WO2009010826A2

WO2009010826A2 - Novel genes and methods of producing carotenoids

Info

Publication number: WO2009010826A2
Application number: PCT/IB2007/004554
Authority: WO
Inventors: Mark A. Scaife; Adam M. Burja; Philip C. Wright
Original assignee: Ocean Nutrition Canada Ltd.
Priority date: 2007-07-13
Filing date: 2007-10-31
Publication date: 2009-01-22
Also published as: WO2009010826A3

Abstract

The present invention relates to the biosynthesis of β-carotene derived carotenoid compounds. This invention is in the field of microbiology. More specifically, this invention pertains to nucleic acids and nuclei acid fragments encoding enzymes useful for production of β-carotene derived carotenoid compounds.

Description

NOVEL GENES AND METHODS OF PRODUCING CAROTENOIDS

FIELD OF INVENTION The present invention relates to the biosynthesis of β-Carotene derived carotenoid compounds. This invention is in the field of microbiology. More specifically, this invention pertains to nucleic acids and nuclei acid fragments encoding enzymes useful for production of β-Carotene derived carotenoid compounds.

BACKGROUND Carotenoids are naturally occurring lipid soluble compounds, synthesised from the ubiquitous C₅ isopentenyl pyrophosphate precursor, and its structural isomer dimethyallyl pyrophosphate. Carotenoids compose an abundant, highly diverse group of compounds. Within this group of compounds two classes exist, the hydrocarbon carotenoids, carotenes, and oxygenated derivatives of carotenes, xanthophylls. Carotenoids are also one of the most abundant and diverse groups of naturally occurring compounds on earth, being responsible for the vibrancy of the world in which we live. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. They are produced ubiquitously by photosynthetic organisms, including cyanobacteria, but are also wide spread among non-photosynthetic bacteria and fungi (Goodwin, The Biochemistry of Carotenoids. New York: Chapman and Hall (1980)). Although carotenoids are classed as secondary metabolites, they are known to be vital for plant growth and photosynthesis. Carotenoids are also essential to many higher organisms, including humans, many of which are not able to synthesise these compounds. Thus dietary intake is the sole source of carotenoids for many organisms. As a result research into dietary sources, and actual consumption of carotenoids, has received much interest in recent years. In 2006, the global market for carotenoids was estimated to be worth US$ 954.7 million, with expectations for this to increase to US $1069.2 million by 2010 (Carotenoids: A Global Strategic Business Report, 2006).

One role of dietary carotenoids is that of a precursor to vitamin A (retinoids). Significant as vitamin A deficiency is the leading cause of preventable blindness in children, while also increasing the risks of disease and death from sever infection (WHO). As dietary antioxidants, carotenoids such as β-carotene, lycopene, astaxanthin, canthaxanthin and lutein, exhibit significant anti-cancer activities, and play an important role in the prevention of chronic diseases (Edge, B-Biology 4, 41, 189-200 (1997); Singh, Oncology-New York, 12, 1643 (1998); Smith, British Journal of Biomedical Science, 55, 268-275 (1998)). Further, consumption of increased amounts of carotenoids has been related to reduced occurrences of degenerative disease such as age-related macular degeneration, the main cause of age related blindness, as well as some cancers and chronic heart disease (Basu, JAOCS, 78 (7), 665-675 (2001); Mares-Perlman et al., JNutr, 132 (3), 518S-524S (2002)).

Owed to their backbone of conjugated double bonds, carotenoids are potent biological antioxidants that can absorb the excited energy of singlet oxygen onto the carotenoid chain, leading to the degradation of the carotenoid molecule, but preventing other molecules or tissues from being damaged (Schagerl and Muller, J Plant Physiol 2005). Carotenoids can be divided into two structurally distinct classes, the carotenes and xanthophylls. Carotenes, possess a hydrocarbon backbone, while xanthophylls contain one or more oxygen-containing functional group (Armstrong and Hearst, Faseb J, 10 (2), 228-3 (1996); Armstrong, Annu Rev Microbiol, 51, 629-59 (1997)). Carotenes are common to many organisms, with a highly conserved mode of biosynthesis. Xanthophylls on the other hand, are produced by a small clutch of organisms. Final products and routes of synthesis are often species specific. Of the Carotenoids, it is the xanthophylls that attract most interest, specifically β-carotene, canthaxanthin and astaxanthin. With β-carotene demanding the largest portion of the global market, while canthaxanthin and astaxanthin command

14.92% and 21.58% of the market, respectively (Carotenoids: A Global Strategic Business Report, 2006).

The xanthophylls encompass various compounds such as the ketocarotenoids, echinenone and canthaxanthin, synthesised by the addition of one or two carbonyl groups at the 4 and 4" positions of β-inone ring of β-carotene, respectively. A reaction catalysed by the class of genes collectively known as β-carotene ketolase genes, β-crytpoxanthin and zeaxanthin are also classed as xanthophylls, these are synthesised by the direct replacement of a hydrogen atom at the 3 and 3" positions of the β-ionone ring of β-carotene with a hydroxyl group (Tian and DellaPenna, Arch Biochem Biophys, 430 (1), 22-9 (2004)). Additionally, there exists a plethora of combined keto and hydroxylated carotenoids, such as 3-hydroxyechinenone, 3^Λ-hydroxyechinenone, adonixanthin, and adonirubin (For example, see Figure 1). All compounds listed here are valuable compounds in their own right with important properties, but significantly they constitute important precursors for the biosynthesis of astaxanthin. Astaxanthin is the final product of this pathway, being both hydroxylated, and ketolated at the 3, 3 ^λ and 4,4' carbons of the β-ionone rings of the β- carotene skeleton. Yet the actual pathway from β-carotene to astaxanthin is not well characterised, and may prove to be species specific (Tao et al., Metab Eng. (2006)).

The specific biosynthetic pathway leading to β-carotene has been studied extensively, thus it is well characterised. It is known that from the isopentenyl pyrophosphate and dimethyallyl pyrophosphate precursors, four intermediate compounds are biosynthesised prior to β-carotene. These compounds are farnesyl diphosphate, geranylgeranyl diphosphate, phytoene and lycopene. As with the biosynthesis of β-carotene, much interest has also been shown in the pathway leading to astaxanthin. However this pathway appears more complex, often presented as a web, with multiple possible intermediates and directions in which biosynthesis may proceed. Although all of these permutations are theoretically possible, little work has been completed to determine exactly which routes are correct, and which are not.

However, it is known that just two enzymes are required for the conversion of β- carotene to astaxanthin, β-carotene hydroxylase and β-carotene ketolase. To date, two types of β-carotene ketolase gene have been characterised which both act upon β-inone rings. These are widely distributed among cyanobacteria. These are the di-keto (canthaxanthin) producing, CrtW type, and the mono-keto (echinenone plus some canthaxanthin) producing, CrtO type. Although catalysing similar reactions, these two types of β-carotene ketolase share no sequence similarities, and so are believed to be the result of convergent evolution (Mochimaru et al., FEBS Lett, 579 (27), 6111-4 (2005)). Additionally there are two types of β-carotene hydroxylases known, β- and ε- hydroxylases, which add a hydroxyl group to either the β- or ε- rings, respectively (Tian and DellaPenna, Arch Biochem Biophys, 430 (1), 22-9 (2004)). The β-carotene hydroxylase genes, although completing the same reaction, can be separated into three groups, based on primary structure. These are the plant and algal, non-photosynthetic bacteria and cyanobacterial β-carotene hydroxylases (Tian and DellaPenna, 2004). β-carotene hydroxylase genes are less well represented amongst cyanobacteria. To date, cyanobacterial β-carotene hydroxylase genes have only been functionally characterised in Synechocystis sp. PCC 6803 (Masamoto et al., Plant Cell Physiol, 39 (5), 560-4 (1998)).

Higher non-photosynthetic organisms, mammals, including humans, lack the genetic machinery for the biosynthesis of carotenoids. Thus carotenoids must be obtained heterotrophically, from dietary sources, via consumption of fruits and vegetables, rich in these compounds. Arguably the most important biological function of carotenoids is their antioxidant potential, demonstrated in their ability to inactivate or quench various free radicals. Owing to this, it is suggested that carotenoids play a vital role in human health (Wang et al, Biotechnol Adv. (2006)). Benefits related to carotenoid consumption including; diminished risk of various cancers, cardiovascular and ophthalmological diseases (Mayne et al., Faseb J 10:690-701 (1996)). As well as enhanced immune response and cell signalling. Additionally, carotenoids are known precursors of vitamin A, which has been implicated in inhibition of tumour development.

SUMMARY OF THE INVENTION

In accordance with the purpose of this invention, as embodied and broadly described herein, this invention relates to β-carotene ketolases and β-carotene hydroxylases and their use in producing carotenoids. Thus provided herein are methods and compositions of matter for producing β-Carotene derived carotenoids. Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention. These are non-limiting examples.

Figure 1 shows a proposed biosynthetic pathway for conversion of beta-carotene to Astaxanthin.

Figure 2 shows the relative catalytic activates (μg g._! DCW) of cyanobacterial CrtO type β-carotene ketolase genes, at 3O⁰C, investigated via co-expression in TOPlO E. coli engineered to accumulate β-carotene (p AC-BET A).

Figure 3 shows the relative catalytic activates (μg g._\ DCW) of cyanobacterial CrtW type β-carotene ketolase genes, at 3O⁰C, investigated via co-expression in TOPlO E. coli engineered to accumulate β-carotene (pAC-BETA). Figure 4 shows the relative activates (μg g^"1 DCW) of cyanobacterial CrtW type β- carotene ketolase genes investigated via co-expression in TOPlO E. coli engineered to accumulate β-carotene (pAC-ZEAX IDI), not producing astaxanthin.

Figure 5 shows Astaxanthin accumulation (μg g^"1 DCW) in E. coli due to co- expression of cyanobacterial CrtW type β-carotene ketolase genes and plasmids pAC- ZEAX IDI.

Figure 6 shows Carotenoid biosynthesis (μg g^"1 DCW) in response to β-carotene ketolase expression, induced with varying concentrations of L-arabinose.

Figure 7 shows Carotenoid extraction from Lyngbya sp. CCAP 1446/5 carotenoids; Zeaxanthin (1), β-cryptoxanthin (2), Echinenone (3), β-carotene (4).

Figure 8 shows Carotenoid accumulation, in zeaxanthin accumulating E. coli due to expression of different cyanobacterial b-carotene ketolase genes from pBAD24.

Figure 9 shows Zeaxanthin biosynthesis in various E. coli strains, cultured at 30oC for 24 hours. Figure 10 shows Zeaxanthin biosynthesis in response to over expression of

Isopentenyl Diphosphate Isomerase (IDI from Er. herbicola) and l-deoxy-D-xylulose-5- phosphate synthase (DXS from Anabaena variabilis ATCC 29413 or E. coli JMlOl).

Figure 11 shows Comparison of astaxanthin biosynthesis in E. coli using the previously reported (Figure 3) single expression vector and dual expression (b-carotene ketolase and b-carotene hydroxylase) vector.

Figure 12 shows Zeaxanthin biosynthesis in response to carbon supplementation of growth media (Luria Bertani (LB)).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and the Tables and their previous and following description.

All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific nucleic acids or to particular methods, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It is to be understood that this invention is not limited to specific synthetic methods, or to specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, to specific pharmaceutical carriers, or to particular pharmaceutical formulations or administration regimens, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Definitions and Nomenclature The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes mixtures of nucleic acids, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the phrase

"optionally obtained prior to treatment" means obtained before treatment, after treatment, or not at all.

"Polypeptide" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules. By "isolated polypeptide" or "purified polypeptide" is meant a polypeptide (or a fragment thereof) that is substantially free from the materials with which the polypeptide is normally associated in nature. The polypeptides of the invention, or fragments thereof, can be obtained, for example, by extraction from a natural source (for example, a mammalian cell), by expression of a recombinant nucleic acid encoding the polypeptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide. In addition, polypeptide fragments may be obtained by any of these methods, or by cleaving full length polypeptides. In addition, as used herein, the term "polypeptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins - Structure and Molecular Properties 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)). As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, characters or words representing amino acid residues.

The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

As used herein, "peptidomimetic" means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance some property of the original peptide, such as increase stability, increased efficacy, enhanced delivery, increased half life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include β-alanine, L-α- amino butyric acid, L-γ-amino butyric acid, L-α-amino isobutyric acid, L-ε-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc- N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc- hydroxyproline, and Boc-L-thioproline.

The word "or" as used herein means any one member of a particular list and also includes any combination of members of that list.

The phrase "nucleic acid" as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single- stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

By "isolated nucleic acid" or "purified nucleic acid" is meant DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, such as an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote (e.g., a transgene); or which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR, restriction endonuclease digestion, or chemical or in vitro synthesis). It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term "isolated nucleic acid" also refers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components, for example, other types of RNA molecules or polypeptide molecules. For example, a naturally-occurring polynucleotide is isolated if it is separated from some or all of the coexisting materials in the natural system.

By a "transgene" is meant a nucleic acid sequence that is inserted by artifice into a cell and becomes a part of the genome of that cell and its progeny. Such a transgene may be (but is not necessarily) partly or entirely heterologous (for example, derived from a different species) to the cell.

The term "cell" as used herein also refers to individual cells, or cultures derived from such cells. For example, the term "cell" can refer to to individual microbial cells, or cultures derived from such cells. A "culture" refers to a composition comprising isolated cells of the same or a different type.

By "transgenic animal" is meant an animal comprising a transgene as described above. Transgenic animals can be made by techniques that are well known in the art.

By "transgenic plant" is meant a plant comprising a transgene as described above. Transgenic plants can be made by techniques that are well known in the art.

By "transgenic cell" is meant a cell comprising a transgene as described above. Transgenic cells can be made by techniques that are well known in the art.

By "knockout mutation" is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift, or missense mutation. A "knockout animal," for example, a knockout mouse, is an animal containing a knockout mutation. The knockout animal or plant may be heterozygous or homozygous for the knockout mutation. Such knockout animals or plants are generated by techniques that are well known in the art. A preferred form of knockout mutation is one where the biological activity of a β-carotene ketolase or β-carotene hydroxylase is not completely eliminated.

By "specifically binds" is meant that an antibody recognizes and physically interacts with its cognate antigen (for example, a β-carotene ketolase polypeptide) and does not significantly recognize and interact with other antigens; such an antibody may be a polyclonal antibody or a monoclonal antibody, which are generated by techniques that are well known in the art.

By "probe," "primer," or oligonucleotide is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target"). The stability of the resulting hybrid depends upon the extent of the base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes or primers specific for β-carotene ketolase or hydroxylase nucleic acids (for example, genes and/or mRNAs) have at least 80%-90% sequence complementarity, preferably at least 91%-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the β-carotene ketolase or hydroxylase nucleic acid to which they hybridize. Probes, primers, and oligonucleotides may be detectably- labeled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art. Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).

By "specifically hybridizes" is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a β-carotene ketolase or hydroxylase nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.

By "high stringency conditions" is meant conditions that allow hybridization comparable with that resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65⁰C, or a buffer containing 48% formamide, 4.8X SSC, 0.2 M Tris-Cl, pH 7.6, IX Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42⁰C. Other conditions for high stringency hybridization, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well-known by those skilled in the art of molecular biology. (See, for example, F. Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998).

By "Open reading frame" or "ORF" is meant a portion of an organism's genome which contains a sequence of bases that could potentially encode a protein. By "Polymerase chain reaction" or "PCR" is meant a biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment of DNA, via enzymatic replication.

The term "isoprenoid" or "terpenoid" refers to the compounds or any molecule derived from the isoprenoid pathway including 10 carbon terpenoids and their derivatives, such as carotenoids and xanthophylls.

The term "carotenoid" refers to a compound composed of a polyene backbone which is condensed from five-carbon isoprene unit. Carotenoids can be acyclic or terminated with one (monocyclic) or two (bicyclic) cyclic end groups. The term "carotenoid" may include both carotenes and xanthophylls. A "carotene" refers to a hydrocarbon carotenoid.

Carotene derivatives that contain one or more oxygen atoms, in the form of hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups, or within glycosides, glycoside esters, or sulfates, are collectively known as "xanthophylls". Carotenoids that are particularly suitable in the present invention are monocyclic and bicyclic carotenoids. The term "carotenoid ketolase" or "ketolase" or "cyclic carotenoid ketolase" refers to the group of enzymes that can add keto groups to the ionone ring of either monocyclic or bicyclic carotenoids.

The term "carotenoid hydroxylase" or "hydroxylase" or "cyclic carotenoid hydroxylase" refers to the group of enzymes that can add hydroxyl groups to the ionone ring of either monocyclic or bicyclic carotenoids.

The term "ketocarotenoid" refers to a keto group-containing carotenoid.

The term "β-Carotene derived carotenoid" refers to both carotenoids and xanthophyll compounds derived from carotene and intermediates thereof. The intermediates can be both naturally occurring and chemically synthesized intermediates. The term "motif refers to short conserved amino acid sequences found in a group of protein sequences. Motifs frequently form a recognition sequence or are highly conserved parts of domains. Motif may also refer to all localized homology regions, independent of their size. A motif descriptor could be used to describe the short sequence motifs, consisting of amino acid characters and other characters represent ambiguities and length insertions. The term "keto group" or "ketone group" will be used interchangeably and refers to a group in which a carbonyl group is bonded to two carbon atoms: R₂CO (neither R may be H).

As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the invention encompasses more than the specific exemplary sequences.

For example, it is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein are common. For the purposes of the present invention substitutions are defined as exchanges within one of the following five groups: 1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, GIy); 2. Polar, negatively charged residues and their amides: Asp, Asn, GIu, GIn; 3. Polar, positively charged residues: His, Arg, Lys; 4. Large aliphatic, nonpolar residues: Met, Leu, lie, VaI (Cys); and 5. Large aromatic residues: Phe, Tyr, Trp.

A "substantial portion" of an amino acid or nucleotide sequence comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. MoI. Biol. 215:403 410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence often or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20 30 contiguous nucleotides may be used in sequence- dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular microbial proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above. The term "complementary" is used to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.

The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the

LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151 153) with the default parameters (GAP PENALTY=IO, GAP LENGTH PENALTY=IO). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1 , GAP PENALTY=3, WIND0W=5 and DIAGONALS SAVED=5.

"Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. T hese building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. "Chemically synthesized", as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available. "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.

"Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

"RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. "Sense" RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. "Antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO 9928508). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

"Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

The term "carbon substrate" refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.

The term "altered biological activity" will refer to an activity, associated with a protein encoded by a microbial nucleotide sequence which can be measured by an assay method, where that activity is either greater than or less than the activity associated with the native microbial sequence. "Enhanced biological activity" refers to an altered activity that is greater than that associated with the native sequence. "Diminished biological activity" is an altered activity that is less than that associated with the native sequence.

"Isolating" and any form such as "isolate" refer to a situation where something is in a form wherein it can be manipulated or further purified. Isolated and its forms indicates that something is in a current state which is different than a previous state. For example, a ribosomal RNA molecule can be "isolated" if it is, for example removed from an organism, synthesized or recombinantly produced. Often, the "isolation" of one thing is in relation to something else. For example, a eukaryote as discussed herein can be isolated as discussed herein, by, for example, culturing the eukaryote, such that the eukaryote survives in the absence of appreciable amounts (detectable) of other organisms. It is understood that unless specifically indicated otherwise, any of the disclosed compositions can be isolated as disclosed herein.

"Purify" and any form such as "purifying" refers to the state in which a substance or compound or composition is in a state of greater homogeneity than it was before. It is understood that as disclosed herein, something can be, unless otherwise indicated, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure. For example, if a given composition A was 90% pure, this would mean that 90% of the composition was A, and that 10% of the composition was one or more things, such as molecules, compounds, or other substances. For example, if a disclosed eukaryotic microorganism, for example, produces 35% DHA, this could be further "purified" such that the final lipid composition was greater than 90% DHA. Unless otherwise indicated, purity will be determined by the relative "weights" of the components within the composition. It is understtod that unless specifically indicated otherwise, any of the disclosed compositions can be purified as disclosed herein.

Compositions The disclosed compositions are related to β-carotene ketolases and β-carotene hydroxylases. For example, disclosed are nucleic acid sequences capable of encoding a β- carotene ketolase or a β-carotene hydroxylase.

It was previously proposed by Ye, et al (2006) ( Ye, et al., Appl Environ Microbiol 72:5829-37 (2006)) stated, referencing the works by Fraser, et al. (Fraser, et al., J Biol Chem 272:6128-35 (1997)), that reactions that produce β-carotene derived carotenoids can proceed with either the ketolation or hydroxylation reaction first, followed by the alternate reaction, resulting in the biosynthesis of astaxanthin. Implying that a β-carotene ketolase enzyme is not only able to utilise β-carotene as a substrate in the biosynthesis of canthaxanthin, via echinenone, but that it is also able to accept hydroxylated compounds, β- cryptoxanthin and zeaxanthin. Similarly, a β-carotene hydroxylase enzyme must be able to accept β-carotene, echinenone and canthaxanthin as substrates. Analysis of the work by Fraser, et al (1997) reveals that this indeed demonstrates the ability of β-carotene hydroxylase enzymes to accept canthaxanthin as a substrate in the biosynthesis of astaxanthin. While also showing that β-carotene ketolase enzymes are not able to accept hydroxylated compounds as substrates in the biosynthesis of astaxanthin. Thus the interpretation of Ye, and co workers, appears to be incorrect. With Fraser, et al (1997) suggesting in the biosynthesis of astaxanthin, the β-carotene ketolase must initially catalyse the conversion of β-carotene to canthaxanthin, and the β-carotene hydroxylase will subsequently convert canthaxanthin into astaxanthin. Several cyanobacteria and their ability to produce β-carotene derived carotenoids have been previously studied. For example, Synechocystis sp. PCC 6803, has been shown to accumulate β-carotene, echinenone, 3 -Hydroxy echinenone and zeaxanthin, in addition to myxol-2-dimethyl fucoside (Takaichi et al., Plant Cell Physiol 42:756-62 (2001)). The presence of functional β-carotene ketolase and hydroxylase genes within its genome have also been confirmed (Fernandez-Gonzalez et al., J Biol Chem 272:9728-33 (1997);

Masamoto et al., Plant Cell Physiol 39:560-4 (1998)). However, co-expression of the Synechocystis sp. PCC 6803 derived β-carotene ketolase enzyme in β-carotene accumulating E. coli resulted in the accumulation of both echinenone and canthaxanthin (Fernandez-Gonzalez et al., J Biol Chem 272:9728-33 (1997)). Canthaxanthin was not reported to accumulate in the native organism. Also Synechocystis sp. PCC 6803 was previously believed to lack a putative CrtW type β-carotene ketolase gene.

In addition, the genome of Nostoc punctiforme PCC 73102 encodes four β-carotene ketolase genes, two CrtW and two CrtO type. Nostoc punctiforme PCC 73102 has been shown to accumulate various carotenoids, including β-carotene, β-cryptoxanthin, echinenone and canthaxanthin, along with ketomyxol-2' -fucoside and myxol 2'-fucosied (Takaichi et al., Plant Cell Physiol 42:756-62 (2001)). Of the four putative β-carotene ketolase genes encoded, only the two CrtW type have been investigated. Expression of each of the CrtW genes separately resulted in the accumulation of both echinenone and canthaxanthin, but with different levels of activity (Steiger et al., Biotechnol Lett 26:813-7 (2004)). Additionally, over expression of the most active ketolase resulted in accumulation of astaxanthin, within a zeaxanthin accumulating E. coli strain (Steiger et al., Biotechnol Lett 26:813-7 (2004)). A single putative β-carotene hydroxylase gene is also encoded by the genome of Nostoc punctiforme PCC 73102, however this is yet to be characterised. A lack of hydroxylated carotenoids in the native organism, previously suggested this may inactive. Anabaena variables ATCC 29413 is able to accumulate β-carotene, echinenone and canthaxanthin as it major carotenoids, in addition to 4-hydroxymyxol and (3R,2'S)-myxol ( Takaichi et al., Plant Cell Physiol 46:497-504 (2005)). Present within its genome are single copies of putative CrtW and CrtO β-carotene ketolase genes, and a single putative β- carotene hydroxylase gene.

Nostoc (Anabaena) sp. PCC 7120 is know to accumulate various carotenoids, including β-carotene and echinenone, as major compounds, with canthaxanthin, 3- hydroxyechinenone, zeaxanthin, myxol-2-fuciside and 4-ketomyxol as a minor compounds ( Mochimaru et al., FEBS Lett 579:6111-4. (2005); Takaichi et al., Plant Cell Physiol 46:497-504 (2005)). Present within the genome of Nostoc sp. PCC 7120 are putative copies of both CrtW and CrtO β-carotene ketolase genes, and a putative β-carotene hydroxylase gene. Deletion of the CrtO gene, resulted in total depletion of canthaxanthin, and the accumulation of small amounts of echinenone, despite the presence of a putative CrtW gene ( Takaichi et al., Plant Cell Physiol 46:497-504 (2005)), while deletion of the CrtW gene implicated it in the conversion of myxol 2'-fucoside to ketomyxol 2'-fucoside, as well as conversion of echinenone to canthaxanthin (Mochimaru et al., FEBS Lett 579:6111-4 (2005)).

The carotenoid biosynthetic pathway of Gleobacter violaceus PCC 7421 has also been studied (Steiger et al., Arch Microbiol 184:207-14 (2005); Tsuchiya et al., FEBS Lett 579:2125-9 (2005)). From the studies it was shown that the carotenoid compounds of

Gleobacter are β-carotene, oscillol difucoside and echinenone (Id.). It is also shown that the genome encodes single putative copies of the CrtO and CrtW type β-carotene ketolase gene, and lacks a putative β-carotene hydroxylase.

Disclosed herein are compositions comprising a nucleic acid that encodes a polypeptide, wherein the polypeptide has at least 80% identity to SEQ ID NO. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60.

Also disclosed are compositions comprising a nucleic acid that encodes a polypeptide, wherein the polypeptide has at least 80% identity to SEQ ID NO. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, wherein the nucleic acid comprises a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47 or a complement thereof.

Also disclosed are compositions comprising a polypeptide wherein the polypeptide is encoded by a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

Also disclosed are compositions comprising a polypeptide wherein the polypeptide is encoded by a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof, wherein the polypeptide comprises the sequence provided in SEQ ID NOs: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, or a complement thereof.

The compositions described herein can further comprise a vector and/or a host cell. Also disclosed are isolated polypeptides, wherein the polypeptide is encoded by a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof. Also disclosed are compositions comprising an isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof. Also disclosed are compositions comprising an isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof, wherein the composition consists essentially of an isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

Also disclosed are compositions comprising a β-Carotene derived carotenoid isolated from a bacteria.

Also disclosed are compositions comprising a β-Carotene derived carotenoid, wherein the composition is produced by the methods described below. As mentioned above, there are two types of β-carotene ketolase, CrtO and CrtW.

Despite catalysing the same reaction, these classes of enzyme share no sequence homology. Additionally these classes of enzyme result in the predominant accumulation of two different compounds. With CrtO type enzymes catalysing the predominant accumulation of the mono-ketolated compound, echinenone, while CrtW type enzymes catalysing the accumulation of the di-ketolated compound, canthaxanthin, with echinenone as a minor intermediate, β-carotene ketolase enzymes have been previously investigated with respect to function and involvement in astaxanthin production. This includes enzymes from Haematococcus pulvialis and various Paracoccus strains. Yet cyanobacterial derived β- carotene ketolase enzymes represent a distantly related group of enzymes. Disclosed herein are nucleic acid sequences capable of encoding either a CrtO or a CrtW type β-carotene ketolase. CrtO type β-carotene ketolase genes were isolated from Lyngbya sp. CCAP 1446/5, Anabaena variabilis ATCC 29413, Nostoc punctiforme PCC 73102.1 and 73102.2 (denoting two CrtW from this organism) and Nostoc sp. PCC 7120. CrtW type β-carotene ketolase genes were isolated from Anabaena variabilis ATCC

29413.1 and 29413.2 (denoting two CrtW from this organism) and Nostoc sp. PCC 7120.

Also disclosed are isolated nucleic acids comprising a nucleic acid sequence at least about 60, 65, 70, 75, 08, 85, 90, 95, 96, 97, 98, 99, or 100% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-9, 45-47, or a complement thereof.

Also disclosed are isolated nucleic acids comprising a nucleic acid sequence, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1- 9, 45-47, or a complement thereof

Also disclosed herein are isolated nucleic acids comprising a nucleic acid sequence at least about 90% identical to the nucleic acid sequnece of SEQ ID NO: 10, or a complement thereof.

Also disclosed herein are isolated nucleic acids comprising a nucleic acid sequence, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 10, or a complement thereof. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell that the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.

The nucleic acids, such as, the polynucleotides described herein, can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer. Synthetic methods useful for making oligonucleotides are also described by Ikuta et al, Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester methods), and Narang et al, Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al, Bioconjug. Chem. 5:3-7 (1994).

The nucleotides of the invention can comprise one or more nucleotide anaologs or substitutions. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl (ψ), hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. A modified base includes but is not limited to 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional base modifications can be found for example in U.S. Pat. No. 3,687,808, Englisch et al , Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine can increase the stability of duplex formation. Often time base modifications can be combined with for example a sugar modifcation, such as 2'-O-methoxyethyl, to achieve unique properties such as increased duplex stability. There are numerous United States patents such as 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091 ;

5,614,617; and 5,681,941, which detail and describe a range of base modifications. Each of these patents is herein incorporated by reference.

Nucleotide analogs can also include modifications of the sugar moiety.

Modifications to the sugar moiety would include natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Ci₀, alkyl or C₂ to Ci₀ alkenyl and alkynyl. 2' sugar modiifcations also include but are not limited to -O[(CH₂)_n O]_m CH₃, -

O(CH₂)_n OCH₃, -O(CH₂)_n NH₂, -O(CH₂)_n CH₃, -O(CH₂)_n -ONH₂, and -O(CH₂)_nON[(CH₂)_n CH₃)]₂, where n and m are from 1 to about 10.

Other modifications at the 2' position include but are not limted to: C₁ to C]₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures such as 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811 ; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety for their teaching of modifications and methods related to the same.

Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate linkage between two nucleotides can be through a 3 '-5' linkage or a 2'-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361 ; and 5,625,050, each of which is herein incorporated by reference in its entirety for their teaching of modifications and methods related to the same.

Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid

(PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. Numerous United States patents disclose how to make and use these types of phosphate replacements and include but are not limited to 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference in its entirety for their teaching of modifications and methods related to the same.

It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). United States patents 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference in its entirety for their teaching of modifications and methods related to the same. (See also Nielsen et al, Science, 254, 1497-1500 (1991)). It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al, Bioorg. Med. Chem. Let., 1994, 4, 1053- 1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al , Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al, Bioorg. Med. Chem. Let, 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J., 1991, 10, 111 1-1118; Kabanov et al, FEBS Lett., 1990, 259, 327-330; Svinarchuk et al, Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al, Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al. , Tetrahedron Lett., 1995, 36, 3651 -3654), a palmityl moiety (Mishra et al , Biochim. Biophys. Acta, 1995, 1264,

229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277, 923-937.

Numerous United States patents teach the preparation of such conjugates and include, but are not limited to U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731 ; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481 ; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference in its entirety for their teaching of modifications and methods related to the same. Also, disclosed are compositions including primers and probes, which are capable of interacting with the polynucleotide sequences disclosed herein. For example, disclosed are primers/probes capable of amplifying a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1- 10, 45-47, or a complement thereof. Also disclosed are primers/probes capable of amplifying the nucleic acid sequences set forth in SEQ ID NOs: 1-10 and 45-47. Examples of such primers/probes are disclosed in Tables 4 - 7.

The disclosed primers can used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non- enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the polynucleotide sequences disclosed herein or region of the polynucleotide sequences disclosed herein or they hybridize with the complement of the polynucleotide sequences disclosed herein or complement of a region of the polynucleotide sequences disclosed herein.

The size of the primers or probes for interaction with the polynucleotide sequences disclosed herein in certain embodiments can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification or the simple hybridization of the probe or primer. A typical primer or probe would be at least 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,

475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long or any length inbetween.

Also disclosed are functional nucleic acids that can interact with the disclosed polynucleotides. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de no vo activity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA of polynucleotide sequences disclosed herein or the genomic DNA of the polynucleotide sequences disclosed herein or they can interact with the polypeptide encoded by the polynucleotide sequences disclosed herein. Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.

Disclosed herein are antisense molecules that interact with the disclosed polynucleotides. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (kd) less than or equal to 10^"6, 10^"8, 10^"10, or 10^"12. A representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non-limiting list of United States patents: 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317,

5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437 each of which is herein incorporated by reference in its entirety for their teaching of modifications and methods related to the same.

Also disclosed are aptamers that interact with the disclosed polynucleotides. Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP (United States patent 5,631,146) and theophiline (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293). Aptamers can bind very tightly with k_dS from the target molecule of less than 10^"12 M. It is preferred that the aptamers bind the target molecule with a k_d less than 10^"6, 10^"8, 10^"10, or 10^"12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (United States patent 5,543,293). It is preferred that the aptamer have a k_d with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the k_d with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide. For example, when determining the specificity of aptamers, the background protein could be ef- lα. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698.

Also disclosed are ribozymes that interact with the disclosed polynucleotides. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (for example, but not limited to the following United States patents: 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463,

5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following United States patents: 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, but not limited to the following United States patents: 5,595,873 and 5,652,107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following United States patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non- limiting list of United States patents: 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.

Also disclosed are triplex forming functional nucleic acid molecules that interact with the disclosed polynucleotides. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson- Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k_d less than 10^"6, 10^"8, 10^"10, or 10^"12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.

Also disclosed are external guide sequences that form a complex with the disclosed polynucleotides. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altman, Science 238:407-409 (1990)). Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells. (Yuan et al, Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, EMBO J 14:159-168 (1995), and Carrara et al, Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)). Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules can be found in the following non- limiting list of United States patents: 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.

Also disclosed are polynucleotides that contain peptide nucleic acids (PNAs) compositions. PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997; 7(4) 431-37). PNA is able to be utilized in a number of methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. A review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (Trends Biotechnol 1997 June; 15(6):224-9). As such, in certain embodiments, one may prepare PNA sequences that are complementary to one or more portions of an mRNA sequence based on the disclosed polynucleotides, and such PNA compositions may be used to regulate, alter, decrease, or reduce the translation of the disclosed polynucleotides transcribed mRNA, and thereby alter the level of the disclosed polynucleotide's activity in a host cell to which such PNA compositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al, Science Dec. 6, 1991; 254(5037):1497-500; Hanvey et al, Science. Nov. 27, 1992; 258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 January; 4(1): 5-23). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achirial, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used. PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al , Bioorg Med Chem. 1995 April; 3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNAs can incorporate any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In expectation of this difficulty, it is suggested that, in producing PNAs with adjacent purines, one should repeat the coupling of residues likely to be added inefficiently. This should be followed by the purification of PNAs by reverse- phase high-pressure liquid chromatography, providing yields and purity of product similar to those observed during the synthesis of peptides. Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNAs (for example, Norton et al, Bioorg Med Chem. 1995 April; 3(4):437-45; Petersen et al, J Pept Sci. 1995 May- June; l(3):175-83; Oram et al, Biotechniques. 1995 September; 19(3):472-80; Footer et al., Biochemistry. Aug. 20, 1996; 35(33): 10673-9; Griffith et al, Nucleic Acids Res. Aug. 11, 1995; 23(15):3003-8; Pardridge et al, Proc Natl Acad Sci USA. Jun. 6, 1995; 92(12):5592-6; Boffa etal, Proc Natl Acad Sci USA. Mar. 14, 1995; 92(6):1901-5; Gambacorti-Passerini et al, Blood. Aug. 15, 1996; 88(4):1411-7; Armitage et al, Proc Natl Acad Sci USA. Nov. 11, 1997; 94(23): 12320-5; Seeger et al, Biotechniques. 1997 September; 23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA- DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs are discussed in Rose (Anal Chem. Dec. 15, 1993; 65(24):3545-9) and Jensen et al. (Biochemistry. Apr. 22,

1997; 36(16):5072-7). Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by Jensen et al. using BIAcore™ technology.

Other applications of PNAs that have been described and will be apparent to the skilled artisan include use in DNA strand invasion, antisense inhibition, mutational analysis, enhancers of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome cleavage, biosensors, in situ hybridization, and the like.

Optionally, isolated nucleotides or isolated polypeptides can also be purified, e.g., are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.

Also disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular polynucleotide is disclosed and discussed and a number of modifications that can be made to a number of molecules including the polynucleotide are discussed, specifically contemplated is each and every combination and permutation of polynucleotide and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment.

For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).

The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5⁰C to 20°C below the Tm. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987: 154:367, 1987 which is herein incorporated by reference in its entirety and at least for material related to hybridization of nucleic acids). As used herein "stringent hybridization" for a DNA:DNA hybridization is about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art. Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k_d, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k_d.

Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein. It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein. Optionally, one or more of the isolated polynucleotides of the invention are attached to a solid support. Solid supports are disclosed herein. Also disclosed herein are arrays comprising polynucleotides capable of specifically hybridizing to a nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof. Also disclosed herein are solid supports comprising one or more polypeptides encoded by a nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1- 10, 45-47, or a complement thereof, attached to the solid support.

Solid supports are solid-state substrates or supports with which molecules, such as analytes and analyte binding molecules, can be associated. Analytes, such as calcifying nano-particles and proteins, can be associated with solid supports directly or indirectly. For example, analytes can be directly immobilized on solid supports. Analyte capture agents, such a capture compounds, can also be immobilized on solid supports. For example, disclosed herein are antigen binding agents capable of specifically binding to a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1- 10, 45-47, or a complement thereof.

A preferred form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which multiple different capture compounds or detection compounds have been coupled in an array, grid, or other organized pattern.

Solid-state substrates for use in solid supports can include any solid material to which molecules can be coupled. This includes materials such as acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Solid-state substrates can have any useful form including thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers, particles, beads, microparticles, or a combination. Solid-state substrates and solid supports can be porous or non-porous. A preferred form for a solid-state substrate is a microtiter dish, such as a standard 96-well type. In preferred embodiments, a multiwell glass slide can be employed that normally contain one array per well. This feature allows for greater control of assay reproducibility, increased throughput and sample handling, and ease of automation. Different compounds can be used together as a set. The set can be used as a mixture of all or subsets of the compounds used separately in separate reactions, or immobilized in an array. Compounds used separately or as mixtures can be physically separable through, for example, association with or immobilization on a solid support. An array can include a plurality of compounds immobilized at identified or predefined locations on the array. Each predefined location on the array generally can have one type of component (that is, all the components at that location are the same). Each location will have multiple copies of the component. The spatial separation of different components in the array allows separate detection and identification of the polynucleotides or polypeptides disclosed herein. Although preferred, it is not required that a given array be a single unit or structure.

The set of compounds may be distributed over any number of solid supports. For example, at one extreme, each compound may be immobilized in a separate reaction tube or container, or on separate beads or microparticles. Different modes of the disclosed method can be performed with different components (for example, different compounds specific for different proteins) immobilized on a solid support.

Some solid supports can have capture compounds, such as antibodies, attached to a solid-state substrate. Such capture compounds can be specific for calcifying nano-particles or a protein on calcifying nano-particles. Captured calcifying nano-particles or proteins can then be detected by binding of a second, detection compound, such as an antibody. The detection compound can be specific for the same or a different protein on the calcifying nano-particle.

Methods for immobilizing antibodies (and other proteins) to solid-state substrates are well established. Immobilization can be accomplished by attachment, for example, to aminated surfaces, carboxylated surfaces or hydroxylated surfaces using standard immobilization chemistries. Examples of attachment agents are cyanogen bromide, succinimide, aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable agents, epoxides and maleimides. A preferred attachment agent is the heterobifunctional cross-linker N- [γ- Maleimidobutyryloxy] succinimide ester (GMBS). These and other attachment agents, as well as methods for their use in attachment, are described in Protein immobilization: fundamentals and applications, Richard F. Taylor, ed. (M. Dekker, New York, 1991);, Johnstone and Thorpe, Immunochemistry In Practice (Blackwell Scientific Publications, Oxford, England, 1987) pages 209-216 and 241-242, and Immobilized Affinity Ligands; Craig T. Hermanson et al., eds. (Academic Press, New York, 1992) which are incorporated by reference in their entirety for methods of attaching antibodies to a solid-state substrate. Antibodies can be attached to a substrate by chemically cross-linking a free amino group on the antibody to reactive side groups present within the solid-state substrate. For example, antibodies may be chemically cross-linked to a substrate that contains free amino, carboxyl, or sulfur groups using glutaraldehyde, carbodiimides, or GMBS, respectively, as cross- linker agents. In this method, aqueous solutions containing free antibodies are incubated with the solid-state substrate in the presence of glutaraldehyde or carbodiimide.

A preferred method for attaching antibodies or other proteins to a solid-state substrate is to functionalize the substrate with an amino- or thiol-silane, and then to activate the functionalized substrate with a homobifunctional cross-linker agent such as (Bis-sulfo- succinimidyl suberate (BS³) or a heterobifunctional cross-linker agent such as GMBS. For cross-linking with GMBS, glass substrates are chemically functionalized by immersing in a solution of mercaptopropyltrimethoxysilane (1% vol/vol in 95% ethanol pH 5.5) for 1 hour, rinsing in 95% ethanol and heating at 120 ⁰C for 4 hrs. Thiol-derivatized slides are activated by immersing in a 0.5 mg/ml solution of GMBS in 1% dimethylformamide, 99% ethanol for 1 hour at room temperature. Antibodies or proteins are added directly to the activated substrate, which are then blocked with solutions containing agents such as 2% bovine serum albumin, and air-dried. Other standard immobilization chemistries are known by those of skill in the art.

Each of the components (compounds, for example) immobilized on the solid support preferably is located in a different predefined region of the solid support. Each of the different predefined regions can be physically separated from each other of the different regions. The distance between the different predefined regions of the solid support can be either fixed or variable. For example, in an array, each of the components can be arranged at fixed distances from each other, while components associated with beads will not be in a fixed spatial relationship. In particular, the use of multiple solid support units (for example, multiple beads) will result in variable distances.

Components can be associated or immobilized on a solid support at any density. Components preferably are immobilized to the solid support at a density exceeding 400 different components per cubic centimeter. Arrays of components can have any number of components. For example, an array can have at least 1,000 different components immobilized on the solid support, at least 10,000 different components immobilized on the solid support, at least 100,000 different components immobilized on the solid support, or at least 1 ,000,000 different components immobilized on the solid support. Optionally, at least one address on the solid support is the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein. Also disclosed are solid supports where at least one address is the sequences or portion of sequences set forth in any of the polynucleotide or polypeptide sequences disclosed herein. Solid supports can also contain at least one address is a variant of the sequences or part of the sequences set forth in any of the polynucleotide or polypeptide sequences disclosed herein.

Also disclosed are antigen microarrays for multiplex characterization of antibody responses. For example, disclosed are antigen arrays and miniaturized antigen arrays to perform large-scale multiplex characterization of antibody responses directed against the polypeptides, polynucleotides and antibodies described herein, using submicroliter quantities of biological samples as described in Robinson et al, Autoantigen microarrays for multiplex characterization of autoantibody responses, Nat Med., 8(3):295-301 (2002), which in herein incorporated by reference in its entirety for its teaching of contructing and using antigen arrays to perform large-scale multiplex characterization of antibody responses directed against structurally diverse antigens, using submicroliter quantities of biological samples.

Protein variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Polypeptide variants generally encompassed by the present invention will typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below), along its length, to a polypeptide sequences set forth herein.

Also disclosed are expression vectors comprising the polynucleotides described elsewhere herein. For example, disclosed are expression vectors comprising the polynucleotides described elsewhere herein, operably linked to a control element. Also disclosed herein are host cells transformed or transfected with an expression vector comprising the polynucleotides described elsewhere herein.

Also disclosed are host cells comprising the expression vectors described herein. For example, disclosed is a host cell comprising an expression vector comprising the polynucleotides described elsewhere herein, operably linked to a control element. Host cells can be eukayotic or prokaryotic cells.

Host cells for expression of the instant genes and nucleic acid fragments can also be microbial hosts that can be found broadly within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances. For example, it is contemplated that any of bacteria, yeast, and filamentous fungi will be suitable hosts for expression of the present nucleic acid fragments. Because of transcription, translation and the protein biosynthetic apparatus is the same irrespective of the cellular feedstock, functional genes are expressed irrespective of carbon feedstock used to generate cellular biomass. Large-scale microbial growth and functional gene expression may utilize a wide range of simple or complex carbohydrates, organic acids and alcohols, saturated hydrocarbons such as methane or carbon dioxide in the case of photosynthetic or chemoautotrophic hosts. However, the functional genes may be regulated, repressed or depressed by specific growth conditions, which may include the form and amount of nitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrient including small inorganic ions. In addition, the regulation of functional genes may be achieved by the presence or absence of specific regulatory molecules that are added to the culture and are not typically considered nutrient or energy sources. Growth rate may also be an important regulatory factor in gene expression. Examples of host strains include but are not limited to bacterial, lower eukaryote, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Nostoc, Lyngbya, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus. Examples of host strains also include but are not limited to lower eukaryotes, including Thraustochytrid species such as Schizochytrium, Ulkenia and Thrausochytrium. Host cells that can be used in the methods described elsewhere herein can also be a transgenic cell or transgenic cell line. Host cells can also be cells that have been transfected with one or more of the disclosed vectors described elsewhere herein. For example, a host cell can be an E. coli cell transfected with one or more of pAC-BETA (providing beta- carotene), p AC-ZE AX (encoding an additional beta-carotene hydroxylase gene, so allowing zeaxanthin biosynthesis) and pAC-ZEAX IDI (same as pAC-ZEAX but also encoding the IDI gene).

The pAC-BETA vector has been previously described by Cunningham et al. (See

Cunningham et al., Plant Cell, 8 (9), 1613-26 (1996) which is hereby incorporated by reference in its entirety for their teaching of pAC-BETA and methods related to the same. pAC-ZEAX and pAC-ZEAX IDI have also been previously described. (See Sun et al., J Biol Chem, 271 (40), 24349-52 (1996) which is hereby incorporated by reference in its entirety for their teaching of p AC-ZEAX and pAC-ZEAX IDI and methods related to the same.

There are a number of compositions and methods which can be used to deliver the disclosed nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A., et al. , Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modifed to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

Expression vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)). For example, disclosed herein are vectors comprising an isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof. Also disclosed are vectors comprising an isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof, wherein the nucleic acid is operably linked to a control element.

Vectors that can be used to produce the compositions described herein include, but are not limited to, pTrcHis2 (Invitrogen) and pBAD24 (See Guzman et al., J Bacteriol, 177 (14), 4121-30 (1995) which is hereby incorporated by reference in its entirety for their teaching of pBAD24 and methods related to the same). Vectors that can be used in the disclosed methods also include, but are not limited to, pAC-ZEAX (SEQ ID NO: 61), pTrc- Av.2W3-pBAD-CrtZ-E.herb, pBAD24-CrtW 7120 (SEQ ID NO: 62), pBAD24-CrtW 73102.1 (SEQ ID NO: 63), pBAD24-CrtW Av.2 (SEQ ID NO: 64), pBAD24-CrtZ Er. Herbicola (SEQ ID NO: 65), pTrc CrtZ Er herbicola pBAD 7120W (SEQ ID NO: 66), pTrc CrtZ Er herbicola pBAD 73102.1 (SEQ ID NO: 67), pTrcCrtZ Er herbicola-pBAD Av.2W (SEQ ID NO: 68), pTrc7120W-pBAD CrtZ Er Herbicola (SEQ ID NO: 69), and pTrc73102.1W-pBAD CrtZ Er herbicola (SEQ ID NO: 70).

The "control elements" present in an expression vector are those non-translated regions of the vector— enhancers, promoters, 5' and 3' untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORTl plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker. Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters (e.g. beta actin promoter). The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ. et al., Gene 18: 355-360 (1982)). Additionally, promoters from the host cell or related species can also be used. Additional promoters that can be used include, but are not limited to, include pTrc and pBAD.

Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al, Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al, MoI. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al. , Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al, MoI. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

The promotor or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

Optionally, the promoter or enhancer region can act as a constitutive promoter or enhancer to maximize expression of the polynucleotides of the invention. In certain constructs the promoter or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. The expression vectors can include a nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. CoIi lacZ gene, which encodes β-galactosidase, and the gene encoding the green fluorescent protein. In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are CHO DHFR-cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media. The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1 : 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al, MoI. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin. As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as an isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-9, 45-47, or a complement thereof into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. In some embodiments the isolated polynucleotides disclosed herein are derived from either a virus or a retrovirus. Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non- proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction abilities (i.e., ability to introduce genes) than chemical or physical methods of introducing genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.

Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer. In Microbiology-1985, American Society for Microbiology, pp. 229-232, Washington, (1985), which is incorporated by reference herein. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference in their entirety for their teaching of methods for using retroviral vectors for gene therapy. A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serves as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61:1213-1220 (1987); Massie et al, MoI. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology 51:261 -21 A (1986); Davidson et al, J. Virology 61 :1226- 1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome- mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4:154- 159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)) the teachings of which are incorporated herein by reference in their entirety for their teaching of methods for using retroviral vectors for gene therapy. Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al, J. Virol. 51 :650-655 (1984); Seth, et al, MoI. Cell. Biol. 4:1528-1533 (1984); Varga et al, J. Virology 65:6061-6070 (1991); Wickham et al, Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. Optionally, both the El and E3 genes are removed from the adenovirus genome.

Another type of viral vector that can be used to introduce the polynucleotides of the invention into a cell is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, or a marker gene, such as the gene encoding the green fluorescent protein, GFP. In another type of AAV virus, the AAV contains a pair of inverted terminal repeats

(ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus. Typically the AAV and Bl 9 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site- specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United States Patent No. 6,261,834 is herein incorproated by reference in its entirity for material related to the AAV vector.

The disclosed vectors thus can provide DNA molecules that are capable of integration into a mammalian chromosome without substantial toxicity.

The inserted genes in viral and retroviral vectors usually contain promoters, or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

Other useful systems include, for example, replicating and host-restricted non- replicating vaccinia virus vectors.

In addition, the disclosed polynucleotides can be delivered to a target cell in a non- nucliec acid based system. For example, the disclosed polynucleotides can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosed expression vectors, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood, to a target organ, or inhaled into the respiratory tract to target cells of the respiratory tract. For example, a composition comprising a polynucleotide described herein and a cationic liposome can be administered to a subjects lung cells. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. MoI. Biol. 1 :95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage. In the methods described herein, delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN™, LIPOFECTAMINE™ (GIBCO-BRL, Gaithersburg, MD), SUPERFECT™ (Qiagen, Hilden, Germany) and TRANSFECTAM™ (Promega Biotec, Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics (San Diego, CA) as well as by means of a SONOPORATION™ machine (ImaRx Pharmaceutical Corp., Tucson, AZ). The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral intergration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can be come integrated into the host genome. Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art.

As described herein, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject's cells in vivo or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject. The invention also provides polypeptides related to β-carotene ketolases and β- carotene hydroxylases. As used herein, the term "polypeptide" is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences capable of functioning as a β-carotene ketolase or hydroxylase.

For example, disclosed herein are polypeptides comprising an amino acid sequence encoded by the polynucleotides described elsewhere herein. For example, disclosed herein are isolated polypeptides comprising an amino acid sequence encoded by a nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof. Also disclosed are isolated polypeptides comprising the sequence provided in SEQ ID NOS: 48-60, or a complement thereof. The polypeptides of the present invention are sometimes herein referred to as a β- carotene ketolase or hydroxylase proteins or β-carotene ketolase or hydroxylase polypeptides, as an indication that their identification has been based at least in part upon their ability to function as a β-carotene ketolase or hydroxylase. Additionally, polypeptides described herein may be identified based on identified conserved domains within other β- carotene ketolase or hydroxylase sequences.

Also disclosed herein are antigen binding agents capable of specifically binding to a polypeptide comprising an amino acid sequence encoded by a nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

Also disclosed are isolated polypeptides comprising an amino acid sequence encoded by a nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1- 10, 45-47, or a complement thereof, with substituted, inserted or deletional variations. Insertions include amino or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion.

Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.

Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M 13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 1 and 2 and are referred to as conservative substitutions.

TABLE 1: Amino Acid Abbreviations

Amino Acid Abbreviations alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys C glutamic acid GIu E glutamine GIn K glycine GIy G histidine His H isolelucine lie I leucine Leu L lysine Lys K phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tyrosine Tyr Y tryptophan Trp W valine VaI V methionine Met M

TABLE 2:Amino Acid Substitutions

Original Residue Exemplary Conservative Substitutions, others are known in the art. ala; ser arg; lys; gin asn; gin; his asp; glu cys; ser gin; asn; lys glu; asp gly; pro his; asn; gin ile; leu; val

Leu; ile; val lys; arg; gin;

Met; leu; ile phe; met; leu; tyr ser; thr thr; ser tip; tyr tyr; tip; phe val; ile; leu

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Tables 1 and 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation or glycosylation.

For example, the replacement of one amino acid residue with another that is biologically and chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues.

Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N- terminal amine and, in some instances, amidation of the C-terminal carboxyl.

Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444

(1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Tables 1 and 2. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al. , Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348- 354 (1992); Ibba, Biotechnology & Genetic Enginerring Reviews 13:197-216 (1995), Cahill et al, TIBS, 14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs). Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH₂NH-, -CH₂S-, -CH₂-CH₂ --, -CH=CH- (cis and trans), -COCH₂ --, - CH(OH)CH₂-, and -CHH₂SO- (These and others can be found in Spatola, A. F. in

Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al, Int J Pept Prot Res 14:177-185 (1979) (-CH₂NH-, CH₂CH₂-); Spatola et al. Life Sci 38:1243-1249 (1986) (-CH H₂-S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (--CH--CH-, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (-COCH₂-); Jennings- White et al. Tetrahedron Lett 23:2533 (1982) (-COCH₂-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-- CH(OH)CH₂-); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH₂-); and Hruby Life Sci 31 :189-199 (1982) (--CH₂-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is -CH₂NH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b- alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad- spectrum of biological activities), reduced antigenicity, and others. D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61 :387 (1992), incorporated herein by reference).

As this specification discusses various polypeptides and polypeptide sequences it is understood that the nucleic acids that can encode those polypeptide sequences are also disclosed. This would include all degenerate sequences related to a specific polypeptide sequence, i.e. all nucleic acids having a sequence that encodes one particular polypeptide sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed polypeptide sequences.

Also disclosed herein are isolated antibodies, antibody fragments and antigen- binding fragments thereof, that specifically bind to a polypeptide comprising an amino acid sequence encoded by a nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof. Also disclosed herein are isolated antibodies, antibody fragments and antigen-binding fragments thereof, that specifically bind to a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 48- 60. Optionally, the isolated antibodies, antibody fragments, or antigen-binding fragment thereof can be neutralizing antibodies. The antibodies, antibody fragments and antigen- binding fragments thereof disclosed herein can be identified using the methods disclosed herein. For example, antibodies that bind to the polypeptides of the invention can be isolated using the antigen microarray described above.

The term "antibodies" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also disclosed are antibody fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with the polypeptides disclosed herein.

"Antibody fragments" are portions of a complete antibody. A complete antibody refers to an antibody having two complete light chains and two complete heavy chains. An antibody fragment lacks all or a portion of one or more of the chains. Examples of antibody fragments include, but are not limited to, half antibodies and fragments of half antibodies. A half antibody is composed of a single light chain and a single heavy chain. Half antibodies and half antibody fragments can be produced by reducing an antibody or antibody fragment having two light chains and two heavy chains. Such antibody fragments are referred to as reduced antibodies. Reduced antibodies have exposed and reactive sulfhydryl groups. These sulfhydryl groups can be used as reactive chemical groups or coupling of biomolecules to the antibody fragment. A preferred half antibody fragment is a F(ab). The hinge region of an antibody or antibody fragment is the region where the light chain ends and the heavy chain goes on.

Antibody fragments for use in antibody conjugates can bind antigens. Preferably, the antibody fragment is specific for an antigen. An antibody or antibody fragment is specific for an antigen if it binds with significantly greater affinity to one epitope than to other epitopes. The antigen can be any molecule, compound, composition, or portion thereof to which an antibody fragment can bind. An analyte can be any molecule, compound or composition of interest. For example, the antigen can be a polynucleotide of the invention. The antibodies or antibody fragments can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic or prophylactic activities are tested according to known clinical testing methods. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Also disclosed are "chimeric" antibodies in which a portion of the heavy or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). The disclosed monoclonal antibodies can be made using any procedure which produces monoclonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.

Alternatively, the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4- co-receptor complexes described herein.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al). DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, such as an Fv, Fab, Fab¹, or other antigen-binding portion of an antibody, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566 which is hereby incorporated by reference in its entirety for its teaching of papain digestion of antibodies to prepare monovaltent antibodies. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

The fragments, whether attached to other sequences, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, MJ. Curr. Opin. Biotechnol. 3:348-354, 1992). As used herein, the term "antibody" or "antibodies" can also refer to a human antibody or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

The disclosed human antibodies can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol, 147(l):86-95, 1991). Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al, J. MoI. Biol., 227:381, 1991; Marks et al, J. MoI. Biol, 222:581, 1991).

The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al. , Nature, 362:255-258 (1993); Bruggermann et al, Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.

Optionally, the disclosed human antibodies can be made from memory B cells using a method for Epstein-Barr virus transformation of human B cells. (See, e.g., Triaggiai et al, An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus, Nat Med. 2004 Aug;10(8):871-5. (2004)), which is herein incorporated by reference in its entirety for its teaching of a method to make human monoclonal antibodies from memory B cells). In short, memory B cells from a subject who has survived a natural infection are isolated and immortalized with EBV in the presence of irradiated mononuclear cells and a CpG oligonuleotide that acts as a polyclonal activator of memory B cells. The memory B cells are cultured and analyzed for the presence of specific antibodies. EBV-B cells from the culture producing the antibodies of the desired specificity are then cloned by limiting dilution in the presence of irradiated mononuclear cells, with the addition of CpG 2006 to increase cloning efficiency, and cultured. After culture of the EBV-B cells, monoclonal antibodies can be isolated. Such a method offers (1) antibodies that are produced by immortalization of memory B lymphocytes which are stable over a lifetime and can easily be isolated from peripheral blood and (2) the antibodies isolated from a primed natural host who has survived a natural infection, thus eliminating the need for immunization of experimental animals, which may show different susceptibility and, therefore, different immune responses.

Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody. To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al , Nature, 321 :522-525 (1986), Reichmann et al , Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al, Nature, 321 :522-525 (1986), Riechmann et al, Nature, 332:323-327 (1988), Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.

Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al), U.S. Patent No. 5,565,332 (Hoogenboom et al), U.S. Patent No. 5,721,367 (Kay et al), U.S. Patent No. 5,837,243 (Deo et al), U.S. Patent No. 5, 939,598 (Kucherlapati et al), U.S. Patent No. 6,130,364 (Jakobovits et al), and U.S. Patent No. 6,180,377 (Morgan et al).

The antibodies disclosed herein can also be administered to a subject. Nucleic acid approaches for antibody delivery also exist. The broadly neutralizing antibodies to the polypeptides disclosed herein and antibody fragments can also be administered to subjects or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment.

The microbial isoprenoid pathway is naturally a multi-product platform for production of compounds such as carotenoids, quinones, squalene, and vitamins. These natural products may be from 5 carbon units to more than 55 carbon units in chain length. There is a general practical utility for microbial isoprenoid production as these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol. 70:181 191 (1991)).

The above-described polynucleotides and polypeptides can be used to produce β- carotene derived carotenoids. The gene and gene sequences described herein enable one to incorporate the production of healthful β-carotene derived carotenoids directly into a cell. This aspect makes the cells into which these genes are incorporated a more desirable production host for animal feed due to the presence of carotenoids which are known to add desirable pigmentation and health benefits to the feed. Salmon and shrimp aquacultures are particularly useful applications for this invention as carotenoid pigmentation is critically important for the value of these organisms (F. Shahidi, J. A. Brown, Carotenoid pigments in seafood and aquaculture, Critical Reviews in Food Science 38(1):1 67 (1998)). Specifically, the β-carotene derived carotenoid, astaxanthin, is a powerful antioxidant and has been reported to boost immune functions in humans and reduce carcinogenesis (Jyonouchi et al., Nutr. Cancer (1995) 23:171 183; Tanaka et al., Cancer Res. (1995) 55:4059 4064).

The sequences described above may be used in vitro and in vivo in recombinant hosts for the production of β-carotene derived carotenoids from monocyclic and bicyclic carotenoid compounds. As such, disclosed herein are compositions produced using the disclosed polynucleotides and polypeptides. For example, disclosed are compositions comprising a β-carotene derived carotenoid produced by the methods described below.

Specific β-carotene derived carotenoids that can be produced by the methods disclosed herein include but are not limited to, canthaxanthin, astaxanthin, adonixanthin, adonirubin, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, 4-keto-gamma- carotene, 4-keto-rubixanthin, 4-keto-torulene, 3-hydroxy-4-keto-torulene, deoxyflexixanthin, and myxobactone. Of particular interest is the production of astaxanthin 4-keto-rubixanthin. The specific substrate for the present CrtO enzyme is a monocyclic or bicyclic carotenoid. Cyclic carotenoids are well known in the art and available commercially. Preferred in the present invention as CrtO ketolase substrates are cyclic carotenoid that include but are not limited to β-Carotene, γ-carotene, zeaxanthin, rubixanthin, echinenone, and torulene.

Also disclosed is a delivery device comprising a any of the compositions described above. For example, disclosed is a delivery device comprising a composition comprising a β-carotene derived carotenoid produced by the methods described elsewhere herein. The delivery device can comprise a microcapsule, a microsphere, a nanosphere or nanoparticle, a liposome, a noisome, a nanoerythrosome, a solid-liquid nanoparticle, a leuprolide, a gel, a gel capsule, a tablet, a lotion, a cream, a spray, an emulsion, or a powder. The delivery device can comprise a microcapsule, a microsphere, a nanosphere or nanoparticle, a liposome, a noisome, a nanoerythrosome, a solid-liquid nanoparticle, a leuprolide, a gel, a gel capsule, a tablet, a lotion, a cream, a spray, an emulsion, or a powder.

Also disclosed is a microcapsule, comprising an agglomeration of primary microcapsules and a loading substance, each individual primary microcapsule having a primary shell, wherein the loading substance comprises any of the compositions described above, and is encapsulated by the primary shell, and wherein the agglomeration is encapsulated by an outer shell. For example, disclosed is a microcapsule, comprising an agglomeration of primary microcapsules and a loading substance, each individual primary microcapsule having a primary shell, wherein the loading substance comprises a β-carotene derived carotenoids produced by the methods described elsewhere herein, and is encapsulated by the primary shell, and wherein the agglomeration is encapsulated by an outer shell.

The primary shell and/or outer shell can comprise a surfactant, gelatin, polyphosphate, polysaccharide, or a mixture thereof. The primary shell and/or outer shell can also comprise gelatin type B, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, starch, modified starch, alfa-lactalbumin, beta-lactoglobumin, ovalbumin, polysorbiton, maltodextrin, cyclodextrin, cellulose, methyl cellulose, ethyl cellulose, hydropropylmethylcellulose, carboxymethylcellulose, milk protein, whey protein, soy protein, canola protein, albumin, kosher gelatin, non-kosher gelatin, Halal gelatin, non- Halal gelatin, or a mixture thereof. The primary shell and/or outer shell can also comprise a complex coacervate, gelatin type A, fish gelatin, a gelatin with a Bloom number of from about 0 to about 300, a gelatin with a Bloom number of from about 0 to about 50, a gelatin with a Bloom number of from about 51 to about 300, a gelatin with a Bloom number of about 0, about 210, about 220, or about 240, a coacervate of gelatin and polyphosphate. The loading substance of the disclosed microcapsules can comprise a β-carotene derived carotenoid produced by the methods described elsewhere herein. The loading substance can be from about 20% to about 90% or 50% to about 70% by weight of the microcapsule. The outer shell of the disclosed microcapsules can have an average diameter of from about 1 μm to about 2,000 μm, about 20 μm to about 1,000 μm, about 30 μm to about 80 μm, about 40 ran to about 10 μm, or about 0.1 μm to about 5 μm.

Also disclosed is a nutritional supplement that comprises any of the compositions, delivery devices, or microcapsules described above. The disclosed nutritional supplements can be in the form of a tablet, gel-cap, capsule, liquid, or syrup.

Also disclosed is a foodstuff that comprises any of the compositions, delivery devices, or microcapsules described above. The foodstuff can be a baked good, a pasta, a meat product, a frozen dairy product, a milk product, a cheese product, an egg product, a condiment, a soup mix, a snack food, a nut product, a plant protein product, a hard candy, a soft candy, a poultry product, a processed fruit juice, a granulated sugar, a sauce, a gravy, a syrup, a nutritional bar, a beverage, a dry beverage powder, a jam or jelly, an infant formula, or a baby food. The foodstuff can also be a fish product, a companion pet food, a livestock or an aquaculture feed. The foodstuff can also be bread, tortillas, cereal, sausage, chicken, ice cream, yogurt, milk, salad dressing, rice bran, fruit juice, a dry beverage powder, rolls, cookies, crackers, fruit pies, or cakes.

Also disclosed is a method of delivering a composition to a subject, comprising administering to the subject any of the compositions, delivery devices, microcapsules, or foodstuffs described above. The subject can be a mammal. The subject can also be a human.

Also disclosed is a use of any of the microcapsules described above and to prepare a medicament for delivering a loading substance to a subject.

Also disclosed is a pharmaceutical formulation comprising any of the compositions, delivery devices, or microcapsules described above, and a pharmaceutical carrier.

Omega-3 and/or Omega-6 fatty acids can also be included in any of the delivery devices, microcapsules, nutritional supplements or foodstuffs described elsewhere herein. For example, disclosed are delivery devices, microcapsules, nutritional supplements and foodstuffs comprising a any of the compositions described above, further comprising omega-3 and/or omega-6 fatty acids. Such compositions can also be used in any of the methods described elsewhere herein.

Omega-3 and/or Omega-6 fatty acids can also be included in any of the pharmaceutical formulations described elsewhere herein. The compositions disclosed herein can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the inflammatory disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated herein by reference in its entirety for its teaching of an approach for parenteral administration.

It will be understood that, if desired, a composition as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may further comprise substituted or derivatized RNA or DNA compositions.

Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton,

PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically- acceptable carriers include, but are not limited to, sterile water, saline, Ringer's solution, dextrose solution, and buffered solutions at physiological pH. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the polynucleotide, polypeptide, antibody, T-cell, TCR, or APC compositions disclosed herein. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

Methods Also disclosed herein are methods of producing β-carotene derived carotenoids. For example, disclosed is a method of producing a β-carotene derived carotenoid in a cell, comprising: bringing into contact a cell and a first vector comprising a first nucleic acid, wherein the first nucleic acid encodes a polypeptide comprising a β-carotene sequence; bringing into contact the cell of (a) and a second vector comprising a second nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β-carotene ketolase sequence, incubating the cell under conditions that allow expression of the β- carotene polypeptide and the β-carotene ketolase polypeptide, thereby producing a β- carotene derived carotenoid in the cell.

Also disclosed is a method of producing a β-carotene derived carotenoid in a cell, comprising: bringing into contact a cell and a first vector comprising a first nucleic acid, wherein the first nucleic acid encodes a polypeptide comprising a β-carotene sequence; bringing into contact the cell of (a) and a second vector comprising a second nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β-carotene ketolase sequence, incubating the cell under conditions that allow expression of the β- carotene polypeptide and the β-carotene ketolase polypeptide, thereby producing a β- carotene derived carotenoid in the cell wherein step (a) further comprises bringing into contact the cell and a third vector comprising a third nucleic acid, wherein the third nucleic acid encodes a third polypeptide comprising a β-carotene hydroxylase sequence; wherein step (b) further comprises incubating the cell under conditions that allow expression of the β-carotene hydroxylase polypeptide.

In the methods described above, the second nucleic acid sequence can comprise a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1 -9, 45-47, or a complement thereof or can be selected from the group consisting of SEQ ID NOs: 1-9, 45-47, or a complement thereof.

In the methods described above, the third nucleic acid sequence comprises a nucleic acid sequence at least about 90% identical to the nucleic acid sequnece of SEQ ID NO: 10, a complement thereof, or the nucleic acid sequence of SEQ ID NO: 10, or a complement thereof. Optionally, the third vector can comprise the sequence of SEQ ID NO. 61 or the sequence of SEQ ID NO. 68. Optionally, the third vector can be pTrc-Av.2W3-pBAD- CrtZ-E.herb.

Also disclosed is amethod of producing a β-carotene derived carotenoid in a cell, comprising: (a) bringing into contact a cell and a first vector comprising a nucleic acid, wherein the first nucleic acid encodes a polypeptide comprising a β-carotene sequence; (b) bringing into contact the cell of (a) and a second vector comprising a second and third nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β- carotene ketolase sequence and the third nucleic acid encodes a third polypeptide comprising a β-carotene hydroxylase sequence; (c) incubating the cell under conditions that allow expression of the β-carotene polypeptide, the β-carotene ketolase polypeptide, and the β-carotene hydroxylase polypeptides; (d) thereby producing a β-carotene derived carotenoid in the cell. In this method the second vector can comprise a sequence selected from the group consisting of SEQ ID NOs. 61-71. In this method, the second nucleic acid sequence can comprise a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-9, 45-47, or a complement thereof or can be selected from the group consisting of SEQ ID NOs: 1-9, 45-47, or a complement thereof. In this method, the third nucleic acid sequence comprises a nucleic acid sequence at least about 90% identical to the nucleic acid sequnece of SEQ ID NO: 10, a complement thereof, or the nucleic acid sequence of SEQ ID NO: 10, or a complement thereof.

Also disclosed is a method of producing a β-carotene derived carotenoid in a cell, comprising: (a) bringing into contact a cell and a first vector comprising a first nucleic acid, wherein the first nucleic acid encodes a first polypeptide comprising a a β-carotene ketolase sequence; (b) incubating the cell under conditions that allow expression of the a β-carotene ketolase polypeptide, (c) thereby producing a β-carotene derived carotenoid in the cell. Further disclosed is a method of producing a β-carotene derived carotenoid in a cell, comprising: (a) bringing into contact a cell and a first vector comprising a first nucleic acid, wherein the first nucleic acid encodes a first polypeptide comprising a a β-carotene ketolase sequence; (b) incubating the cell under conditions that allow expression of the a β-carotene ketolase polypeptide, (c) thereby producing a β-carotene derived carotenoid in the cell, wherein step (a) further comprises bringing into contact the cell and a second vector comprising a second nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β-carotene hydroxylase sequence; wherein step (b) further comprises incubating the cell under conditions that allow expression of the β-carotene hydroxylase polypeptide.

Also disclosed is a method of producing a β-carotene derived carotenoid in a cell, comprising: (a) bringing into contact a cell and a first vector comprising a first nucleic acid, wherein the first nucleic acid encodes a first polypeptide comprising a a β-carotene ketolase sequence; (b) incubating the cell under conditions that allow expression of the a β-carotene ketolase polypeptide, (c) thereby producing a β-carotene derived carotenoid in the cell, wherein the first vector of step (a) further comprise a second nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β-carotene hydroxylase sequence; wherein step (b) further comprises incubating the cell under conditions that allow expression of the β-carotene hydroxylase polypeptide.

The β-carotene ketolase if any of the disclosed methods can be, but is not limited to a β-carotene ketolase isolated from Lyngbya sp. CCAP 1446/5, Nostoc punctiforme PCC 73102.1, Nostoc punctiforme 73102.2, Anabaena variabilis ATCC 29413.1, Anabaena variabilis ATCC 29413.2, or Nostoc sp. PCC 7120.

The disclosed methods can further comprise incubating the cell at between 15°C and 42°C, more preferably between 25°C and 37°C and most preferably between 30-37°C in the presence of simple sugars such as, but not limited to, glycerol necessary for biomass accumulation with compound production further induced via supplementation with compounds such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) and L-arabinose.

Alternatively fermentation can occur simply in the presence of simple sugars such as, but not limited to D-sorbitol, glucose and maltose without the need for induction of production. The cells used in the methods disclosed herein can also be in or part of a system. The system can be a bacteria, fungal, algal, plant, animal, or trangenic animal.

Also disclosed are methods of reducing reactive oxygen species in a subject, comprising the step of administering to the subject an effective amount of one or more of the disclosed compositions, delivery devices, microcapsules, nutritional supplements or foodstuffs disclosed herein.

Also disclosed are methods of removing free radicals in a subject, comprising the step of administering to the subject an effective amount of one or more of the disclosed compositions, delivery devices, microcapsules, nutritional supplements or foodstuffs disclosed herein. Where commercial production of β-carotene derived carotenoid compounds is desired, using the disclosed polynucleotides and/or polypetides, a variety of culture methodologies may be applied. For example, large-scale production of a specific gene product, overexpressed from a recombinant microbial host may be produced by both batch or continuous culture methodologies. A classical batch culturing method is a closed system where the composition of the media is set at the beginning of the culture and not subject to artificial alterations during the culturing process. Thus, at the beginning of the culturing process the media is inoculated with the desired organism or organisms and growth or metabolic activity is permitted to occur adding nothing to the system. Typically, however, a "batch" culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase are often responsible for the bulk of production of end product or intermediate in some systems. Stationary or post- exponential phase production can be obtained in other systems.

A variation on the standard batch system is the fed-batch system. Fed-batch culture processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the culture progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO.sub.2. Batch and fed-batch culturing methods are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227, (1992), herein incorporated by reference.

Commercial production of β-carotene derived carotenoids may also be accomplished with a continuous culture. Continuous cultures are an open system where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in log phase growth.

Alternatively continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added, and valuable products, by-products or waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials. Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

Fermentation media in the present invention can nitrogen substrates such as natural nitrogen sources including peptone, casamino acids, casein, yeast extract,malt extract, or an organic nitrogen sources such as sodium glutamate, but not limited thereto, either in their entirety or with supplementation with suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose, glycerol, and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. For example a medium that can be used as a basic medium can be LB broth which consists of 10 g casein protein, 1O g NaCl and 5 g yeast extract without a separate carbon source. The same medium can also be used with the addition of carbon to the medium.

As described elsewhere herein, the addition of carbon sources can be used in the methods described herein to increase production of β-carotene derived carotenoids. For example, D-sorbitol, IDI and DXS can be used with the methods described herein. The additional carbon sources can also be used for induction with both IPTG and L-arabinose.

Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, methane or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th (1993), 415 32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Suiter et al., Arch. Microbiol. 153:485 489 (1990)). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.

Carotenoid biosynthesis can also affected by precursor availability, as determined by both the over expression of key metabolic genes (IDI and DXS), and by media supplementation. As such, disclosed herein are methods of producing β-carotenoid derived carotenoids as disclosed elsewhere herein, further comprising the addition of carbon sources. For example, additional carbon sources, metabolically linked to the synthesis of glyceraldehyde 3-Phosphate (G3P) & pyruvate can be introduced to the cells. As described below, glycerol and D- sorbitol can be added to media used to grow the cells. Also as described below, supplementation of growth media resulted in enhanced carotenoid synthesis both on terms of mg g-1 DCW and mg L-I.

In addition IDI or DXS can be added to reaction mixtures of the disclosed methods of producing β-carotenoid derived carotenoids. IDI or DXS can be provided as either an exogenous substrate or nucleic acid sequences capable of encoding the IDI or DXS peptides can be delivered to the same cells as described in the methods described elsewhere herein via a separate vector or the nucleic acids can be cloned into the ketolase, hydroxylase, or ketolase and hydroxylase vectors described above. DXS catalyses the first reaction of the non-mevalonatepathway whereas IDI catalyses the conversion of isopentenyl pyrophosphate (IPP) into its structural isomer, dimethylallyl pyrophosphate (DMAPP), a process previously identified as a bottle neck in the synthesis of carotenoids. As described below, overexpression of IDI (Er. herbicola (pAC-ZEAX IDI)), in addition to the over expression of either a cyanobacterial (A. variabilis ATCC 29413) or bacterial (E. coli JMlOl) DXS gene from pBAD24 resulted in a 4.9 and 5.9 fold increase in carotenoid biosynthesis, respectively.

Plants and algae are also known to produce carotenoid compounds. The nucleic acids and nucleic acid fragments described herein can also be used to create transgenic plants having the ability to express these microbial proteins, thus increasing their total carotenoid outputs. Preferred plant hosts will be any variety that will support a high production level of the instant proteins. Suitable green plants will include but are not limited to soybean, rapeseed (Brassica napus, B. campestris), pepper, sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), wheat (Triticum sp), barley (Hordeum vulgare), oats (A vena sativa, L), sorghum (Sorghum bicolor), rice (Oryza sativa), Arabidopsis, cruciferous vegetables (broccoli, cauliflower, cabbage, parsnips, etc.), melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses. Algal species include but not limited to commercially significant hosts such as Spirulina, Haemotacoccus, and Dunalliela. Production of the carotenoid compounds may be accomplished by first constructing chimeric genes of present invention in which the coding region are operably linked to promoters capable of directing expression of a gene in the desired tissues at the desired stage of development. For reasons of convenience, the chimeric genes may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non- coding sequences encoding transcription termination signals must also be provided. The instant chimeric genes may also comprise one or more introns in order to facilitate gene expression.

Any combination of any promoter and any terminator capable of inducing expression of a coding region may be used in the chimeric genetic sequence. Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes. One type of efficient plant promoter that may be used is a high level plant promoter. Such promoters, in operable linkage with the genetic sequences or the present invention should be capable of promoting expression of the present gene product. High level plant promoters that may be used in this invention include the promoter of the small subunit (ss) of the ribulose-l,5-bisphosphate carboxylase from example from soybean (Berry-Lowe et al., J. Molecular and App. Gen., 1 :483 498 1982)), and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light-induced in plant cells (see, for example, Genetic

Engineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum, N. Y. (1983), pages 29 38; Coruzzi, G. et al., The Journal of Biological Chemistry, 258:1399 (1983), and Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2:285 (1983)).

Plasmid vectors comprising the instant chimeric genes can then constructed. The choice of plasmid vector depends upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4:2411 2418; De Almeida et al., (1989) MoI. Gen. Genetics 218:78 86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA blots (Southern, J. MoI. Biol. 98, 503, (1975)). Northern analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed. Appl., 618 (1 2) (1993) 133 145), Western analysis of protein expression, or phenotypic analysis.

For some applications it will be useful to direct the instant proteins to different cellular compartments. It is thus envisioned that the chimeric genes described above may be further supplemented by altering the coding sequences to encode enzymes with appropriate intracellular targeting sequences such as transit sequences (Keegstra, K., Cell 56:247 253 (1989)), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels, J. J., Ann. Rev. Plant Phys. Plant MoI. Biol. 42:21 53 (1991)), or nuclear localization signals (Raikhel, N. Plant Phys. 100:1627 1632 (1992)) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future that are useful in the invention.

Disclosed herein are kits that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include primers to perform the amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended. Also disclosed herein are kits comprising one or more vectors comprising nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof

EXAMPLES

Example 1 Strains and culture conditions Cyanobacteria were cultured in a plant growth chamber (Sanyo, Japan), at 25⁰C, 50 μE m^"2 sec^"1 illumination, 60% humidity, on a 12 hour light/dark regime. Cyanobacterial strains and culture media are detailed in Table 3. Culture media was prepared according to the CCAP protocol (www.ccap.ac.uk). E. coli cultures, unless otherwise stated, were cultured on LB (Sigma) at 37⁰C, when broth culture; with shaking at 250 rpm. When required, antibiotics ampicillin and chloramphenicol were used at 50 μg/ml. DNA extraction

Genomic DNA was extracted via a modified version of Tamagnini, et al. (Tamagnini et al., Appl Environ Microbiol, 63 (5), 1801-1807 (1997)). Briefly, 4 week 50 ml cyanobacterial cultures were harvested by centrifugation at 4,800 x g. The cell pellet was then re-suspended in 1 ml of 50 mM Tris, pH 7.4, 100 mM ETDA, pH 8.0, 25% sucrose, vortexed at high speed for 30 seconds and centrifuged at 16,500 x g for 10 minutes, repeated three times. The cell pellet was subsequently re-suspended in 50 mM Tris-HCl, pH 8.0, plus 10 mM EDTA. Lysis was induced by the addition of 0.6 g 0.4-0.6 mm diameter acid-washed glass beads, 25 μl of 10% sodium dodecyl sulfate (SDS), 500 μl phenol-chloroform (1 :1 [vol/vol]), and vortexing at high speed for 60 seconds, followed by a 60 second incubation on ice, repeated 4 times. Liquid phases were separated by centrifugation at 16,500 x g for 15 min, at 4⁰C, and the upper aqueous phase was further extracted with an equal volume of chloroform. Genomic DNA was precipitated with 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold 100% ethanol, incubated at -2O⁰C overnight. Precipitated DNA was pelleted via centrifugation at 16,500 x g for 30 mins, at 4⁰C. DNA pellet was the washed with 500 μl ice cold 70% ethanol and collected by centrifugation at 16,500 x g for 10 minutes. Supernatant was aspirated and the DNA pellet air dried. DNA was re-suspended in 200 μl nuclease free water, quantified via spectrophotometry and diluted to 100 ng/μl, using nuclease free water. 900μl of solution MD3 was added to 20,000 ng genomic DNA (MoBio Ultraclean microbial DNA isolation kit). Solution was mixed 5 seconds by vortex, and loaded onto a spin filter, and centrifuged at 16,500 x g for 30 seconds. DNA was washed with 300μl Solution MD4, and eluted with 50μl of Solution MD5. The final DNA quantification was obtained by spectrophotometry. Table 3 : Cyanobacterial strains and culture media

Degenerate primer design

Degenerate primers were designed using the web based programme Biology Workbench (SDSC), in conjunction with the BLAST programme at the NCBI. Briefly, putative β-carotene ketolase sequences were identified via BLASTN using the Synechocystis sp. PCC 6803 CrtO type β-carotene ketolase nucleotide sequence as the query. Cyanobacterial hits were then used to generate a CLUSTALW alignment, from this, degenerate primers were designed based on conserved regions. Table 4 details the primer sequences along with their location within the gene. Gene specific PCR primers were designed using the previously identified putative cyanobacterial β-carotene ketolase sequences and flanking regions, and the software package DSGene 1.5 (Accerlys), gene specific primer sequences are shown in Table 5.

Table 4: Sequence and location of degenerate primers used to screen the genome of Lyngbya sp. CCAP 1446/5 for putative CrtO genes

Table 5: Oligonucleotide primers used for amplification of putative CrtO type β-carotene ketolase genes.

Target ORF Primer Name Sequence T_m

Av CrtO Ftrc ATGCAAGAGTATGATGTTGTAATGATTGGTGC (SEQ ID

ABA_21204 NO: 13)

62.8

Av CrtO R GTCATCAGTAGTCAACCGTCAACCGTCAAC (SEQIDNO:

14) Asp CrtO Ftrc ATGCAAGAATATGATGTTGTAATGATTGGTGCAG(SEQ

BAB_75443.1 IDNO; 15) 62.8

GTCGTCAGTAGTCAACCGTCAACCGTTAACTGTCAAC

Asp CrtO R (SEQ ID NO: 16)

Np.1 CrtO

ATGCAAGAGTATGATGTTGTGCTGATCG (SEQ ID NO: 17) ZP_00111616 Ftrc 62.8

Np.1 CrtO R CGAATCTCTTGCATCCTTTAAGGTTTGGCTAATAGGATG

(SEQ ID NO: 18)

Gv CrtO Ftrc ATGCCAGGGAGTTCGGACGTGG (SEQ ID NO: 19)

56

BAC 88335 Gv CrtO R CGACCAGCTGCCGGTCAGGCTGCGGTACACC (SEQ ID

NO: 20)

Np.2 CrtO ATGGAATCGTATGACGTTGTAATTATTGGCGCTGGTC Ftrc (SEQ ID NO: 21)

64.3 ZP 00112486

Np.2 CrtO R CAGCCTCTTAGTCTGATTCACTCCTCAAGACAGATG

(SEQ ID NO: 22) ATGGAAAACTACGACGTTGTTATTATTGG (SEQ ID NO: Lyngbya Ftrc

23)

Not yet 60.0 CGCTATCGTTTCGCAGTTCTTAGGACAGCATAGG (SEQ submitted Lyngbya R

ID NO: 24)

Syn pTrcF ATGATCACCACCGATGTTGTC (SEQ ID NO: 25)

NP_442491 56 Syn pTrcR GCTGTACCAAAAACGACGTTG (SEQ ID NO: 26)

5

Genome Walking and Cloning

To elucidate the complete, putative β-carotene ketolase ORF for Lyngbya sp. CCAP 1446/5 the APAgene™ GOLD Genome walking kit (BIO S&T, Quebec, Canada) was used, as per the manufacturer's instructions. Briefly PCR was performed using degenerate PCR primers

10 (Table 4). Gel extraction of the correct PCR product, based on size, using QIAGEN's Gel Extraction Kit, was followed by cloning of the purified PCR product in to vector pCR4- TOPO (Invitrogen). This was transformed into competent TOPlO E. coli cells. Clones were analysed for the desired insert via colony screen PCR, and positive clones were sequenced to confirm the amplification of a novel putative CrtO type ketolase gene. Based 5 on this sequence data Genome walking Primers were designed according to the manufacturer's protocol, and genome walking amplification steps were completed according to the manufacturer's instructions. Table 6: Oligonucleotide primers used for amplification of putative CrtW type β-carotene ketolase genes.

ORF ID Organism Primer Name Sequence Tm

Anabaena ATGACTGAATGTAATTCCATATCTTTGC (SEQ ID NO: variabilis pTrcAv.lWF 27)

YP 322565.1

ATCC GTAATTATTATTATAAACATCTGGTAATCTCC (SEQ ID

29413 pTrcAv.lWR NO: 28) 55

Nostoc pTrc73102.1WF ATGATCCAGTTAGAACAACCACTCAG (SEQ ID NO: 29) punctiforme

ZP 00345866.1

PCC GTATTTTGCTTTGTAAATTTCTGGTAACTGC (SEQ ID

73102.1 pTrc73102.1WR NO: 30) 56

Anabaena ATGGTTCAGTGTCAACCATCATCTCTACG (SEQ ID NO: variabilis pTrcAv.2WF 31)

YP 324388.1

ATCC GTATAAAGATATTTTGTAAGCTTCCGG (SEQ ID NO:

29413 pTrcAv.2WR 32) 55

ATGGTTCAGTGTCAACCATCATCTCTGC (SEQ ID NO:

Nostoc sp. pTrc7120WF 33)

NP 487229.1

PCC 7120 GTATAAAGATATTTTGTGAGCTTCAGG (SEQ ID NO: pTrc7120WR 34) 55

Nostoc ATGAATTTTTGTGATAAACCAGTTAGCTATTATGTTGC

₇P nn_{i i} ms _? punctiforme pTrc73102.2WF (SEQ ID NO: 35)

- ^ ^^^ pcc GTACGAATTGGTTACTGAATTGTTGAATAC (SEQ ID

73102.2 _PTrc73102.2WR NO: 36) 56

ATGATGCGTGGCTCGGCAGTAAAGGAACG (SEQ ID

Gloeobacter pTrc7421WF NO: 37)

NP 924674.1 violaceus GTAGCGGCGCGCCTCGGGCAGCCGATGC (SEQ ID NO:

PCC 7421 38) pTrc7421 WR 65 PCR and Cloning

5 100 ng/μl genomic DNA was added to the following reaction mix. 0.5 μl (2.5 units)

Taq DNA polymerase (Sigma), 6 μl 10 x PCR buffer, 8 μl MgCl₂ (25 mM), 1.25 μl 10 mM dNTP (Promega), 2 μl (20 pmol) of each primer, and nuclease free water to 50 μl (Promega). PCR was performed on a Gene Amp System 9700 (Applied Bio systems) thermocycler, with the parameters set at 94⁰C for 3 minutes, followed by 30 cycles of 94⁰C

10 for 30 seconds, annealing at primer specific temperature (Table 5) for 30 seconds, and 68⁰C for 110 seconds. 30 μl of the PCR reaction was separated on a 1% TAE agarose gel. Positive products were extracted and cleaned using the QIAEX II gel extraction kit (QIAGEN), cloned into the expression plasmid ρTrcHis2 TOPO (Invitrogen), and transformed into TOPlO E. coli cells, according to manufacturer's protocol.

15 Creation of pBAD expression plasmids was achieved as follows; the gene of interest was amplified using oligo-nucleotide linker primers (Table 6). Primers are composed of two regions. One region complementary to the gene of interest, and a second region complementary to the vector, pBAD24, flanking the insertion site. ORF amplification was performed as above, gel extracted PCR product was used in a subsequent PCR cloning reaction. This consisted of, 5 μl 1OX Pfu buffer (plus MgC12), 1 μl 1OmM dNTP, 2μl PCR product, lμl Ncol digested pBAD24, 3U Pfu DNA polymerase and nuclease free water to 50 μl. PCR parameters were 94⁰C for 2 minutes, followed by 35 cycles of 94⁰C for 60 seconds, 55⁰C for 30 seconds, 72⁰C for 12 minutes. Amplification products were Dpnl digest, 37⁰C for 90 minutes, and transformed in to competent TOPlO E. coli cells, according to the manufacturer's protocol. Correct insertion was confirmed via colony screen PCR and sequencing.

Table 7: Oligo-nucleotide primers used for PCR based cloning of CrtW type β-carotene

10 ketolase genes, into pBAD24.

Source Primer

ORF ID Primer Sequence

Organism Name

Nostoc 73102. IWF- GGCTAGCAGGAGGAATTCACCATGATCCAGTT punctiforme LINKER AGAACAACCAC (SEQ ID NO: 39)

ZP 00345866.1

PCC 73102. IWR- CGACTCTAGAGGATCCCCGGGTACGTATTTTGC

73102.1 LINKER TTTGTAAATTTCTG (SEQ ID NO: 40)

7120 WF- GGCTAGCAGGAGGAATTCACCATGGTTCAGTGT

Nostoc sp. LINKER CAACCATC (SEQ ID NO: 41)

NP 487229.1

PCC 7120 7120WR- CGACTCTAGAGGATCCCCGGGTACGTATAAAGA

LINKER TATTTTGTGAGC (SEQ ID NO: 42)

Anabaena AV.2WF- GGCTAGCAGGAGGAATTCACCATGGTTCAGTGT variabilis LINKER

YP 324388.1 CAACCATCATC (SEQ ID NO: 43)

ATCC AV.2WR- CGACTCTAGAGGATCCCCGGGTACGTATAAAGA

29413 LINKER TATTTTGTAAGCTTC (SEQ ID NO: 44)

Functional Characterisation: Co-expression

Each putative cyanobacterial β-carotene ketolase gene was co-expressed in E. coli 15 TOP 10 cells containing either pAC-BETA or pAC-ZEAX IDI (Cunningham et al., 1996) (Kindly provided by FX Cunningham). Expression was induced according to the manufacturer's protocol. Briefly, 100 ml LB medium, containing appropriate antibiotics was inoculated with 3 ml of inoculum, grown at 37⁰C to an OD_δoo of 0.5, and induced with IPTG, ImM final concentration. After induction, cultures were incubated at 3O⁰C 24 hours, 20 in the dark. Cultures were harvested via centrifugation at 5000 x g, 10 minutes, lyophilised and stored at -8O⁰C, until analysis. Carotenoid analysis/HPLC work

To extract carotenoids for analysis, 5 ml of ice-cold acetone (HPLC grade) was added to the lyophilised cell pellets. The solution was then vortexed at high speed for 15 25 seconds, incubated on ice, with shaking at 250 rpm, for 5 minutes, and vortexed again at high speed for 15 seconds. Cell debris was then pelleted via centrifugation at 3, 500 x g for 20 minutes. The supernatant was transferred to an amber glass 20 ml screw top vial, evaporated under a stream of nitrogen and resuspended in 250 μl ice cold acetone. Residual cell debris was again removed by centrifugation at 16, 500 x g for 5 minutes prior to running on HPLC analysis.

HPLC Analysis

Carotenoid pigment analysis was performed using an Agilent 1100 HPLC system equipped with a Phenomenex, Luna Cl 8, 4.6 x 250 mm, 5 μm column, in conjunction with a phenomenex SecurityGuard C18, 4 x 3.0 μm guard, injection volume of 50 μl, UV detection at 474nm and a mobile phase of Ethyl Acetate Methanol: Water (10:88:2), 0.75 ml min^"1 for 10 minutes, increasing to Ethyl Acetate:Methanol:Water (48:50:2), 1.5 ml min^"1 for the remaining 20 minutes. Results β-carotene ketolase genes: CrtO Six CrtO type β-carotene ketolase genes were identified from five cyanobacteria, whose genomes have been previously sequenced. Based on identified conserved domains within these sequences, degenerate PCR primers were designed to allow amplification of putative CrtO type β-carotene ketolases from un-sequence organisms. The cyanobacteria Lyngbya sp. CCAP 1446/5 was used to demonstrate the practical application of this method. Once the complete open reading frame had been elucidated, this gene was functionally characterised along with the other CrtO type β-carotene ketolase genes.

All investigated putative CrtO type β-carotene ketolase genes were found to be active, by co-expression in β-carotene accumulating E. coli (Figure 2). Of the CrtO type β- carotene ketolase genes, that from Synechocystis sp. PCC 6803 demonstrated the highest level of activity towards the β-carotene substrate. This activity included the capacity to function as a di -ketolase, resulting in the accumulation of both echinenone and canthaxanthin. Similar di-ketolase activity was demonstrated by the CrtO type β-carotene ketolases genes derived form Gloeobacter violaceus PCC 7421, Nostoc punctiforme PCC 73102 (.1) and Anabaena variabilis ATCC 29413 (Figure 2). W hile the β-carotene ketolase genes derived from Lyngbya sp. CCAP 1446.5, Nostoc sp. PCC 7120 and Nostoc punctiforme PCC 73102 (.2) lack this function. These mono-ketolase gene's were also the least active of the CrtO type β-carotene ketolase genes investigated (Figure 2). β-carotene ketolase genes: CrtW All CrtW type ketolases were shown to be catalytically active. The direct comparison allowed by this study also demonstrates that this is at a level significantly greater than that of the CrtO type ketolases. All CrtW type ketolase genes also demonstrate the characteristic di-keto capacity, determined by the predominant biosynthesis of canthaxanthin, when co-expressed in β-carotene accumulating E. coli (Figure 3). Of the CrtW type β-carotene ketolase genes investigated, that from Nostoc punctiforme PCC 73102(.l) demonstrated greatest catalytic activity upon over expression in β-carotene accumulating E. coli, while the second CrtW type ketolase from this cyanobacteria is the least catalytically active of the genes investigated. Co-expression in zeaxanthin accumulating E. coli

Co-expression of the investigated β-carotene ketolase genes in zeaxanthin accumulating E. coli further confirmed the activity of the CrtW type β-carotene ketolase genes, while also demonstrating a difference in function between the CrtO and CrtW type ketolases. As co-expression of the CrtO type ketolases in zeaxanthin accumulating E. coli failed to change the carotenoid profile of the host organism. While co-expression of the highly active CrtW type ketolase genes in the zeaxanthin accumulating host, resulted in either; the predominant accumulation of large amounts of the di-keto carotenoid, canthaxanthin, accompanied by reduced levels of hydroxylated carotenoid, zeaxanthin (Figure 4), or accumulation of large amounts of the di-keto, di-hydroxy carotenoid, astaxanthin, accompanied by small amounts of zeaxanthin and canthaxanthin (Figures 5). Thus it was demonstrated that all cyanobacterial derived CrtW type β-carotene ketolase genes, investigated are able to participate a metabolic pathway resulting in the biosynthesis of astaxanthin. However, CrtO type ketolases derived from the same group of organisms are not able to participate in this pathway. True even for the most active of the CrtO type β- carotene ketolases, suggesting that a difference in substrate specificity exists between these two classes of β-carotene ketolase. Application of pBAD24 for Astaxanthin biosynthesis

Insertion of functional CrtW type β-carotene ketolase genes into plasmid pBAD24, allowed gene expression to be tightly regulated by the inducible promoter, pBAD. Achieved using a novel PCR based, ligation independent cloning method in which the PCR amplified template DNA and the plasmid act as both forward and reverse primers, allowing for the PCR based insertion of the specific DNA fragment in to a predetermined location within the plasmid. Failure to induce expression of a heterologous CrtW type β-carotene ketolase gene with L-arabinose, resulted in a significant reduction in the synthesis of ketolated compounds, echinenone and canthaxanthin, accompanied by the absence of astaxanthin (Figure 6). Induction of gene expression resulted in the reproducible and predominant biosynthesis astaxanthin, accompanied by small amounts of zeaxanthin and canthaxanthin (Figure 6). It was also demonstrated that induction with just 0.05% L- arabinose results in the greatest return with respect to astaxanthin biosynthesis. β-carotene hydroxylase genes

A bioinformatics screen of the genomes of five cyanobacteria revealed that putative β-carotene hydroxylase genes are less prevalent within the genomes of this group of organisms, than are putative β-carotene ketolase genes. As of the five cyanobacterial genomes screened four possessed single putative β-carotene hydroxylases, based on sequence homology. Functional characterisation of putative genes, via co-expression in E. coli confirmed the functionality of the previously characterised Synechocystis sp. PCC 6803 β-carotene hydroxylase, and further demonstrated that the putative β-carotene hydroxylase gene from Anabaena variabilis ATCC 29413 is also functional. Further, it was shown that the putative β-carotene hydroxylase genes from Nostoc punctiforme PCC 73102 and Nostoc sp. PCC 7120, were not functional. Direct comparison of the functional β-carotene hydroxylase genes reveals that the Synechocystis sp. PCC 6803 derived β-carotene hydroxylase is approximately 4 times more active than that derived form Anabaena variabilis ATCC 29413, resulting in the biosynthesis of 68.5 μg g^"1 (DCW), compared to 17.9μg g^" (DCW). Expression of the Er. herb icola (Pantoea agglomerans) derived β- carotene hydroxylase, isolated form the vector pAC-ZEAX, within the same expression system, demonstrates that these cyanobacterial β-carotene hydroxylase genes are significantly less active than other known β-carotene hydroxylase genes, as co-expression of the Er. Herbicola β-carotene hydroxylase in β-carotene accumulating E. coli resulted in the accumulation of 279 μg g^"1 (DCW) zeaxanthin, 15.6 and 4.01 fold greater than the cyanobacteria genes investigated. Discussion

Lyngbya sp. CCAP 1446/5 is able to accumulate β-carotene, echinenone, β- cryptoxanthin and zeaxanthin (Figure 7). This study shows both the carotenoid profile of this organism, and the characterisation of a functional CrtO gene, the first biosynthetic gene involved in carotenoid biosynthesis from this organism. When over-expressed in β-carotene accumulating E. coli this putative CrtO is able catalyse the synthesis of echinenone (Figure 3). As the genome of Lyngbya sp. CCAP 1446/5 has not been sequenced, it is currently not possible to determine the presence or absence of other functional β-carotene ketolase genes. However based on the carotenoid profile, Lyngbya sp. CCAP 1446/5 possess a complete carotenoid biosynthetic pathway, which would include at least; one functional β-carotene hydroxylase gene, and due to the presence of a large peak corresponding to astaxanthin, at least one functional CrtW type β-carotene ketolase gene, in addition to the CrtO ketolase identified here.

A bioinformatics screen of the sequenced genomes of five cyanobacteria, Nostoc punctiforme PCC 73102, Anabaena variabilis ATCC 29413, Nostoc sp, PCC 7120, Gloeobacter violaceus PCC 7421 and Synechocystis sp. PCC 6803, revealed the presence of twelve putative β-carotene ketolase genes, six putative CrtO and six putative CrtW type ketolases. Additionally a seventh CrtO type ketolase gene was elucidated from an un- sequenced cyanobacterium, Lyngbya sp. CCAP 1446/5. All putative CrtO type β-carotene ketolase genes investigated contained six conserved motifs, previously reported to depict a functional ketolase gene. No such class specific motifs, known to be essential to function, are found in the CrtW type ketolases. However three conserved histidine rich motifs have been identified that are thought to be vital to function. These motifs were present in all putative CrtW type ketolases identified.

This bioinformatics screen demonstrated that a number of cyanobacteria possess within their genomes multiple genes able to complete the same function. For example, I PCC 73102 encodes within its genome two CrtO type ketolases, and two CrtW type ketolases, while I ATCC 29413 has one CrtO and two CrtW type ketolases. Alternatively, the genome of I PCC 6803 encodes a single CrtO type β-carotene ketolase gene. Coincidentally this was the most active cyanobacterial CrtO type ketolase investigated. In this work it was demonstrated that all of the investigated ketolase genes are functional, contradicting the conclusions of Tsuchiya, et al (2005), who stated that just one β-carotene ketolase gene is able to be functional within a single organism (Tsuchiya et al., FEBS Lett, 579 (10), 2125-9 (2005)).

Previously a number of cyanobacterial β-carotene ketolase genes have been functionally characterised, these include genes from Synechocystis sp. PCC 6803, Nostoc punctiforme PCC 73102 and Gloeobacter violaceus PCC 7421 (Fernandez-Gonzalez et al., J Biol Chem, 272 (15), 9728-33 (1997); Steiger and Sandmann, Biotechnol Lett, 26 (10), 813-7 (2004); Steiger et al., Arch Microbiol, 184 (4), 207-14 (2005)). The carotenoid biosynthetic pathway of Gloeobacter violaceus PCC 7421 has been characterised by two independent research groups, with contradicting results (Steiger et al., 2005; Tsuchiya et al.,

2005). Additionally the function of the β-carotene ketolases derived from Nostoc sp. PCC 7120, have previously been determined based in gene-knock out studies (Mochimaru et al., FEBS Lett, 579 (27), 6111-4 (2005)).

This example shows for the first time the functional characterisation, via co- expression in E. coli, of two CrtW and one CrtO type β-carotene ketolase genes derived from Anabaena variabilis ATCC 29413, two CrtO type ketolases from Nostoc punctiforme PCC 73102, and single CrtW and CrtO ketolase genes from Nostoc sp. PCC 7120.

This example demonstrates that all β-carotene ketolase genes investigated, both CrtO and CrtW type, are functional. Confirming previous works of Fernandez-Gonzales (1997) and Steiger (2004, 2005), while conflicting with the results of Tsuchiya (2005). Further this investigation demonstrates that cyanobacterial CrtO type β-carotene ketolase genes are not able to participate in the astaxanthin biosynthetic pathway. This has previously been reported based on the analysis of CrtO type β-carotene ketolases derived from R. erythropolis strain PR4 and Synechocystis sp. PCC 6803, and their co expression with pACCR25ΔX (this encodes the CrtZ from Pantoea ananatis (Choi et al., Appl Microbiol Biotechnol. (2007); Misawa et al., J Bacteriol, 111 (22), 6575-84 (1995). As such, this example expended this lack of catalytic activity to include CrtO type ketolases derived from Nostoc punctiforme PCC 73102, Anabaena variabilis ATCC 29413, Gloeobacter violaceus PCC 7421 and Nostoc sp. PCC 7120. This was based on co- expression of these CrtO ketolases with genes isolated from Er. herbicola. In the characterisation a number of novel CrtO ketolases, it was noticed that several possess the catalytic potential to produce the di-ketolated compound canthaxanthin. For Nostoc punctiforme PCC 73102(.l), at 3O⁰C, canthaxanthin was the predominant keto-carotenoid synthesised. A feature not previously reported for CrtO type β-carotene ketolases. Previously, all CrtO type ketolases have been collectively referred to as mono-ketolases, reflecting the predominant biosynthesis of echinenone, the mono-ketolated carotenoid. This predominant di-ketolase activity was also observed for the CrtO type ketolase from Synechocystis sp. PCC 6803 when expressed at 4O⁰C.

With respect to astaxanthin biosynthesis, the failure of all CrtO type β-carotene ketolase genes to influence the carotenoid profile, when co-expressed in zeaxanthin accumulating E. coli, provides a number of significant insights. Initially, that the β-carotene ketolase and hydroxylase genes are competing for a single substrate, β-carotene, with the CrtO type ketolase, loosing the competition. As determined by the lack of keto-carotenoids. Second, that the CrtO β-carotene ketolases are not able to utilise hydroxylated carotenoids, β-cryptoxanthin and zeaxanthin, as substrates. Significant as in astaxanthin biosynthesis, at least one of the β-carotene ketolase or β-carotene hydroxylase genes involved in astaxanthin biosynthesis can be bi-functional.

A direct comparison of CrtO type β-carotene ketolase catalytic activity revealed that the Synechocystis sp. PCC 6803 derived ketolase is the most active. While those from Nostoc punctiforme(.2) PCC 73102 and Lyngbya sp. CCAP 1446/5 are the least active. Co- expression of CrtW type β-carotene ketolase genes in β-carotene producing E. coli resulted in the accumulation of large amounts of canthaxanthin. From this investigation, for the first time it is possible to see that the ketolase gene derived from Nostoc punctiforme PCC 73102(.l) is significantly more active than the remaining genes investigated. Producing 2.3 fold more canthaxanthin than the next most active ketolase gene, derived from Anabaena variabilis ATCC 29413(.2), and 16.8 fold more than the least active CrtW type ketolase investigated, which was also isolated from Nostoc punctiforme PCC 73102(.2). Similarly the two genes isolated from Anabaena variabilis ATCC 29413(.l) differ significantly in catalytic activity. Co-expression of CrtW type β-carotene ketolase genes in zeaxanthin producing E. coli demonstrated the ability of all CrtW type ketolase genes investigated to participate in a biosynthetic pathway leading to the production of astaxanthin. Although expression of CrtW type β-carotene ketolase genes from the pTrc promoter results in astaxanthin biosynthesis, it was decided that gene expression should be controlled by a more stringent promoter. For this the plasmid pBAD24 was employed (Guzman et al., JBacteriol, 177 (14), 4121-30 (1995)). The CrtW type β-carotene ketolase genes from Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102 and Anabaena variabilis ATCC 29413 were cloned in to this vector as inframe fusions, using a rapid PCR based cloning method. Using this pBAD expression system it was demonstrated that basal expression of the Anabaena variabilis ATCC 29413(.2) derived CrtW type β-carotene ketolase gene is not sufficient to result in the biosynthesis of detectable levels of astaxanthin. The affect of induction strength was then tested, ranging from 0.05 to 5% L-arabinose. Demonstrating that the addition of 0.05% L-arabinose was sufficient to stimulate gene expression, the result of which was astaxanthin biosynthesis. This astaxanthin biosynthesis was highly reliable and reproducible.

Contrary to the β-carotene ketolase genes, it was found that β-carotene hydroxylase genes are much less prevalent among the cyanobacteria, with single putative copies being detected in just four of the cyanobacteria investigated here, β-carotene hydroxylase genes isolated from Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413 were found to be functional. W hile those isolated form Nostoc sp. PCC 7120 and Nostoc punctiforme PCC 73102 were not active. This concurs with predictions based on the carotenoid profile of these organisms, as both Nostoc punctiforme PCC 73102 and Nostoc sp. PCC 7120 lack hydroxylated carotenoids, while Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413 do not.

Example 2

Screen of Cyanobacterial b-carotene ketolase genes Thirteen putative cyanobacterial β-carotene ketolase genes were screened for catalytic function. All investigated b-carotene ketolase genes were demonstrated to be functional, even when multiple ketolase genes are present within a single genome. In addition, this example reveals that all CrtW type β-carotene ketolase genes are able to participate in astaxanthin biosynthesis, while all investigated CrtO type β-carotene ketolase genes lack this catalytic activity, despite their apparent similarities in function. Construction of a stable & controllable expression system

Three CrtW type ketolases were selected for this example. The three CrtW type ketolases were placed under the control of the pBAD promoter, of expression vector pBAD24, using a PCR based cloning method. This system allowed for controlled and reproducible astaxanthin biosynthesis. Carotenoid accumulation, in zeaxanthin accumulating E. coli due to expression of different cyanobacterial b-carotene ketolase genes from pBAD24 can be seen in Figure 8. Enhanced Astaxanthin Biosynthesis

Carotenoid biosynthesis was also investigated in several E. coli strains. Te results revealed differences in the ability of these strains to biosynthesisize carotenoids. (Figure 9) As shown in Figure 9, carotenoid biosynthesis in TOPlO E. coli was 1.4 times greater than it was in the next strain, DH5a, and is 3 times greater than in HBlOl. Metabolic Engineering

As described above, DXS catalyses the first reaction of the non-mevalonate pathway. It was believed that overexpression of this gene should theoretically pull more substrate into this linear pathway, subsequently increasing carotenoid biosynthesis. IDI catalyses the conversion of isopentenyl pyrophosphate (IPP) into its structural isomer, dimethylallyl pyrophosphate (DMAPP), a process previously identified as a bottle neck in the synthesis of carotenoids. Overexpression of IDI (Er. herbicola (pAC-ZEAX IDI)), in addition to the over expression of either a cyanobacterial (A. variabilis ATCC 29413) or bacterial (E. coli JMlOl) DXS gene from pBAD24 resulted in a 4.9 and 5.9 fold increase in carotenoid biosynthesis, respectively. (Figure 10) Creation of a dual expression vector

Two genes are involved in the synthesis of astaxanthin from β-carotene, a β-carotene ketolase and a β-carotene hydroxylase. For this example a β-carotene hydroxylase gene expressed from co-expression plasmid pAC-ZEAX-IDI, controlled by its native promoter was used. This example demonstrated that expression of this β-carotene hydroxylase from a strong promoter (pBAD) results in increased astaxanthin biosynthesis, within E. coli. (Figure 11). Carbon Supplementation

To further improve carotenoid synthesis, the addition of carbon sources, metabolically linked to the synthesis of glyceraldehyde 3-Phosphate (G3P) & pyruvate, to the growth media were investigated. Revealing glycerol and D- sorbitol as promising additional carbons sources. Supplementation of growth media resulted in enhanced carotenoid synthesis both on terms of mg g-1 DCW and mg L-I. Suggesting precursor availability may be a limiting factor in carotenoid biosynthesis for this system. (Figure 12).

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a nucleic acid that encodes a polypeptide, wherein the polypeptide has at least 80% identity to SEQ ID NO. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60.

2. The composition of claim 1, wherein the nucleic acid comprises a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

3. A composition comprising a polypeptide wherein the polypeptide is encoded by a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

4. The compostion of claim 5, wherein the polypeptide comprises the sequence provided in SEQ ID NOS: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, or a complement thereof.

5. The composition of any of the claims 1-4, further comprising a vector.

6. The composition of any of the claims 1-4 further comprising a host cell.

7. An isolated polypeptide, wherein the polypeptide is encoded by a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

8. The isolated polypeptide of claim 9 comprising the sequence provided in SEQ ID NOS: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, or a complement thereof.

9. A composition comprising an isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

10. The composition of claim 1 wherein the composition consists essentially of an isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

11. An isolated nucleic acid comprising a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

12. The isolated nucleic acid of claim 13, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-10, 45-47, or a complement thereof.

13. A vector comprising the nucleic acid of any of the claims 13-14.

14. The vector of claim 15, wherein the nucleic acid is operably linked to a control element.

15. The vector of claim 16, wherein the control element is a promoter.

16. The vector of claim 17, wherein the promoter is selected from the group consisting of pTrc and pBAD.

17. A host cell comprising the nucleic acid of vector of any of the claims 13-18.

18. The host cell of claim 19, wherein the host cell is a bacterial cell.

19. An isolated polypeptide comprising an amino acid sequence encoded by the nucleic acids of any of the claims 13-14.

20. An isolated polypeptide comprising the sequence provided in SEQ ID NOS: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, or a complement thereof.

21. An islolated nucleic acid encoding the polypeptide of any of claims 21-22.

22. A vector comprising a nucleic acid of any of the claims 13-14.

23. The vector of claim 22 comprising the sequence of SEQ ID NO. 61, 62, 63, 64. 65. 66. 67, 68, 69, 70, or 71.

24. A method of producing a β-Carotene derived carotenoid in a cell, comprising:

(a) bringing into contact a cell and a first vector comprising a first nucleic acid, wherein the first nucleic acid encodes a polypeptide comprising a β-carotene sequence;

(b) bringing into contact the cell of (a) and a second vector comprising a second nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β-carotene ketolase sequence

(c) incubating the cell under conditions that allow expression of the β-carotene polypeptide and the β-carotene ketolase polypeptide,

(d) thereby producing a β-Carotene derived carotenoid in the cell.

25. The method of claim 24, wherein step (a) further comprises bringing into contact the cell and a third vector comprising a third nucleic acid, wherein the third nucleic acid encodes a third polypeptide comprising a β-carotene hydroxylase sequence; wherein step (b) further comprises incubating the cell under conditions that allow expression of the β-carotene hydroxylase polypeptide.

26. The method of any of the claims 24-25, wherein the second nucleic acid sequence comprises a nucleic acid sequence at least about 90% identical to one of the nucleic acid sequnces selected from the group consisting of SEQ ID NOs: 1-9, 45-47, or a complement thereof.

27. The method of any of the claims 24-26, wherein the second nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-9, 45-47, or a complement thereof.

28. The method of claim 25, wherein the third nucleic acid sequence comprises a nucleic acid sequence at least about 90% identical to the nucleic acid sequnece of SEQ ID NO: 10, or a complement thereof.

29. The method of claim 25, wherein the third nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 10, or a complement thereof.

30. A method of producing a β-Carotene derived carotenoid in a cell, comprising: (a) bringing into contact a cell and a first vector comprising a nucleic acid, wherein the first nucleic acid encodes a polypeptide comprising a β-carotene sequence;

(b) bringing into contact the cell of (a) and a second vector comprising a second and third nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β-carotene ketolase sequence and the third nucleic acid encodes a third polypeptide comprising a β-carotene hydroxylase sequence;

(c) incubating the cell under conditions that allow expression of the β-carotene polypeptide, the β-carotene ketolase polypeptide, and the β-carotene hydroxylase polypeptides;

(d) thereby producing a β-Carotene derived carotenoid in the cell.

31. The method of claim 30, wherein the second vector comprises the sequence of SEQ ID NO. 61, 62, 63, 64. 65. 66. 67, 68, 69, 70, or 71.

32. A method of producing a β-Carotene derived carotenoid in a cell, comprising:

(a) bringing into contact a cell and a first vector comprising a first nucleic acid, wherein the first nucleic acid encodes a first polypeptide comprising a a β- carotene ketolase sequence;

(b) incubating the cell under conditions that allow expression of the a β-carotene ketolase polypeptide,

(c) thereby producing a β-Carotene derived carotenoid in the cell.

33. The method of claim 32, wherein step (a) further comprises bringing into contact the cell and a second vector comprising a second nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β-carotene hydroxylase sequence; wherein step (b) further comprises incubating the cell under conditions that allow expression of the β-carotene hydroxylase polypeptide.

34. The method of claim 32 wherein the first vector of step (a) further comprise a second nucleic acid, wherein the second nucleic acid encodes a second polypeptide comprising a β-carotene hydroxylase sequence; wherein step(b) further comprises incubating the cell under conditions that allow expression of the β-carotene hydroxylase polypeptide.

35. The method of any of the claims 24-34 wherein the β-carotene ketolase is a CrtW type or a CrtO type β-carotene ketolase.

36. The method of any of the claims 24-35 wherein the β-carotene ketolase is a β- carotene ketolase isolated from Lyngbya sp. CCAP 1446/5, Nostoc punctiforme PCC 73102.1, Nostoc punctiforme 73102.2, Anabaena variabilis ATCC 29413.1, Anabaena variabilis ATCC 29413.2, or Nostoc sp. PCC 7120.

37. The method of any of claims 24-35 wherein the second vector comprises the sequence of SEQ ID NO. 61, 62, 63, 64. 65. 66. 67, 68, 69, 70, or 71.

38. The method of any of the claims 25-31 or 33-37, wherein the β-carotene hydroxylase is a CrtZ type β-carotene hydroxylase.

39. The method of any of the claims 25-31 or 33-38, wherein the β-carotene hydroxylase is a β-carotene hydroxylase isolated from Anabaena variabilis ATCC 29413.

40. The method of any of the claims 25-29 or 30-39, wherein the second vector comprises the sequence of SEQ ID NO. 61.

41. The method of any of the claims 25-29 or 30-39, wherein the second vector comprises the sequence of SEQ ID NO. 68.

42. The method of any of the claims 24-39 wherein the conditions of step (b) comprise incubating the cell between 30-37°C in the absence of L-arabinose.

43. The method of any of the claims 39-41 wherein the conditions of step (b) comprise incubating the cell between 30-37°C in the presence of L-arabinose.

44. The method of any of the claims 24-43, wherein the β-Carotene derived carotenoid produced is astaxanthin, lutein, canthaxanthin, adonixanthin, β-cryptoxanthin, adonirubin, echinenone, 3-hydroxyechinenone or 3'-hydroxyechinenone.

45. The method of any of the claims 24-44, wherein the cell is an E. coli cell.

46. The method of any of the claims 24-45, wherein the cell is in a system.

47. The method of claim 46, wherein the system is a bacteria, plant, animal, trangenic animal, fungi, or yeast.

48. The method of any of the claims 24-47, further comprising purifying the β-Carotene derived carotenoid.

49. The method of claim 48, further comprising isolating the purified β-Carotene derived carotenoid.

50. A composition comprising the β-Carotene derived carotenoid produced by the methods of claims of any of the claims 24-49.

51. A delivery device comprising the composition of claim 50.

52. The delivery device of claim 51, wherein the delivery device comprises a microcapsule, a microsphere, a nanosphere or nanoparticle, a liposome, a noisome, a nanoerythrosome, a solid-liquid nanoparticle, a leuprolide, a gel, a gel capsule, a tablet, a lotion, a cream, a spray, an emulsion, or a powder.

53. A microcapsule, comprising an agglomeration of primary microcapsules and a loading substance, each individual primary microcapsule having a primary shell, wherein the loading substance comprises the composition of claim 50, and is encapsulated by the primary shell, and wherein the agglomeration is encapsulated by an outer shell.

54. The microcapsule of claim 53, wherein the primary shell and/or outer shell comprises a surfactant, gelatin, polyphosphate, polysaccharide, or a mixture thereof.

55. The microcapsule of claim 53, wherein the primary shell and/or outer shell comprises gelatin type B, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, starch, modified starch, alfa-lactalbumin, beta-lactoglobumin, ovalbumin, polysorbiton, maltodextrin, cyclodextrin, cellulose, methyl cellulose, ethyl cellulose, hydropropylmethylcellulose, carboxymethylcellulose, milk protein, whey protein, soy protein, canola protein, albumin, kosher gelatin, non-kosher gelatin, Halal gelatin, non-Halal gelatin, or a mixture thereof.

56. A nutritional supplement comprising the composition of claim 50, the delivery device of any of claims 51-52, or the microcapsule of any of claims 53-55.

57. The nutritional supplement of claim 56, wherein the supplement is in the form of a tablet, gel-cap, capsule, liquid, or syrup.

58. A foodstuff comprising the composition of claim 50, the delivery device of any of claims 51-52, the microcapsule of any of claims 53-55, or the nutritional supplement of any of claims 56-57.

59. The foodstuff of claim 58, wherein the foodstuff is a baked good, a pasta, a meat product, a frozen dairy product, a milk product, a cheese product, an egg product, a condiment, a soup mix, a snack food, a nut product, a plant protein product, a hard candy, a soft candy, a poultry product, a processed fruit juice, a granulated sugar, a sauce, a gravy, a syrup, a nutritional bar, a beverage, a dry beverage powder, a jam or jelly, an infant formula, or a baby food.

60. The foodstuff of claim 58, wherein the foodstuff is a fish product, a companion pet food, a livestock or an aquaculture feed.

61. The foodstuff of claim 58, wherein the foodstuff is bread, tortillas, cereal, sausage, chicken, ice cream, yogurt, milk, salad dressing, rice bran, fruit juice, a dry beverage powder, rolls, cookies, crackers, fruit pies, or cakes.

62. A method of delivering the composition of claim 50, the delivery device of any of claims 51-52, the microcapsule of any of claims 53-55, the nutritional supplement of any of claims 56-57, or the foodstuff of any of claims 58-61.

63. The method of claim 62, wherein the subject is a mammal.

64. The method of claim 62, wherein the subject is a human.

65. A use of a microcapsule of any of claims 53-55, and to prepare a medicament for delivering a loading substance to a subject.

66. A method of reducing reactive oxygen species in a subject, comprising the step of administering to the subject an effective amount of the composition of claim 50, the delivery device of any of claims 51-52, the microcapsule of any of claims 53-55, the nutritional supplement of any of claims 56-57, or the foodstuff of any of claims 58- 61.

67. A method of removing free radicals in a subject, comprising the step of administering to the subject an effective amount of the composition of claim 50, the delivery device of any of claims 51-52, the microcapsule of any of claims 53-55, the nutritional supplement of any of claims 56-57, or the foodstuff of any of claims 58- 61.

68. A pharmaceutical formulation comprising a the composition of claim 50, the delivery device of any of claims 51-52, the microcapsule of any of claims 53-55, and a pharmaceutical carrier.

69. A kit comprising any one or more of the compositions of claims 1-23 or 12-1 A.

70. The composition of claim 50, the delivery device of any of claims 51-52, the microcapsule of any of claims 53-55, the nutritional supplement of any of claims 56- 57, or the foodstuff of any of claims 58-61, further comprising omega-3 and/or omega-6 fatty acids.

71. The method of any of the claims 70-71, wherein the composition of claim 50, the delivery device of any of claims 51-52, the microcapsule of any of claims 53-55, the nutritional supplement of any of claims 56-57, or the foodstuff of any of claims 58- 61, further comprises omega-3 and/or omega-6 fatty acids.

72. The pharmaceutical formulation of claim 68 further comprising omega-3 and/or omega-6 fatty acids.

73. A composition comprising a β-Carotene derived carotenoid isolated from a bacteria.

74. The composition of claim 73, wherein the composition is produced by the methods of claims of any of the claims 24-49.