Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-12-04
2002-12-10
McElwain, Elizabeth F. (Department: 1638)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023200
Reexamination Certificate
active
06492509
ABSTRACT:
TECHNICAL FIELD
The present invention is directed to enzymes, methods to purify, and obtain such enzymes, amino acid and nucleic acid sequences related thereto, and methods of use for such compositions in genetic engineering applications.
INTRODUCTION
Background
Through the development of plant genetic engineering techniques, it is possible to transform and regenerate a variety of plant species to provide plants which have novel and desirable characteristics. One area of interest for such plant genetic engineering techniques is the production of valuable products in plant tissues. Such applications require the use of various DNA constructs and nucleic acid sequences for use in transformation events to generate plants which produce the desired product. For example, plant functional promoters are required for appropriate expression of gene sequences, such expression being either in the whole plant or in selected plant tissues. In addition, selective marker sequences are often used to identify the transformed plant material. Such plant promoters and selectable markers provide valuable tools which are useful in obtaining the novel plants.
A desirable goal which involves such genetic engineering techniques, is the ability to provide crop plants having a convenient source of wax esters. Wax esters are required in a variety of industrial applications, including pharmaceuticals, cosmetics, detergents, plastics, and lubricants. Such products, especially long chain wax esters have previously been available from the sperm whale, an endangered species, or more recently, from the desert shrub, jojoba. Neither of these sources provides a convenient supply of wax esters. Thus, in order to obtain a reliable source of such compounds, transformation of crop plants, which are easily manipulated in terms of growth, harvest and extraction of products, is desirable.
In order to obtain such transformed plants, however, the genes responsible for the biosynthesis of the desired wax ester products must first be obtained. Wax ester production results from the action of at least two enzymatic activities, fatty acyl reductase and fatty acyl:fatty alcohol acyltransferase, or wax synthase. In addition, a &bgr;-ketoacyl-Coenzyme A synthase may also be involved in wax biosynthesis by providing very long chain fatty acyl-CoA substrates for the reductase and wax synthase enzymatic reaction. Preliminary studies with such enzymes and extensive analysis and purification of a fatty acyl reductase, indicate that these proteins are associated with membranes, however the enzyme responsible for the fatty acyl:fatty alcohol ligation reaction in wax biosynthesis has not been well characterized. Thus, further study and ultimately, purification of this enzyme is needed so that the gene sequences which encode the enzymatic activity may be obtained.
It is desirable, therefore, to devise a purification protocol whereby the wax synthase protein may be obtained and the amino acid sequence determined and/or antibodies specific for the wax synthase obtained. In this manner, library screening, polymerase chain reaction (PCR) or immunological techniques may be used to identify clones expressing a wax synthase protein. Clones obtained in this manner can be analyzed so that the nucleic acid sequences corresponding to wax synthase activity are identified. The wax synthase nucleic acid sequences may then be utilized in conjunction with fatty acyl reductase proteins, either native to the transgenic host cells or supplied by recombinant techniques, for production of wax esters in host cells.
Relevant Literature
Cell-free homogenates from developing jojoba embryos were reported to have acyl-CoA fatty alcohol acyl transferase activity. The activity was associated with a floating wax pad which formed upon differential centrifugation (Pollard et al. (1979) supra; Wu et al. (1981) supra).
Solubilization of a multienzyme complex from
Euglena gracilis
having fatty acyl-CoA transacylase activity is reported by Wildner and Hallick (Abstract from
The Southwest Consortium Fifth Annual Meeting
, Apr. 22-24, 1990, Las Cruces, N.Mex.).
Ten-fold purification of jojoba acyl-CoA: alcohol transacylase protein is reported by Pushnik et al. (Abstract from
The Southwest Consortium Fourth Annual Meeting
, Feb. 7, 1989, Riverside, Calif.).
An assay for jojoba acyl-CoA:alcohol transacylase activity was reported by Garver et al. (
Analytical Biochemistry
(1992) 207:335-340).
WO 93/10241 is directed to plant fatty acyl-CoA:fatty alcohol O-acyltransferases. A jojoba 57 kD protein is identified as the jojoba fatty acyl-CoA:fatty alcohol O-acyltransferase (wax synthase). The present inventors later reported that the 57 kD protein from jojoba is a &bgr;-ketoacyl-CoA synthase involved in the biosynthesis of very long chain fatty acids (Lassner et al. (
The Plant Cell
(1996) 8:281-292).
Photoaffinity labeling of a 57 kD jojoba seed polypeptide postulated to be an acyl-CoA:fatty alcohol acyltransferase was also reported by Shockey et al. (
Plant Phys
. (1995) 107:155-160).
U.S. Pat. No. 5,728,412 describes the isolation of genes encoding soluble wax synthase enzymes which are active on short chain alcohols and acetyl-CoA to produce an acetate ester.
SUMMARY OF THE INVENTION
By this invention, nucleic acid sequences encoding fatty acyl-CoA: fatty alcohol O-acyltransferase protein (fatty alcohol acyltransferase, E.C.2.3.1.75), are provided, wherein said protein is active in the formation of wax esters from fatty alcohol and fatty acyl substrates. This fatty acyl-CoA: fatty alcohol O-acyltransferase is also referred to herein as “wax synthase”. The wax synthase of this invention may be active with a variety of fatty acyl and fatty alcohol substrates, including acyl-CoAs and acyl-ACPs. The carbon chain length of these substrates may vary, although a given wax synthase may show preference for acyl and alcohol substrates having a specific chain length or may be active with acyl and alcohol substrates having a wide range with respect to carbon chain length.
In general, the wax synthase of this invention has activity towards at least those acyl and alcohol substrates having a chain length of from 8 to 26 carbons, although other acyl or alcohol substrates may be tested and further activities discovered. In addition, having obtained the wax synthase protein of this invention, further manipulations are now possible as described in further detail below. These manipulations may lead to production or discovery of other related wax synthases.
In one important aspect of this invention, nucleic acid sequences are provided which encode for wax synthase. Methods are described whereby these sequences may be identified and obtained from the amino acid sequences of the wax synthase proteins of this invention. Uses of the structural gene sequences for isolation of other wax synthase sequences, as well as in recombinant constructs for transcription of wax synthase nucleic acid sequences and/or expression of wax synthase proteins in host cells are described. Uses of other nucleic acid sequences associated with wax synthase protein are also considered, such as the use of 5′ and 3′ noncoding regions.
Thus, this invention encompasses plant wax synthase nucleic acid sequences and the corresponding amino acid sequences, and the use of these nucleic acid sequences in the preparation of oligonucleotides containing wax synthase encoding sequences for analysis and recovery of plant wax synthase gene sequences. The plant wax synthase encoding sequence may encode a complete or partial sequence depending upon the intended use. All or a portion of the genomic sequence, or cDNA sequence, is intended.
Of special interest are recombinant DNA constructs which provide for transcription or transcription and translation (expression) of the plant wax synthase sequences. In particular, constructs which are capable of transcription or transcription and translation in plant host cells are preferred. For some applications a reduction in plant wax synthase may be desired. Thus, recombinant constructs
Lardizabal Kathryn Dennis
Lassner Michael W.
Metz James George
Arnold & Porter
Calgene LLC
Eagle Alissa M.
McElwain Elizabeth F.
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