Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing a carotene nucleus
Reexamination Certificate
1999-06-02
2003-11-04
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing a carotene nucleus
Reexamination Certificate
active
06642021
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention describes nucleic acid sequences for eukaryotic genes encoding ∈ lycopene ∈-cyclase (also known as ∈-cyclase and ∈ lycopene cyclase), isopentenyl pyrophosphate isomerase (IPP) and &bgr;-carotene hydroxylase as well as vectors containing the same and hosts transformed with said vectors. The present invention also provides methods for augmenting the accumulation of carotenoids, changing the composition of the carotenoids, and producing novel and rare carotenoids. The present invention provides methods for controlling the ratio or relative amounts of various carotenoids in a host. The invention also relates to modified lycopene ∈-cyclase, IPP isomerase and &bgr;-carotene hydroxylase. Additionally, the present invention provides a method for screening for genes and cDNAs encoding enzymes of carotenoid biosynthesis and metabolism.
1. Background of the Invention
Carotenoid pigments with cyclic endgroups are essential components of the photosynthetic apparatus in oxygenic photosynthetic organisms (e.g., cyanobacteria, algae and plants; Goodwin, 1980). The symmetrical bicyclic yellow carotenoid pigment &bgr;-carotene (or, in rare cases, the asymmetrical bicyclic &agr;-carotene) is intimately associated with the photosynthetic reaction centers and plays a vital role in protecting against potentially lethal photooxidative damage (Koyama, 1991). &bgr;-carotene and other carotenoids derived from it or from &agr;-carotene also serve as light-harvesting pigments (Siefermann-Harms, 1987), are involved in the thermal dissipation of excess light energy captured by the light-harvesting antenna (Demmig-Adams & Adams, 1992), provide substrate for the biosynthesis of the plant growth regulator abscisic acid (Rock & Zeevaart, 1991; Parry & Horgan, 1991), and are precursors of vitamin A in human and animal diets (Krinsky, 1987). Plants also exploit carotenoids as coloring agents in flowers and fruits to attract pollinators and agents of seed dispersal (Goodwin, 1980). The color provided by carotenoids is also of agronomic value in a number of important crops. Carotenoids are currently harvested from a variety of organisms, including plants, algae, yeasts, cyanobacteria and bacteria, for use as pigments in food and feed.
The probable pathway for formation of cyclic carotenoids in plants, algae and cyanobacteria is illustrated in FIG.
1
. Two types of cyclic endgroups or rings are commonly found in higher plant carotenoids, these are referred to as the &bgr; (beta) and ∈ (epsilon) rings (FIG.
3
). The precursor acyclic endgroup (no ring structure) is referred to as the &PSgr; (psi) endgroup. The &bgr; and ∈ endgroups differ only in the position of the double bond in the ring. Carotenoids with two &bgr; rings are ubiquitous, and those with one &bgr; and one ∈ ring are common, but carotenoids with two ∈ rings are uncommon. &bgr;-carotene (
FIG. 1
) has two &bgr;-endgroups and is a symmetrical compound that is the precursor of a number of other important plant carotenoids such as zeaxanthin and violaxanthin (FIG.
2
).
Genes encoding enzymes of carotenoid biosynthesis have previously been isolated from a variety of sources including bacteria (Armstrong et al., 1989, Mol. Gen. Genet. 216, 254-268; Misawa et al., 1990, J. Bacteriol., 172, 6704-12), fungi (Schmidhauser et al., 1990, Mol. Cell. Biol. 10, 5064-70), cyanobacteria (Chamovitz et al., 1990, Z. Naturforsch, 45c, 482-86; Cunningham et al., 1994) and higher plants (Bartley et al., Proc. Natl. Acad. Sci USA 88, 6532-36; Martinez-Ferez & Vioque, 1992, Plant Mol. Biol. 18, 981-83). Many of the isolated enzymes show a great diversity in structure, function and inhibitory properties between sources. For example, phytoene desaturases from the cyanobacterium Synechococcus and from higher plants and green algae carry out a two-step desaturation to yield &zgr;-carotene as a reaction product. In plants and cyanobacteria a second enzyme (&zgr;-carotene desaturase), similar in amino acid sequence to the phytoene desaturase, catalyzes two additional desaturations to yield lycopene. In contrast, a single desaturase enzyme from
Erwinia herbicola
and from other bacteria introduces all four double bonds required to form lycopene. The Erwinia and other bacterial desaturases bear little amino acid sequence similarity to the plant and cyanobacterial desaturase enzymes, and are thought to be of unrelated ancestry. Therefore, even with a gene in hand from one source, it may be difficult to identify a gene encoding an enzyme of similar function in another organism. In particular, the sequence similarity between certain of the prokaryotic and eukaryotic genes encoding enzymes of carotenoid biosynthesis is quite low.
Further, the mechanism of gene expression in prokaryotes and eukaryotes appears to differ sufficiently such that one cannot expect that an isolated eukaryotic gene will be properly expressed in a prokaryotic host.
The difficulties in isolating genes encoding enzymes with similar functions is exemplified by recent efforts to isolate the gene encoding the enzyme that catalyzes the formation of &bgr;-carotene from the acyclic precursor lycopene. Although a gene encoding an enzyme with this function had been isolated from a bacterium, it had not been isolated from any photosynthetic procaryote or from any eukaryotic organism. The isolation and characterization of the enzyme catalyzing formation of &bgr;-carotene in the cyanobacterium Synechococcus PCC7942 was described by the present inventors and others (Cunningham et al., 1993 and 1994). The amino acid sequence similarity of the cyanobacterial enzyme to the various bacterial lycopene &bgr;-cyclases is so low (ca. 18-25% overall; Cunningham et al., 1994) that there is much uncertainty as to whether they share a common ancestry or, instead, represent an example of convergent evolution.
The need remains for the isolation of eukaryotic and prokaryotic genes and cDNAs encoding polypeptides involved in the carotenoid biosynthetic pathway, including those encoding a lycopene ∈-cyclase, IPP isomerase and &bgr;-carotene hydroxylase. There remains a need for methods to enhance the production of carotenoids, to alter the composition of carotenoids, and to reduce or eliminate carotenoid production. There also remains a need in the art for methods for screening for genes and cDNAs encoding enzymes of carotenoid biosynthesis and metabolism.
SUMMARY OF THE INVENTION
Accordingly, a first object of this invention is to provide purified and/or isolated nucleic acids which encode enzymes involved in carotenoid biosynthesis; in particular, lycopene ∈-cyclase, IPP isomerase and &bgr;-carotene hydroxylase.
A second object of this invention is to provide purified and/or isolated nucleic acids which encode enzymes which produce novel or uncommon carotenoids.
A third object of the present invention is to provide vectors containing said genes.
A fourth object of the present invention is to provide hosts transformed with said vectors.
Another object of the present invention is to provide hosts which accumulate novel or uncommon carotenoids or which accumulate greater amounts of specific or total carotenoids.
Another object of the present invention is to provide hosts with inhibited and/or altered carotenoid production.
Another object of this invention is to secure the expression of eukaryotic carotenoid-related genes in a recombinant prokaryotic host.
Yet another object of the present invention is to provide a method for screening for eukaryotic and prokaryotic genes and cDNAs which encode enzymes involved in carotenoid biosynthesis and metabolism.
An additional object of the invention is to provide a method for manipulating carotenoid biosynthesis in photosynthetic organisms by inhibiting the synthesis of certain enzymatic products to cause accumulation of precursor compounds.
Another object of the invention is to provide modified lycopene ∈-cyclase, IPP isomerase and &bgr;carotene hydroxylase.
These
Cunningham, Jr. Francis X.
Sun Zairen
Achutamurthy Ponnathapu
Arent Fox Kintner Plotkin & Kahn
Kerr Kathleen
University of Maryland
LandOfFree
Methods of producing carotenoids by the expression of plant... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods of producing carotenoids by the expression of plant..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods of producing carotenoids by the expression of plant... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3136605