MXPA00011969A - Genes of carotenoid biosynthesis and metabolism and methods of use thereof - Google Patents

Abstract

Nucleic acid sequences encoding&egr;-cyclase, isopentenyl pyrophosphate isomerase and&bgr;-carotene hydroxylase as well as vectors containing the same and hosts transformed with the vectors. Methods for controlling the ratio of various carotenoids in a host and for the production of novel carotenoid pigments. The present invention also provides a method for screening for eukaryotic genes encoding carotenoid biosynthesis, and for modifying the disclosed enzymes.

Description

BIOSYNTHESIS AND METABOLISM OF CAROTENOID GENES AND METHODS FOR USING THEM FIELD OF THE INVENTION The present invention describes nucleic acid sequences from eukaryotic genes that encode lycopene e-cyclase (also known as e-cyclase and e-lycopene cyclase), isopentyl pyrophosphate isomerase (IPP) and β-carotene hydroxylase as well as vectors that contain them and hosts transformed by the vectors. The present invention also provides methods to increase the accumulation of carotenoids, to change the composition of carotenoids and to produce rare and novel carotenoids. The present invention provides methods for controlling the relative proportion or amounts of various carotenoids in a host. The invention also relates to lycopene e-cyclase, IPP isomerase and modified β-carotene hydroxylase. Additionally, the present invention provides a method for classifying genes and cDNAs that encode enzymes for the biosynthesis and metabolism of carotenoids.

BACKGROUND OF THE INVENTION Carotenoid pigments with cyclic end groups are essential components of the photosynthetic apparatus of oxygenic photosynthetic organisms (eg, cyanobacteria, algae and plants; Goodwin, 1980). The yellow bicyclic and symmetrical carotenoid pigment β-carotene (or in rare cases, the asymmetric bicyclic α-carotene) is intimately associated with the photosynthetic reaction center and plays a vital role in protecting against potentially lethal photo-oxidative damage (Koyama, 1991). The ß-carotenes and other carotenoids derived from it or from -carotene, also serve as pigments that collect light (Siefermann-Harms, 1987), are involved in the thermal dissipation of excess light energy captured by the antenna that collects light (Demming-Adams &Adams, 1992), provide a substrate for the biosynthesis of abscisic acid regulator of plant growth (Rock &Zeevaart, 1991; Parry &Horgan, 1991) and are precursors of vitamin A in plants. diets of humans and animals (Krinsky, 1987). Plants also take advantage of carotenoids as coloring agents in flowers and fruits to attract pollinators and agents that disperse seeds (Goodwin, 1980). The color supplied by the carotenoids also has agronomic value in several important crops. Carotenoids are normally harvested from a variety of organisms, including plants, algae, yeasts, cyanobacteria and bacteria, to be used as pigments in food and meals. The probable route of formation of cyclic carotenoids in plants, algae and cyanobacteria is illustrated in figure 1. In the carotenoids of higher plants there are usually two types of cyclic end groups or rings, these are referred to as the beta (beta) rings ye (epsilon) (figure 3). The precursor acyclic terminal group (without ring structure) is referred to as the terminal group? (psi). The terminal groups ß and e differ only in the position of the double bond of the ring. Carotenoids with two ß rings are ubiquitous and those that have a ß ring and an e ring are common although carotenoids with two e rings are not common. Β-carotene (Figure 1) has two β-terminal groups and is a symmetric compound that is a precursor to numerous other carotenoids of important plants, such as zeaxanthin and violaxanthin (Figure 2). Genes coding for carotenoid biosynthesis enzymes have been previously isolated from a variety of sources including bacteria (Armstrong et al., 1989, Mol.Gen, Genet., 216, 254-268; Misawa et al. ., 1990, J. Bacteriol., 172, 6704-12), to fungi (Schmidhauser et al., 1990, Mol.Cell. Biol., 10, 5064-70), to cyanobacteria (Chamovitz et al., 1990, Z. Naturforsch, 45c, 482-86; Cunningham et al., 1994) and higher plants (Bartley et al., Proc. Natl. Acad. Sci. USA 88, 6532.36; Martinez-Ferez & Vioque, 1992, Plant. Mol. Biol. 18, 981 83). Many of the isolated enzymes show a great diversity in structure, functions and inhibitory properties between sources. For example, the desaturases phytoenas from Synechococcus cyanobacterium and higher plants and green algae carry out desaturation in two steps to produce? -carotenes as a reaction product. In plants and cyanobacteria, a second enzyme (β-carotene desaturase), with a similar amino acid sequence to phytoena desaturase, catalyzes two additional desaturations to produce lycopene. In contrast, a single desaturase enzyme from Erwinia herbicola and other bacteria introduces the four double bonds required to form lycopene. Erwinia and other bacterial desaturases have little similarity in the amino acid sequence with plant desaturases and cyanobacteria enzymes and are thought to have an unrelated ancestry. Therefore, even if you have a gene from a source, it may be difficult to identify a gene that codes for an enzyme of similar function in another organism. In particular, the sequence similarity between certain prokaryotic and eukaryotic genes that code for carotenoid biosynthesis enzymes is very low. Furthermore, the mechanism for the expression of genes in prokaryotes and in eukaryotes seems to differ enough in such a way that we can not expect that an isolated eukaryotic gene will be expressed properly in a prokaryotic host. The difficulties in isolating genes encoding enzymes with similar functions is exemplified by recent efforts to isolate the gene encoding the enzyme that catalyzes the formation of β-carotene from the lycopene acyclic precursor. Although the gene coding for an enzyme with this function had been isolated from a bacterium, it had not been isolated from any photosynthetic prokaryote organism or from any eukaryotic organism. The isolation and characterization of the enzyme that catalyzes the formation of β-carotene in the cyanobacterium Synechococcus PCC7942, were described by the inventors of the present and by others (Cunningham et al., 1993 and 1994). The similarity in the amino acid sequence of the cyanobacterial enzyme to the various bacterial lycopene β-cyclases is so low (approximately 18-25% overall, Cunningham et al., 1994) that there is much uncertainty as to whether they share a common ancestor , or instead, represent an example of a convergent evolution. There remains a need to isolate eukaryotic and prokaryotic genes and cDNAs that encode polypeptides involved in the biosynthetic pathway of carotenoids, including those encoding lycopene e-cyclase, IPP isomerase and β-carotene hydroxylase. There continues to be a need for methods that increase the production of carotenoids; that alter the composition of carotenoids and that reduce or eliminate the production of carotenoids. There also remains a need in the art for methods for screening genes and cDNAs that encode enzymes for the biosynthesis and metabolism of carotenoids.

SUMMARY OF THE INVENTION In accordance with the foregoing, a first object of this invention is to provide purified and / or isolated nucleic acids encoding enzymes involved in the biosynthesis of carotenoids, in particular, lycopene e-cyclase, IPP isomerase and β- carotene hydroxylase. A second object of this invention is to provide purified and / or isolated nucleic acids encoding enzymes that produce novel or unusual carotenoids.

A third object of the present invention is to provide vectors containing said genes. A fourth object of the present invention is to provide hosts transformed with those vectors. Another object of the present invention is to provide hosts that accumulate novel or unusual carotenoids or that accumulate large amounts of specific or total carotenoids. Another object of the present invention is to provide hosts with inhibited and / or altered carotenoid production. Another object of this invention is to ensure the expression of genes related to eukaryotic carotenoids in a recombinant prokaryotic host. Still another object of the present invention is to provide a method for screening eukaryotic and prokaryotic genes and cDNAs that encode enzymes involved in the biosynthesis and metabolism of carotenoids. A further object of the invention is to provide a method for manipulating the biosynthesis of carotenoids in photosynthetic microorganisms, by inhibiting the synthesis of certain enzyme products to cause the accumulation of precursor compounds. Another object of the invention is to provide lycopene e-cyclase, IPP isomerase and modified β-carotene hydroxylase. These and other objects of the present invention have been achieved by the inventors herein, as described below. The subject of the present invention is an isolated and / or purified nucleic acid sequence encoding a protein having lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase enzymatic activity and having the amino acid sequence SEQ ID NOS: 2, 4, 14 to 21 or 23 to 27. The invention also includes vectors comprising any of the nucleic acid sequences listed above and host cells transformed with these vectors. Another subject of the present invention is a method for producing or increasing the production of a carotenoid in a host cell, comprising inserting into the host cell a vector comprising a heterologous nucleic acid sequence, which codes for a protein having enzymatic activity of lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase, wherein the heterologous nucleic acid sequence is operatively linked to a promoter and expresses the heterologous nucleic acid sequence to produce the protein.

Another subject of the present invention is a method for modifying the production of carotenoids in a host cell, the method comprising inserting into the host cell a vector comprising a heterologous nucleic acid sequence, which produces an RNA and / or which codes for a protein that modifies the enzymatic activity of lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase, with respect to an untransformed host cell, wherein the heterologous nucleic acid sequence is operatively linked to a promoter and expresses the nucleic acid sequence heterologous in the host cell to modify the production of the carotenoids in the host cell, with respect to the non-transformed host cell. The present invention also includes a method for expressing, in a host cell, a heterologous nucleic acid sequence, which codes for a protein having lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase enzymatic activity, the method comprises inserting into the host cell a vector comprising the heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence is operatively linked to a promoter and which expresses the heterologous nucleic acid sequence. Also included is a method for expressing, in a host cell, a heterologous nucleic acid sequence encoding a protein that modifies the enzymatic activity of lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase, in the host cell, with respect to to a non-transformed host cell, the method comprises inserting into the host cell a vector comprising the heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence is operatively linked to a promoter.; and expressing the heterologous nucleic acid sequence. Another subject of the present invention is a method for screening genes and cDNAs that encode enzymes involved in the biosynthesis and metabolism of carotenoids.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and of the many attendant advantages thereof, will be readily obtained as it is better understood with reference to the following detailed description, when taken together with the accompanying drawings, wherein : Figure 1 is a schematic representation of the putative route of β-carotene biosynthesis in cyanobacteria, algae and plants. Enzymes that catalyze different stages are indicated on the left. The white sites of the NFZ and MPTA bleaching herbicides are also indicated on the left. Abbreviations: DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; GPP, geranyl pyrophosphate; IPP, isopentyl pyrophosphate; LCY, lycopene cyclase; MVA, mevalonic acid; MPTA, 2- (4-methylphenoxy) triethylamine hydrochloride; NFZ, norflurazon; PDS, phytoena desaturase; PSY, phytoena synthase; ZDS,? -carotene desaturase; PPPP, prefitoena pyrophosphate. Figure 2 represents possible routes of synthesis of cyclic carotenoids and xanthophylls (oxycarotenoids) common to plants and algae from neurosporene. The demonstrated activities of the ß- and e-cyclase enzymes of A. thaliana are indicated by bold arrows labeled with β or e, respectively. A bar below the arrow leading to e-carotene indicates that enzyme activity was examined but no products were detected. The steps marked by an arrow with a dotted line have not been specifically examined. The conventional numbering of carbon atoms is provided for neurosporene and a-carotene. The inverted triangles (1) 3 mark the positions of the double bonds introduced as a consequence of the desaturation reactions. Figure 3 represents the carotene end groups found in plants.

Figure 4 is a DNA sequence and the predicted amino acid sequence of a lycopene c-cyclase cDNA, isolated from A. thaliana (SEQ ID NOS: 1 and 2). These sequences were deposited with the access number Genbank USO738. This cDNA is incorporated in the pATeps plasmid. Figure 5 is a DNA sequence coding for ß-carotene hydroxylase isolated from A. thaliana (SEQ ID NO: 3). This cDNA is incorporated in the pATOHB plasmid. Figure 6 is an alignment of the predicted amino acid sequences of ß-carotene hydroxylase (SEQ ID NO: 4) of A. thaliana with those of the bacterial β-carotene hydroxylase enzymes of Alicalgenes sp. (SEQ ID NO 5) (Genbank D58422), Erwinia herbicola Eho 10 (SEQ ID NO .: 6) (GenBank M872280), Erwinia uredovora (SEQ ID NO: 7) (GenBank D90087) and Agrobacterium aurianticum (SEQ ID NO: 8) (GenBank D58420). A consensus sequence is also shown. The five genes are identical where, in the consensus, a capital letter appears. A lowercase letter indicates that three of the five including A. thaliana, have identical residue, TM; transmembrane Figure 7 is a DNA sequence of a cDNA encoding an isolated IPP isomerase of A. thaliana (SEQ ID NO: 9). This cDNA is incorporated in the plasmid pATDP5. Figure 8 is a DNA sequence of a second cDNA encoding another IPP isomerase isolated from A. thaliana (SEQ ID NO: 10). This cDNA is incorporated in the plasmid pATDP7. Figure 9 is a DNA sequence of a cDNA coding for an IPP isomerase isolated from Haematococcus pluvialis (SEQ ID NO: 11). This cDNA is incorporated in the plasmid pHP04. Figure 10 is a DNA sequence of a second cDNA encoding another IPP isomerase isolated from Haematococcus pluvialis (SEQ ID NO: 12). This cDNA is incorporated in the plasmid pHP05. Figure 11 is an alignment of the amino acid sequences predicted by the IPP isomerase cDNAs isolated from A. thaliana (SEQ ID NO .: 16 and 18), H. pluvialis (SEQ ID NOS .: 14 and 15), Clarkia breweri (SEQ ID NO .: 17) (see, Blanc &Pichersky, Plant Physiol. (1995) 108; 855; accession number Genbank X82627), and Saccharomyces cerevisiae (SEQ ID NO.: 19) (access number Genbank J05090). Figure 12 is a DNA sequence of the cDNA encoding an IPP isomerase isolated from Tagetes erecta (marigold, SEQ ID NO: 13). This cDNA is incorporated in the plasmid pPMDPI. The xxx denote a region not originally sequenced. Figure 21A shows the complete sequence of the calendula.

Figure 13 is an alignment of the consensus sequence of four β-cyclases (SEQ ID NO .: 20) of plants with the lycopene e-cyclase (SEQ ID NO: 21) of A. Thaliana. A capital letter is used in the ß plant consensus where the four genes of β-cyclase predict the same amino acid residue in this position. A lowercase letter indicates that an identical residue was found in three of the four. The dashed lines indicate that the amino acid residue was not conserved and the points in the sequence denote a vacuum. The consensus of the aligned sequences is provided, in uppercase letters under the alignment, where the β- and e-cyclases have the same amino acid residue. The arrows indicate some of the conserved amino acids that will be used as binding sites for the construction of chimeric cyclases with novel enzymatic activities. Several regions of interest include a sequence signature indicative of a dinucleotide binding motif and the two predicted transmembrane (TM) helical regions are indicated below the alignment and are underlined. Figure 14 shows the nucleotide (SEQ ID NO: 22) and the amino acid sequences (SEQ ID NO: 23) of cDNA # 5 of Adonis palaestina e-cyclase (pheasant eye). Figure 15A shows the nucleotide (SEQ ID NO: 24) and the amino acid sequences (SEQ ID NO: 25) of cDNA e-cyclase from potato. Figure 15B shows the amino acid sequence (SEQ ID NO: 26) of a chicic lettuce / potato lycopene e-cyclase. The amino acids with lower case letters come from the lettuce cDNA and those that appear with a capital letter come from the potato cDNA. The product of this Chimeric cDNA has an e-cyclase activity and converts lycopene to monocyclic d-carotene. Figure 16 shows a comparison between the amino acid sequences of e-cyclase (SEQ ID NO: 27) of Arabidopsis and e-cyclase (SEQ ID NO: 25). Figure 17A shows the nucleotide sequence of Ipil (SEQ ID NO: 28) of Adonis palaestina and the figure 17B shows the nucleotide sequence of Ipi2 (SEQ ID DO NOT. : 29) of Adonis palaestina. Figure 18A shows the nucleotide sequence of Ipil (SEQ ID NO .: 11) of Haemotoccus pluvialis and Figure 18B shows the nucleotide sequence of Ipi2 (SEQ.

ID NO. : 30) of Haemotoccus pluvialis. Figure 19A shows the nucleotide sequence of Ipil (SEQ ID NO: 31) of Lactuca sativa (romaine lettuce) and Figure 19B shows the nucleotide sequence of Ipi2 (SEQ ID NO: 32) of Lactuca sativa. Figure 20 shows the nucleotide sequence of Ipil (SEQ ID NO: 33) of Chlamydomonas reinhardrii. Figure 21a shows the nucleotide sequence of Ipil (SEQ ID NO: 34) of Tagetes erecta (marigold) and Figure 2IB shows the nucleotide sequence of Ipil (SEQ ID NO: 15) of Oryza sativa (rice). Figure 22 shows an alignment of amino acid sequences of several isopentyl isomerases (IPI) (SEQ ID NO: 16, 36-45) of plants and green algae. Figure 23 shows a comparison between nucleotide sequences # 3 e-cyclase cDNA and # 5 Adonis palaestina cDNA. Figure 24 shows a comparison between predicted amino acid sequences # 3 e-cyclase cDNA and # 5 Adonis palaestina cDNA. Figure 25 shows an alignment of sequences of several ß- and plant e-cyclases. The sequences represented with gray denote identical sequences between the e-cyclases, the sequences represented in black denote identical sequences between both cyclases, the β- and the e. Figure 26 shows an alignment of sequences of the plant e-cyclases of Figure 25. The sequences depicted in black denote identical sequences between the e-cyclases. Figure 27 is a dendrogram or "tree" illustrating the degree of similarity in the amino acid sequence of various β-lycopene and e-cyclases.

Figure 28 shows a comparison between predicted amino acid sequences of Arabidopsis e-cyclases and lettuce e-cyclases.

DESCRIPTION OF THE PREFERRED MODALITIES The present invention includes an isolated and / or purified nucleic acid sequence encoding a protein having lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase enzymatic activity and having the amino acid sequences of SEQ. ID NO. : 2, 4, 14 to 21, 23 or 2 to 27. The nucleic acids encoding lycopene e-cyclase, β-carotene hydroxylase and IPP isomerases have been isolated from various genetically distant sources. The inventors of the present invention have isolated nucleic acids encoding the enzyme IPP isomerase, which catalyzes the reversible conversion of isopentyl pyrophosphate (IPP) in dimethylallyl pyrophosphate (DMAPP). The IPP isomerase cDNAs were isolated from the plants A. thaliana, Tagetes erecta (marigold), Adonis palaestina (pheasant eye), Lactuca sativa (romaine lettuce) and the green algae H. pluvialis and Chlamydomonas reinhardrii. The alignments of the amino acid sequences predicted in some of these cDNAs are shown in Figures 12 and 22. The plasmids containing some of these cDNAs were deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852 on March 4, 1996, with access numbers ATCC 98000 (pHP05 - H. pluvialis); 98001 (pMDPl - (cord) 98002 (pATDP7 - A. thaliana) and 98004 (pHP04 -H. pluvialis) The inventors have also isolated nucleic acids encoding the enzyme β-carotene hydroxylase, which is responsible for hydroxylating to the terminal group ß in the carotenoids The nucleic acid of the present invention is shown in SEQ ID NO: 3 and in Figure 5. The full-length cDNA product hydroxylates both terminal groups of β-carotene as well as the products of the cDNAs encoding truncated proteins in up to 50 N-terminal amino acids The products of genes encoding proteins truncated between about 60 and 110 amino acids from the N terminus preferably hydroxylate only one ring A plasmid containing this gene was deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852 on March 4, 1996 with accession number ATCC 98002 (pATDP7 - A. thaliana). Researchers have also isolated nucleic acids encoding the enzyme lycopene e-cyclase, which is responsible for the formation of terminal e groups in carotenoids. The e-cyclase of A. thaliana adds an e-ring only at one end of the symmetrical lycopene, while the related β-cyclase adds a ring at both ends. The A. thaliana cDNA of the present invention is shown in Figure 4 and in SEQ ID NO. : 1. A plasmid containing this gene was deposited in American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852 on March 4, 1996, with accession number ATCC 98005 (pATeps A. thaliana). In addition, lycopene e-cyclases have been identified in lettuce and in Adonis palaestina (cDNA # 5) which codes for enzymes that convert lycopene into bicyclic e-carotene (e, e-carotene). An additional Adonis palaestina cDNA (cDNA # 3) codes for a lycopene e-cyclase that converts lycopene to d-carotene (e,? -carotene) and differs from the lycopene e-cyclase which forms bicyclic e-carotene (e , e-carotene) only through 5 amino acids. One or more of these amino acids can be modified by altering the nucleotide sequence in cDNA # 5 to obtain an enzyme that forms e, bicyclic e-carotene. The sequences of the e-cyclases of Adonis palaestina and Arabidopsis thaliana have approximately 70% nucleotide identity and approximately 72% amino acid identity. Initial experiments of the inventors with chimeric genes indicated that the part of the e-cyclase which is responsible for the addition of 2 e-rings to form the e, e-carotene is the carboxy terminal portion of the gene. Lettuce e-cyclase adds two e-rings to form e, e-carotene. A DNA encoding a partial potato e-cyclase (which lost its amino-terminal portion), when combined with an amino terminal region of the lettuce e-cyclase gene, produces a monocyclic d-carotene (e,? Carotene) . With the discovery of the differences between clone # 3 and clone # 5 of Adonis palaestina, the specific amino acids responsible for the addition of an additional e-ring have been identified (figure 24). Specifically, in clone # 3 amino acid 55 is Thr and Ser in clone # 5, amino acid 210 is Asn in clone # 3 and Asp in clone # 5, amino acid 231 is Asp in clone # 3 and Glu in clone # 5, amino acid 352 is lie in clone # 3 and Val in clone # 5 and amino acid 524 is Lys in clone # 3 and Arg in clone # 5. It can be seen that these changes are very conservative, since it is only a change, at amino acid 210, the load of the protein changes. Thus, it is clear that the nucleic acids of the invention that code for the enzymes as presently disclosed, can be altered to increase a particularly desirable property of the enzyme, to change a property of the enzyme or to reduce an undesirable property of the enzyme. These modifications can be effected by deletion or deletion, substitution or insertion of one or more amino acids and can be performed by routine enzymatic manipulation of the nucleic acid encoding the enzyme (such as, for example, by digesting restriction enzymes, eliminating nucleotides by mung bean nuclease or Bal31, the insertion of nucleotides by the Klenow fragment and by religation of the ends), by cycle-directed mutagenesis or can be accidental, such as, for example, by low fidelity PCR or those obtained by means of mutations in the hosts that are producers of the enzymes. These techniques as well as other suitable techniques are well known in the art. The mutations can be made in the nucleic acids of the invention, such that a particular codon is changed in a codon encoding a different amino acid. This mutation is generally done by making as few changes as possible in the nucleotide. A substitution mutation of this type can be performed to change an amino acid in the resulting protein in a non-conservative form (i.e. by changing the codon of an amino acid belonging to a group of amino acids having a particular size or characteristic in an amino acid). which belongs to another group) or in a conservative form (ie, changing the codon of an amino acid belonging to a group of amino acids that has a particular size or characteristic in an amino acid belonging to the same group). This conservative change generally leads to a minor change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. It should be considered that the present invention includes sequences containing conservative changes, which do not significantly alter the activity or binding characteristics of the resulting protein. The following is an example of several amino acid groups: Amino acids with non-polar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan and Methionine. Amino acids with polar R groups without charge: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine and Glutamine. Amino acids with charged polar R groups: (negatively charged at pH 6.0): aspartic acid and glutamic acid. Basic amino acids: (positively charged at pH 6.0): Lysine, Arginine and Histidine.

Another group may be the amino acids with phenyl groups: phenylalanine, tryptophan and tyrosine. Another grouping can be in accordance with the molecular weight (i.e., the size of the R groups). Particularly preferred substitutions are: - Lys by Arg and vice versa, in such a way that the positive charge can be maintained; - Glu for Asp and vice versa, in such a way that the negative charge can be maintained; - Being by Thr, in such a way that a free -OH can be maintained; and - Gln by Asn in such a way that a free NH2 can be maintained. Substitutions of amino acids can also be introduced to substitute an amino acid with a particularly preferred property. For example, a Cys can be introduced to provide a potential site for the disulfide bridges with another Cys. A His can be introduced as a particularly "catalytic" site (ie, His can act as an acid or a base and is the most common amino acid in biochemical catalysis). Pro can be introduced, due to its particularly flat structure that induces ß turns or turns in the structure of the protein.

It is clear that certain modifications of SEQ ID NOS .: 2, 4, 14 to 21, 23 or 25 a may occur. 27 without destroying the activity of the enzyme. It will be especially noted that the truncated versions of the nucleic acids of the invention are functional. For example, of the N terminus of the lycopene e-cyclases of the invention several amino acids can be deleted (from 1 to about 120) and a functional protein can still be produced. This fact is especially clarified with figure 25, which shows an alignment of sequences of several plant e-cyclases. As can be seen from Figure 25, there is a great disparity of the sequences between the amino acid sequences of 2 and about 50 to 70 (depending on the particular sequence since there are gaps). There is less sequence dissimilarity, but also in a significant amount, between about 50 to 70 and about 90 to 120 (depending on the particular sequence). After this, the sequences are conserved very well, except for small niches of dissimilarity between approximately 275 to 295 and approximately 285 a 305 (depending on the particular sequence) and between approximately 395 to 415 and approximately 410 to 430 (depending on the particular sequence). The inventors of the present have found that the amount of the 5 'region present in the nucleic acids of the invention can alter the activity of the enzyme. Instead of reducing the activity, the truncation of the 5 'region of the nucleic acids of the invention can result in an enzyme with a different specificity. Thus, the present invention relates to nucleic acids and enzymes encoded in this manner that are truncated in a range of 0 to 50, preferably 0 to 25, codons of the 5 'start codon of their prokaryotic counterparts, as determined by the alignment maps discussed below. For example, when the cDNA coding for β-carotene hydroxylase from A. Thaliana is truncated, the resulting enzyme catalyzes the formation of β-cryptoxanthin as the main product and zeaxanthin as the minor product; contrasting with its normal production of zeaxanthin. It is intended that the present invention include those nucleic acids and amino acid sequences in which substitutions, deletions, additions or other modifications have occurred, as compared to SEQ ID NO: 2, 4, 14 to 21, 23 or 25 to 27 , without destroying the activity of the enzyme. Preferably, substitutions, deletions, additions or other modifications occur at the 5 'end or at any other of those positions that already showed dissimilarity between any of the amino acid sequences currently revealed (see also Figure 25) or other amino acid sequences which are known in the art and which code for the same enzyme (i.e., lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase). In each case, the similarity and identity of the nucleic acid and the amino acid sequence were measured using the sequence analysis software, for example, Sequence Analysis, Gap, or BestFit software packages from Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705), MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wisconsin 53715) or MacVector (Oxford Molecular Group, 2105 S. Bascom Avenue, Suite 200, Campbell, California 95008). This software uses algorithms to match similar sequences by assigning degrees of identity to the various substitutions, deletions or other modifications and includes detailed instructions regarding useful parameters, etc., so that those of ordinary skill in the art can easily compare similarities and sequence identities. An example of a useful algorithm in this respect is the Needleman and Wunsch algorithm, which is used in the Gap program mentioned above. This program finds the alignment of two complete sequences, which maximizes the number of matches and minimizes the number of holes. Another useful algorithm is the Smith and Waterman algorithm, which is used in the BestFit program mentioned above. This program creates optimal alignments of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using Smith and Waterman's local homology algorithm. Conservative (ie, similar) substitutions usually include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid, glutamic acid, asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Substitutions may also be made on the basis of preserving hydrophobicity or hydrophilicity (see Kyte and Doolittle, J. Mol. Biol. 157: 105-132 (1982)) or on the basis of the ability to adopt a secondary structure of similar polypeptide. (see Chou and Fasman, Adv. Enzymol 47: 45-148 (1978)). If comparison is made between nucleotide sequences, preferably the stretch or comparison length of the sequences is at least 50 nucleotides, more preferably at least 60 nucleotides, at least 75 nucleotides or at least 100 nucleotides. What is most preferred is that the comparison be made between the nucleic acid sequences which code for the enzyme coding regions necessary for the activity of the enzyme. If the comparison is made between amino acid sequences, preferably the comparison length is at least 20 amino acids, more preferably at least 30 amino acids, at least 40 amino acids or at least 50 amino acids. What is most preferred is that the comparison be made between the amino acid sequences in the enzyme coding regions necessary for the activity of the enzyme. It should be appreciated that also within the scope of the present invention are nucleic acid sequences encoding lycopene e-cyclases, IPP isomerases and β-carotene hydroxylases encoding enzymes having the same amino acid sequence as SEQ ID NOS .: 2, 4, 14 to 21, 23 or 25 to 27, but which degenerate into the nucleic acids specifically disclosed herein. It is preferred that the amino acid residues described herein be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the polypeptide retains the desired functional property of immunoglobulin binding. In accordance with the present invention, conventional techniques of molecular biology, microbiology and recombinant DNA can be utilized within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-111 [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook "Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J.E., ed. (1994)]; "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Harnes & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Harnes & S.J. Higgins, Eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide to Molecular Cloning" (1984). The present invention also includes vectors.

Suitable vectors according to the present invention comprise a nucleic acid of the invention which codes for an enzyme involved in the biosynthesis or metabolism of carotenoids and a promoter suitable for the host and can be constructed using techniques well known in the art (e.g. Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, Ausubel et al., Current Protocole in Molecular Biology, Greene Publishing and Wiley Interscience, New York, 1991). Suitable vectors for eukaryotic expression in plants are described in Frey et al., Plant J. (1995) 8 (5): 693 and in Misawa et al, 1994a; incorporated as reference in the present. Suitable vectors for prokaryotic expression include pACYC184, pUC119, and pBR322 (which are available from New England BioLabs, Bevery, MA) and pTrcHis (invitrogen) and pET28 (novagen and derivatives thereof).

The vectors of the present invention may additionally contain regulatory elements such as promoters, repressors, selectable markers, such as antibiotic resistance genes, etc. The nucleic acids encoding the carotenoid enzymes as described above, when cloned into a suitable expression vector, can be used to overexpress these enzymes in a plant expression system or to inhibit the expression of these enzymes. For example, a vector containing the gene encoding lycopene e-cyclase can be used to increase the amount of -carotene and carotenoids derived from α-carotene (for example lutein and α-cryptoxanthin) in an organism and thereby alter the value nutritional, pharmacology and the value of the body's visual appearance. Therefore, the present invention includes a method for producing or reinforcing the production of a carotenoid in a host cell, relative to an untransformed host cell, wherein the method comprises inserting within the host cell a vector comprising a sequence of heterologous nucleic acid encoding a protein having lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase activity, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and expressing the heterologous nucleic acid sequence to produce the protein. This invention also includes a method for modifying the production of carotenoids in a host cell, the method comprising inserting within the host cell a vector comprising a heterologous nucleic acid sequence that produces an RNA and / or codes for a protein that modifies the enzymatic activity of lycopene e-cyclase, IPP isomerase or β-carotene hydroxylase, relative to an untransformed host cell, wherein the heterologous nucleic acid sequence is operably linked to a promoter, and expressing the heterologous nucleic acid sequence in the host cell to modify the production of the carotenoids in the host cell, in relation to the non-transformed host cell. The term "modify production" means that the amount of carotenoids produced in the host cell can be reinforced, reduced or remain the same, as compared to the non-transformed host cell. According to one embodiment of the present invention, the composition of the carotenoids (ie, the specific carotenoids produced) change one against the other, and this change is compensated by the result of either a net gain, a net loss or no change in the total amount of carotenoids produced in the cell. According to another embodiment of the present invention, the production or biochemical activity of the carotenoids (or the enzymes that catalyze their formation) is reinforced by the insertion of a nucleic acid encoding an enzyme, according to the invention. In another embodiment of the invention, the production of biochemical activity of the carotenoids (or the enzymes that catalyze their formation) can be reduced or inhibited by different approaches available to those skilled in the art, among which include methodologies or approaches concerning: antisense (eg Gray et al (1992) Plant Mol. Biol. 19: 69-87), ribozymes (eg, Wegener et al (1994) Mol. Gen. Genet. 245: 465-470) -suppression (eg, Fray and Grierson (1993) Plant Mol. Biol. 22: 89-602), directed disruption of genes (eg, Schaefer et al. (1997) Plant J. 11: 1195-1206), intracellular antibodies (for example, Rondón and Marasco (1997) Ann. Rev. Microbiol. 51: 257-283) or any other approach based on knowledge or availability of the amino acid or nucleic acid sequences of the invention and / or the portions thereof, in order to reduce the accumulation of carotenoids with anil the e and compounds derived from them (for the inhibition of e-cyclase) or carotenoids with β-hydroxylated rings and compounds derived therefrom. For inhibition of β-hydroxylase) or, in the case of IPP isomerase, accumulation of any isoprenoid compound. Preferably, at least a portion of the nucleic acid sequences used in the methods, vectors and host cells of the invention codes for an enzyme having an amino acid sequence that is at least 85% identical, preferably at least 90%, or at least 95% identical or identical, complementary to SEQ ID NOS .: 2, 4, 14 to 21, 23 or 25 to 27). The identity of sequences is determined as previously observed. Preferably, additions of sequences, deletions or other modifications are made in the manner indicated above, so as not to affect the function of the particular enzyme. In a preferred embodiment, vectors containing DNA encoding an eukaryotic IPP isomerase are made to the 5 'end of a DNA encoding a second eukaryotic carotenoid enzyme. The inventors have discovered that by including an IPP isomerase gene the supply of substrate for the carotenoid pathway is increased, thus reinforcing the production of the final carotenoid products, as compared to the host cell that is not transformed with this vector. It is evident when observing the deep pigmentation in E. coli colonies that accumulate carotenoid, which also contain one of the aforementioned IPP isomerase genes, when a comparison is made with colonies lacking the additional IPP isomerase gene. Similarly, a vector comprising an IPP isomerase gene can be used to enhance the production of any secondary metabolite of dimethylallyl pyrophosphate and / or isopentenyl pyrophosphate (eg, isoprenoids, steroids, carotenes, etc.). The term "isoprenoid" is intended to refer to a member of the naturally occurring class of compound whose carbon skeletons are composed, in whole or in part, of C5 isopentyl units. Preferably, the carbon skeleton is of an essential oil, a fragrance, a rubber, a carotenoid or a therapeutic compound, for example paclitaxel. A vector containing the cDNA encoding the lycopene e-cyclase of the invention, preferably the lycopene e-cyclase from lettuce or Adonis e-cyclase # 5, can be used to increase the amount of bicyclic e-carotene in an organism and in this way alter the nutritional value, pharmacology and the value of visual appearance of organisms. In addition, the transformed organism can be used in the formulation of therapeutic agents, for example in the treatment of cancer (see, Mayne et al (1996) FASEB J. 10: 690-701; Tsushima et al. (1995) Biol. Pharm. Bull, 18: 227-233). An antisense strand of a nucleic acid can be inserted into a vector. For example, the lycopene gene e-cyclase can be inserted into a vector and incorporated into the genomic DNA of a host, thus inhibiting the synthesis of e, β-carotenoids (lutein and -carotene) and reinforcing the synthesis of β, β -carotenoids (zeaxanthin and ß-carotene). The present invention also relates to novel enzymes that code for the amino acid sequences of the invention or portions thereof. This invention also relates to novel enzymes that can transform known carotenoids into novel or non-common products. Currently, e-carotene (see figure 2) and? -carotene are commonly produced only in minor amounts. As already described below, an enzyme can be produced that transforms lycopene into β-carotene and lycopene into e-carotene. With these products in the hand, the bulk synthesis of other carotenoids derived from them is possible. For example e-carotene can be hydroxylated to form lactucaxanthin, an isomer of lutein (an e-ring and a β-ring) and zeaxanthin (two β-rings), where the two end groups are e-rings. In addition, novel enzymes produced by truncating the 5 'region of the known enzymes, as discussed above, novel enzymes that can participate in the formation of unusual carotenoids can be formed by replacing portions of a gene with an analogous sequence from a structurally related gene. For example, β-cyclase and e-cyclase are structurally related (see Figure 13). By replacing a portion of the β-cyclase with the analogous portion of the e-cyclase, an enzyme producing β-carotene will be produced (an β-end group). In addition, replacing a portion of the lycopene e-cyclase with the analogous portion of the β-cyclase will produce an enzyme that produces e-carotene (with some exceptions, eg, lettuce e-cyclase, the plant e-cyclases they normally produce a compound with a terminal group e, d-carotene). Similarly, ß-hydroxylase could be modified to produce enzymes of novel function by creating hybrids with e-hydroxylase. The host systems according to this invention can comprise any organism that already produces carotenoids or that has been genetically modified to produce carotenoids. The IPP isomerase genes can be applied more widely to enhance the production of any DMAPP-dependent product and / or IPP as a precursor. Organisms that already produce carotenoids include vegetables, algae, some yeasts, fungi, and cyanobacteria and other photosynthetic bacteria. The transformation of these hosts with the vectors according to the present invention can be done using standard techniques, for example those described in Misawa et al., (1990) supra; Hundle et al., (1993) supra; Hundle et al., (1991) supra; Misawa et al., 1991 supra, Sandmann et al., Supra; and Schnurr et al., (supra). Transgenic organisms can be constructed to include nucleic acid sequences of the present invention (Bird et al, 1991, Bramley et al, 1992, Misawa et al, 1994a, Misawa et al, 1994b, Cunningham et al, 1993). The incorporation of these sequences can allow the control of carotenoid biosynthesis, content or composition in the host cell. These transgenic systems can be constructed to incorporate sequences that permit over-expression of the nucleic acids of the present invention. Transgenic systems can also be constructed with antisense expression of the nucleic acid sequences of the present invention. This antisense expression would cause the accumulation of substrates of the enzyme encoded by the homosentide chain. A method for classifying eukaryotic genes encoding enzymes involved in carotenoid biosynthesis comprises transforming a host cell with a nucleic acid that may contain a bicarbent eukaryotic or prokaryotic biosynthetic gene; culture the transformed host to obtain colonies; and classify the colonies that exhibit a different color to the colonies of the untransformed host. Suitable hosts include E. coli, cyanobacteria such as Synechococcus and Synechocystis, algae cells and plant cells. E. coli is preferred. In a preferred embodiment, the above "color complementation" classification protocol can be improved by using mutants that are either: (1) deficient in at least one carotenoid biosynthetic gene or (2) overexpressed by at least one biosynthetic gene of carotenoid In any case, these mutants will accumulate carotenoid precursors. The DNA or cDNA libraries, prokaryotic and eukaryotic, can be classified in total to determine the presence of genes for carotenoid biosynthesis, metabolism and degradation of carotenoids. Preferred organisms to be classified include photosynthetic organisms. E. coli can be transformed with these eukaryotic cDNA libraries using conventional methods, such as those described in Sambrook et al, 1989 and according to the protocols described by sellers of cloning vectors. For example, cDNA libraries in bacteriophage vectors, for example ZAP Lambda (Stratagene) or ZIPLOX Lambda (Gibco BRL) can be extracted in bulk and used to transform E. Coli. The transformed E. coli can be grown using conventional techniques. The culture broth preferably contains antibiotics to select and preserve plasmids. Suitable antibiotics include penicillin, ampicillin, chloramphenicol, etc. The cultivation is typically carried out at 15 to 40 ° C, preferably at room temperature or slightly higher (18 to 28 ° C), for 12 hours to 7 days. The cultures are seeded in plates and the plates are visually classified to differentiate colonies with different color from the host E. coli colonies transformed with the empty plasmid cloning vector. For example, him __. coli transformed with the plasmid, pAC-BETA (described below) produces yellow colonies that accumulate β-carotene. After transformation with a cDNA library, colonies containing a hue different from that formed by E. coli / pAC-BETA are expected to contain enzymes that modify the structure or accumulation of β-carotene. Similar strains of E. coli can be genetically engineered to accumulate the early products in carotenoid biosynthesis, for example lycopene,? -carotene, etc. Having generally described the invention, certain specific examples are provided for further compression for the sole purpose of illustrating the invention, and not for the purpose of limiting it in any way.

EXAMPLE I. Isolation of β-carotene hydroxylase Construction of the plasmid A fragment of 8.6 kb BglII containing the carotenoid biosynthetic genes of Erwinia herbicola was first cloned into the BamH1 site of the plasmid vector pACYC184 (resistant to chloramphenicol) and then a fragment of 1 was deleted. lkb BamHl containing ß-carotene hydroxylase from E. Herbicola (CrtZ). The E. coli strains containing the resulting plasmid, pAC-BETA, accumulate β-carotene and form yellow colonies (Cunningham et al., 1994). A full-length cDNA encoding the IPP isomerase of Haematococcus pluvialis (HP04) was first excised with BamHl and Kpnl pBluescript SK, and then ligated into the corresponding sites of the pTreHisA vector with high level expression of the tre promoter (Invitrogen, Inc. .). a fragment containing the IPP isomerase and the tre promoter was subsequently excised with EcoKV and Kpnl, treated with the Kleenow fragment of DNA polymerase to produce blunt ends and ligated into the Kleenow-treated HindIII site of pAC-BETA. The E. coli cells transformed with this new plasmid pAC-BETA-04 form orange colonies on LB plates (in comparison with the yellow colonies containing pAC-BETA) and the cultures accumulate essentially more β-carotene (approximately twice as much) as those that contain pAC-BETA.

Classification of an Arabidopsis cDNA library Several expression libraries of? Arabidopsis cDNAs were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH) (Kleber et al., 1993). The libraries of? CDNAs were extracted in vivo using the Ex Assist SOLR system from Stratagene to produce a fagomid cDNA library, where each fagomid also contains a gene that confers resistance to antibiotic ampicillin. The strain of E. coli DH10BZIP was chosen as the host cell for the classification and production of pigment, although we have also used TOP10F1 and XLl-Blue for this purpose. The DH10B cells were transformed with the plasmid pAC-BETA-04 and plated on LB agar plates containing chloramphenicol at 50 μg / ml. (from United States Biochemical Corporation). The Arabidopsis fagomid cDNA library was then introduced into the DH10B cells which already contained pAC-BETA-04.

Transformed cells containing pAC-BETA-04 and the fagomids from the Arabidopsis cDNA library were selected on agar layers with chloramphenicol plus ampicillin (150 μg / ml). The maximum color development occurred after 3 to 7 incubation at room temperature and the uncommon bright yellow colonies were selected from a background of several thousand orange colonies of each agar plate. The selected colonies were inoculated in 3 ml of the liquid LB medium containing ampicillin and chloramphenicol and the cultures were incubated at room temperature for 1 to 2 days, with shaking. The cells were then harvested by centrifugation and extracted with acetone in microcentrifuge tubes. After centrifugation, the pigmented extract was applied on silica gel in thin layer chromatography (TLC) and revealed with mobile phases: ether (1: 1 by volume). The cDNAs coding for β-carotene hydroxylase were identified based on the appearance of a yellow pigment co-migrating with zeaxanthin on the TLC plates.

Subcloning and sequencing Plasmids containing β-carotene hydroxylase cDNA were recovered and analyzed by standard procedures (Sambrook et al., 1989). The β-carotene hiroxylase from Arabidopsis was completely sequenced in both chains, in an automatic sequencer (Applied Biosystems, Model 373A, Version 2.0.1S). The 0.95kb cDNA insert was also excised and ligated into the pTrcHis vector. A BglII restriction site within the cDNA was used to remove that portion of the cDNA encoding the region of the N-terminal sequence of the predicted polypeptide, which is also found in the bacterial β-carotene hydroxylase (Figure 6). A BglII-XhoI fragment was directionally cloned into TrcHis vectors digested with BamHI-XhoI.

Pigment analysis A single colony was used to inoculate 50 ml of the LB medium containing ampicillin and chloramphenicol in a 250 ml flask. The cultures were incubated at 28 ° C for 36 hours with gentle agitation and then harvested at 5000 rpm in a SS-34 rotor. The cells were washed once with distilled H20 and resuspended with 0.5 ml of water. The extraction and HPLC methods are as described essentially in the foregoing (Cunningham et al., 1994).

II. Biochemical analysis and isolation of a lycopene e-cyclase from Arabidopsis. Construction of the plasmid The construction of plasmids pAC-LYC, pAC-NEUR, and pAC-ZETA is described in Cunningham et al., (1994). Briefly, suitable carotenoid biosynthetic genes from Erwinia herbicola, Rhodobacter capsulatus, and Synechococcus sp., Strain PCC7942 were cloned into the plasmid vector pACYC184 (New England BioLabs, Beverly, MA). The E. coli cultures containing the plasmids pAC-ZETA, pAC-NEUR and pAC-LYC, accumulate β-carotene, neurosporene and lycopene, respectively. Plasmid pAC-zeta was constructed in the following way: a 8.6 kb BglII fragment containing the carotenoid biosynthetic genes of __ .. herbicola (GenBank M87280, Hundle et al., 1991) was obtained after partial digestion of the plasmid pPL376 (Perry et al., 1986; Tuveson et al., 1986) and cloned into the BamHI-BamHI site of pACYC184 to give the plasmid pAC-EHER. Deletion of the adjacent 0.8- and 1.1-kB fragments of BamHI-BamHI (deletion Z in Cunningham et al., 1994) and a fragment of 1. 1 ikB Sall-Sall (X deletion) served to remove most of the coding regions of ß-carotene hydroxylase from __. herbicola (crtZ gene) and zeaxanthin glucocil transferase (crtX gene), respectively. The resulting plasmid, pAC-BETA, retains the functional genes for geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene of saturase (crtl) and lycopene cyclase (crtY). The E. coli cells containing this plasmid form yellow colonies and accumulate β-carotene. A plasmid containing the lycopene cDNAs e- and β-cyclase from A. thaliana was constructed by removing the e-cyclase in clone y2 as a PvuI-PvuII fragment and binding this piece to the SnaBI site of a plasmid (pSPORT 1 of GIBCO-BRL) that already contained the β-cyclase (Cunningham et al., 1996) Organisms and growth conditions Strains of E. coli TOP10 and TOP10 F * (obtained from Invitrogen Corporation, San Diego, CA) and XLl-Blue (Stratagene) were cultured in Luria-Bertani (LB) medium (Sambrook et al., 1989) at 37 ° C in the dark on a platform agitator at 225 cycles per minute. The components of the medium were obtained from Difco (extract of yeast and tryptone) or from Sigma (NaCl). Ampicillin at 150 μg / ml and / or chloramphenicol at 50 μg / ml (both from United States Biochemical Corporation) was used, as appropriate, for the selection and preservation of the plasmids.

Mass excision and complementation color complementation classification of a A. thaliana cDNA library A 1.2 kb cDNA library, fractionated by size, of A. thaliana, in ZAPII lambda (Kieber et al., 1993) was obtained from the Arabidopsis Biological Resource Center at the University of Ohio (Material number CD4-14). Other libraries fractionated by size were obtained (material numbers CD4-13, CDA-15 and CDA-16). An aliquot of each library was treated to produce a mass excision of the cDNAs and thus produce a fagomid library according to the instructions provided by the cloning vector (Stratagene), the E. coli strain XLl-Blue and the phage were used. auxiliary R408). The cleaved fagomid titer was determined and the library was introduced to the E. coli strain TOP10 F 'which accumulates lycopene (this strain contains the plasmid pAC-LYC) by incubation of the fagomid with the E. coli cells for 15 minutes at 37 ° C. The cells had already been cultured overnight at 30 ° C in LB medium supplemented with 2% maltose (w / v) and 10 mM MgSO4 (final concentration) and collected in 1.5 ml microcentrifuge tubes., in setting number 3, of an Eppendor microcentrifuge (5415C) for 10 minutes. The granules were resuspended in 10 mM MgSO 10 to a volume equal to half the initial volume of the culture. The transformants were dispersed in large petri dishes with LB agar (diameter of 150 mm) containing antibiotics to provide selection of the cDNA clones (ampicillin) and maintenance of pAC-LYC (chloramphenicol). Approximately 10,000 colony forming units were dispersed in each plate. The petri dishes were incubated at 37 ° C for 16 hours and then at room temperature for 2 to 7 days to allow maximum color development. The boxes were visually classified with the help of an illuminated 3x magnifying glass and a dissection microscope in the low power setting, for the unusual yellow-pink to bright yellow colonies, which could be seen in the background of the pink colonies. A colony color from yellow to pinkish-yellow was taken as presumptive evidence of a cyclization activity. These yellow colonies were harvested with sterile sticks and used to inoculate 3ml of the LB medium in culture tubes, growing overnight at 37 ° C and shaking at 225 cycles / minute. The cultures were divided into two aliquots in microcentrifuge tubes and collected by centrifugation at set point No. 5 of an Eppendorf 5415C microcentrifuge. After discarding the liquid, a granule was frozen for further purification of the plasmid DNA. To the second granule 1.5 ml EtOH was added and the granule was resuspended by vortex mixing and the extraction was allowed to proceed in the dark for 15 to 30 minutes with occasional remixing. The insoluble materials were subjected to high speed centrifugation for 10 minutes in a microcentrifuge, to obtain granules. The absorption spectra of the supernatant fluids were recorded between 350-550 nm with a six Perkin Elmer lambda spectrometer.

Analysis of isolated clones Eight of the yellow colonies containing β-carotene indicated that a single gene product catalyses the two cyclizations required to form the two β-end groups of symmetric β-carotene from the symmetric lycopene precursor. One of the yellow colonies contained a pigment with the characteristic spectrum of d-carotene, a monocyclic carotenoid with a single e-end group. Unlike β-cyclase, this e-cyclase is apparently unable to carry out a second cyclization at the other end of the molecule. The observation that e-cyclase is unable to form two e-cyclic end groups (e.g., bicyclic e-carotene) sheds light on the mechanism by which plants can coordinate and control the flow of substances to carotenoids derived from β-carotene compared to those derived from α-carotene and can also prevent the formation of carotenoids with two extreme groups e. The availability of gene A. Thaliana coding for e-cyclase allows targeted manipulation of plant species and algae, for modification of the carotenoid content and composition. Through the inactivation of e-cyclase, either at the gene level by deletion of the gene or by insertional inactivation or by reduction of the amount of enzyme formed (for example by antisense technology), the formation of β-carotene can be increased and other pigments derived therefrom. As vitamin A derives only from carotenoids with the ß-end groups, a reinforcement of the production of ß-carotene with respect to a-carotene can improve the nutritional value of the harvested vegetables. The reduction of carotenoids with e-end groups can also be valuable in modifying the color properties of harvested vegetables and the specific tissues of these vegetables. Alternatively, when the production of a-carotene or the pigments, for example lutein that are derived from a-carotene, is what is desired, regardless of the properties and color, nutritional value or other situations, the e-can be overexpressed. cyclase or express it in specific tissues. When an agronomic value of a crop is related to the pigmentation provided by the carotenoids, the targeted manipulation of the expression of the e-cyclase gene and / or the production of the enzyme may be of commercial value. The predicted amino acid sequences of the enzyme e-cyclase from A. Thaliana was determined. A comparison of the amino acid sequences of the ß- and e-cyclase enzymes of Arabidopsis thaliana (figure 13), as predicted by the DNA sequence of the respective cDNAs (Figure 4 for the e-cyclase cDNA sequence) indicates that these two enzymes have many regions of sequence similarity, but are only about 37% identical at a general level of the amino acids. The degree of sequence identity at the base level of DNA, only about 50%, is sufficiently low that we and others have been able to detect this gene by hybridization, using β-cyclase as a probe in the application experiments in DNA gel.

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Having fully described the invention, it is evident that anyone experienced in this field will be able to make many changes and modifications to it without departing from the spirit or scope of the inventions herein established.

Claims (8)

1. CLAIMS; An isolated and / or purified nucleic acid sequence encoding a protein having an enzyme activity of lycopene e-cyclase and having an amino acid sequence that is at least 85% identical to one of SEQ ID NOS .: 23, 25 or 26.
2. 2. The nucleic acid sequence of claim 1, wherein the protein has the amino acid sequence of one of SEQ ID NOS .: 23, 25 or 26.
3. 3. A vector comprising the sequence of nucleic acid of claim 1, wherein the nucleic acid sequence is operably linked to a promoter.
4. 4. A host cell containing the vector of claim 3.
5. The host cell of claim 4, wherein the host cell is selected from the group consisting of: a bacterial cell, an algae cell, a yeast cell and a plant cell.
6. 6. The host cell according to the claim 4, wherein the host cell is a photosynthetic cell.
7. 7. An isolated and / or purified protein having lycopene e-cyclase enzymatic activity and having an amino acid sequence that is at least 85% identical to one of SEQ ID NOS .: 23, 25 or 26.
8. 8 The protein according to claim 7, wherein the protein has the amino acid sequence of one of SEQ ID NOS .: 23, 25 or 26.