Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
2000-04-12
2004-03-09
Reynolds, Deborah J. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S006120, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06703240
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to and benefit of provisional application USSN 60/129,009, filed Apr. 13, 1999, pursuant to 35 USC 119 (e).
COPYRIGHT NOTICE
Pursuant to 37 C.F.R. 1.71 (e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The invention relates to methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that encode proteins having enzyme activities involved in starch metabolism which are useful for introduction into plant species, and other hosts, and related aspects.
BACKGROUND OF THE INVENTION
Genetic engineering of agricultural organisms dates back thousands of years to the dawn of agriculture. The hand of man has selected the agricultural organisms having the phenotypic traits that were deemed desirable, e.g., taste, high yield, caloric value, ease of propagation, resistance to pests and disease, and appearance. Classical breeding methods to select for germplasm encoding desirable agricultural traits had been a standard practice of the world's farmers long before Gregor Mendel and others identified the basic rules of segregation and selection. For the most part, the fundamental process underlying the generation and selection of desired traits was the natural mutation frequency and recombination rates of the organisms, which are quite slow compared to the human lifespan and make it difficult to use conventional methods of breeding to rapidly obtain or optimize desired traits in an organism.
The recent advent of non-classical, or “recombinant” genetic engineering techniques has provided new means to expedite the generation of agricultural organisms having desired traits that provide an economic, ecological, nutritional, or aesthetic benefit. To date, most recombinant approaches have involved transferring a novel or modified gene into the germline of an organism to effect its expression or to inhibit the expression of the endogenous homologue gene in the organism's native genome. However, the currently used recombinant techniques are generally unsuited for substantially increasing the rate at which a novel or improved phenotypic trait can be evolved. Essentially all recombinant genes in use today for agriculture are obtained from the germplasm of existing plant and microbial specimens, which have naturally evolved coordinately with constraints related to other aspects of the organism's evolution and typically are not optimized for the desired phenotype(s). The sequence diversity available is limited by the natural genetic variability within the existing specimen gene pool, although crude mutagenic approaches have been used to add to the natural variability in the gene pool.
Unfortunately, the induction of mutations to generate diversity often requires chemical mutagenesis, radiation mutagenesis, tissue culture techniques, or mutagenic genetic stocks. These methods provide means for increasing genetic variability in the desired genes, but frequently produce deleterious mutations in many other genes. These other traits may be removed, in some instances, by further genetic manipulation (e.g., backcrossing), but such work is generally both expensive and time consuming. For example, in the flower business, the properties of stem strength and length, disease resistance and maintaining quality are important, but often initially compromised in the mutagenesis process.
STARCH METABOLISM IN PLANTS
The biosynthesis of starches in higher plants occurs in three steps, the first of which involves synthesis of ADP-glucose from ATP and glucose-l-phosphate and is catalyzed by ADP-glucose pyrophosphorylase (“ADPGPP”; EC 2.4.7.27). The second step of starch biosynthesis is transfer of a glucosyl moiety of ADP-glucose to a maltodextrin or starch to give rise to a new 1,4-glucosyl linkage; the reaction is catalyzed by a starch synthase (“SS”; EC 2.4.1.21), of which there are several forms present either as soluble enzymes or bound to starch particles as particulate enzymes. The third reaction is catalyzed by branching enzymes (“BE”; EC 2.4.1.18) and is responsible for synthesis of 1,6-glucosyl linkages. An exemplary starch biosynthetic pathway is illustrated in FIG.
1
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Starch metabolism is a dynamic process wherein catabolic activities antagonize the synthetic (anabolic) processes which form starch. Examples of catabolic activities include amylase (alpha and beta), two categories of debranching enzymes, isoamylases and pullulanases (limit dextrinases; R enzymes), and starch phosphorylase. The composition of starches that are produced result from the relative actions of the anabolic and catabolic activities.
The enzymatic activities of the various enzymes involved in starch metabolism control the properties and types of the starches which are present in the plant, typically in the form of storage granules. Various commercial native starches produced in a variety of plants differ dramatically in important physical, mechanical, and chemical properties, and are important for foodstuff and industrial uses (Swinkels, J.(1985) Starch 37:1). It is theoretically possible to alter the composition of starches made in a plant cell or plant storage organ by introducing heterologous or modified genes encoding enzymes that can alter starch metabolism. U.S. Pat. Nos. 5,750,875 and 5,824,790 disclose methods that reportedly modify starch metabolizing ability by introducing foreign genes into a plant genome or by suppressing endogenous gene expression. However, both of these methods are severely limited by the small pool of naturally occurring genes in various organisms that are useful for the methods. It would be highly desirable for the art to have methods for producing novel starch compositions by engineering gene sequences encoding modified starch biosynthetic enzymes, and introducing these gene sequences into plant cells, thereby creating novel plant cells that produce a desirable starch composition, particularly of types which are industrially useful and not available or obtainable only by laborious purification and chemical modification methods.
As noted, the advent of recombinant DNA technology has provided agriculturists with additional means of modifying plant genomes. While certainly practical in some areas, to date genetic engineering methods have had limited success in transferring or modifying important biosynthetic or other pathways, including certain naturally-occurring genes encoding starch metabolizing enzymes into photosynthetic organisms and bacteria. The creation of plants and other photosynthetic organisms having improved starch biosynthetic pathways can provide increased yields of certain types of starchy foodstuffs, enhanced industrial feedstocks, improved chemical compositions and clothing, and may alter the types and properties of polyglucan polymers available for a wide range of industrial and pharmaceutical uses, among other desirable phenotypes.
Thus, there exists a need for improved methods for producing plants and agricultural photosynthetic microbes comprising heterologous gene sequences which encode one or more enzyme(s) that result in production of an improved starch composition. In particular, these methods should provide general means for producing novel starch metabolic enzymes, including increasing the diversity of the starch metabolic enzyme gene pool and the rate at which genetic sequences encoding one or more starch metabolic enzyme species having desired properties are evolved. It is particularly desirable to have methods which are suitable for rapid evolution of genetic sequences to function in one or more plant species and confer an improved starch phenotype (e.g., increased control over branching structures, improved physioc
Huisman Gjalt
Raillard Sun Ai
Stemmer Willem P. C.
Subramanian Venkitswaran
Holmer Christopher
Kruse Norman J.
Maxygar, Inc.
Reynolds Deborah J.
Townsend & Townsend & Crew
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