials production from genetically modified green plant platforms.
SUMMARY OF THE INVENTION
The present invention provides a method for the production of malate conjugated aromatic acids comprising: contacting a glycosylated aromatic acid with an effective amount of sinapoylglucose:malate sinapoyltransferase which catalyzes the substitution of a glucose moiety on the glycosylated aromatic acid with a malate moiety to form a malate conjugated aromatic acid. Suitable aromatic acids are described by the formula
wherein
R
1
-R
6
are each independently H, or OH, or COOH or OR
7
or R
7
COOH and R
7
is C
1
to C
20
substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene;
providing at least one of R
1
-R
6
is COOH
In an alternate embodiment the invention provides a method for the production of carboxylic acid conjugated aromatic acids comprising: contacting a glycosylated aromatic acid with an &agr;-hydroxycarboxylic acid of the general formula:
R—COOH, where R is C
1
to C
20
substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene;
and an effective amount of sinapoylglucose:malate sinapoyltransferase which catalyzes the subs
Flint Dennis
Meyer Knut
Viitanen Paul
Achutamurthy Ponnathapu
E. I. Du Pont de Nemours and Company
Fronda Christian L
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