| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 09.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 26.09.00 |
| PATENT TITLE |
Fermentative carotenoid production |
| PATENT ABSTRACT |
Novel proteins of Flavobacrerium sp. R1534 and the DNA sequences which encode these proteins are disclosed which provide an improved biosynthetic pathway from farnesyl pyrophosphate and isopentyl pyrophosphate to various carotenoid precursors and carotenoids, especially .beta.-carotene, lycopene, zeaxanthin and cantaxanthin. Processes are also provided for preparing zeaxanthin by culturing a transformed host cell containing an expression cassette that includes a polynucleotide having a DNA sequence which encodes the GGPP synthase of Flavobacterium sp. R1534 (crtE), the prephytoene synthase of Flavobacterium sp. R1534 (crtB), the phytoene desaturase of Flavobacterium sp. R1534 (crtI), the lycopene cyclase of Flavobacterium sp. R1534 (crtY), or the .beta.-carotene hydroxylase of Flavobacterium sp. R1534 (crtZ). The polynucleotide is substantially free of other polynucleotides of Flavobacterium sp. R1534. The process further includes isolating the zeaxanthin from such cells or the culture medium. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 23.04.99 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Armstrong, et al., Mol. Gen. Genet., 216:254-268 (1989). Misawa, et al., J. Bacteriol., 172, No. 12: 6704-6712 (1990). Armstrong, et al., PNAS, 87:9975-9979 (1990). Carratoli, et al., J. Biol. Chem., 266, No. 9: 5854-5859 (1991). Schmidhauser, et al., J. Biol. Chem., 269, No. 16:12060-12066 (1994). Schmidhauser, et al., Mol. Cell. Biol., 10, No. 10: 5064-5070 (1990). Hoshino, et al., Appl. Environ. Microbiol., 59, No. 9: 3150-3153 (1993). Chamovitz, et al., Plant Mol. Biol., 16: 967-974 (1991). Cunningham, et al., Plant Cell, 6: 1107-1121 (1994). Martinez-Ferez, et al., Biochim. Biophys. Acta, 1218:145-152 (1994). Martinez-Ferez, et al., Plant Mol. Biol., 18: 981-983 (1992). Bartley, et al., Journal of Biological Chemistry, 268, No. 4: 25718-25721 (1993). Bartley, et al., PNAS, 88: 6532-6536 (1991). Schmidt, A., Gene, 91:113-117 (1990). Fontes, et al., EMBO Journal, 12: 1265-1275 (1993). Romer, et al., Biochem. Biophys. Res. Commun., 196: 1414-1421 (1993). Bouvier, et al., Plant Journal, 6(1): 45-54 (1994). Misawa, et al., Biochem. and Biophys. Res. Comm., vol. 39, No. 3, pp. 867-876 (1995). Derwent Abstract No. AN 94-230229. Pasamontes, et al., "Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534" Gene, 185: 35-41 (1997). Misawa, et al., "Structure and Functional Analysis of a Marine Bacterial Carotenoid Gene Cluster and Astaxanthin Biosynthetic Pathway Proposed at the Gene Level," J. Bacteriol., 177(22), 6575-6584 (1995). Derwent English language abstract of CH 595 444 A. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A process for preparing zeaxanthin comprising the steps of: a) culturing, in a suitable medium containing farnesyl pyrophosphate and isopentyl pyrophosphate under culture conditions sufficient for the expression of enzymes which catalyze the conversion of the farnesyl pyrophosphate and isopentyl pyrophosphate to zeaxanthin, a transformed host cell containing an expression cassette comprising a polynucleotide having the following DNA sequences: i) a DNA sequence which encodes the geranylgeranyl pyrophosphate (GGPP) synthase of Flavobacterium sp. R1534 (crtE), ii) a DNA sequence which encodes the prephytoene synthase of Flavobacterium sp. R1534 (crtB), iii) a DNA sequence which encodes the phytoene desaturase of Flavobacterium sp. R1534 (crtI), iv) a DNA sequence which encodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY), and v) a DNA sequence which encodes the .beta.-carotene hydroxylase of Flavobacterium sp. R1534 (crtZ), the polynucleotide being substantially free of other polynucleotides of Flavobacterium sp. R1534; and b) isolating the zeaxanthin from such cells or the culture medium. 2. The process of claim 1 wherein the expression cassette further comprises a regulatory region. 3. The process of claim 1 wherein the polynucleotide comprises: a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQ ID NO:1), b) the prephytoene synthase has the amino acid sequence of FIG. 9 (SEQ ID NO:3), c) the phytoene desaturase has the amino acid sequence of FIG. 10 (SEQ ID NO:5), d) the lycopene cyclase has the amino acid sequence of FIG. 11 (SEQ ID NO:7), and e) the .beta.-carotene hydroxylase has the amino acid sequence of FIG. 12 (SEQ ID NO:9). 4. The process of claim 3 wherein the expression cassette further comprises a regulatory region. 5. The process of claim 3 therein: a) the DNA sequence encoding the GGPP synthase comprises residues 2521-3408 of FIG. 7 (SEQ ID NO:2), b) the DNA sequence encoding the prephytoene synthase comprises residues 4316-3405 of FIG. 7 (SEQ ID NO:4), c) the DNA sequence encoding the phytoene desaturase comprises residues 4313-5797 of FIG. 7 (SEQ ID NO:6), d) the DNA sequence encoding the lycopene cyclase comprises residues 5794-6942 of FIG. 7 (SEQ ID NO:8), and e) the DNA sequence encoding the .beta.-carotene hydroxylase comprises residues 6939-7448 of FIG. 7 (SEQ ID NO:10). 6. The process of claim 5 wherein the expression cassette further comprises a regulatory region. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION Over 600 different carotenoids have been described from carotenogenic organisms found among bacteria, yeast, fungi and plants. Currently only two of them, .beta.-carotene and astaxanthin are commercially produced in microorganisms and used in the food and feed industry. .beta.-carotene is obtained from algae and astaxanthin is produced in Pfaffia strains which have been generated by classical mutation. However, fermentation in Pfaffia has the disadvantage of long fermentation cycles and recovery from algae is cumbersome. Therefore, it is desiderable to develop production systems which have better industrial applicability, e.g., can be manipulated for increased titers and/or reduced fermentation times. Two such systems using the biosynthetic genes form Erwinia herbicola and Erwinia uredovora have already been described in WO 91/13078 and EP 393 690, respectively. Furthermore, three .beta.-carotene ketolase genes (.beta.-carotene .beta.-4-oxygenase) of the marine bacteria Agrobacterium aurantiacum and Alcaligenes strain PC-1 (crtW) [Misawa, 1995, Biochem. Biophys. Res. Com. 209, 867-876] [Misawa, 1995, J. Bacteriology 177, 6575-6584] and from the green algae Haematococcus pluvialis (bkt) [Lotan, 1995, FEBS Letters 364, 125-128] [Kajiwara, 1995, Plant Mol. Biol. 29, 343-352] have been cloned. E. coli carrying either the carotenogenic genes (crtE, crtB, crtY and crtI) of E. herbicola [Hundle, 1994, MGG 245, 406-416] or of E. uredovora and complemented with the crtW gene of A. aurantiacum [Misawa, 1995] or the bkt gene of H. pluvialis [Lotan, 1995][Kajiwara, 1995] resulted in the accumulation of canthaxanthin (.beta.,.beta.-carotene-4,4'-dione), originating from the conversion of .beta.-carotene, via the intermediate echinenone (.beta.,.beta.-carotene-4-one). Introduction of the above mentioned genes (crtW or bkt) into E. coli cells harbouring besides the carotenoid biosynthesis genes mentioned above also the crtZ gene of E. uredovora [Kajiwara, 1995][Misawa, 1995], resulted in both cases in the accumulation of astaxanthin (3,3'-dihydroxy-.beta.,.beta.-carotene-4,4'-dione). The results obtained with the bkt gene are in contrast to the observation made by others [Lotan, 1995], who using the same experimental set-up, but introducing the H. pluvialis bkt gene in a zeaxanthin (.beta.,.beta.-carotene-3,3'-diol) synthesising E. coli host harbouring the carotenoid biosynthesis genes of E. herbicola, a close relative of the above mentioned E. uredovora strain, did not observe astaxanthin production. However, functionally active combinations of the carotenoid biosynthesising genes of the present invention with the known crtW genes have not been shown so far and even more importantly there is a continuing need in even more optimized fermentation systems for industrial application. SUMMARY OF THE INVENTION Novel proteins of Flavobacterium sp. R1534 and the DNA sequences which encode these proteins have been discovered which provide an improved biosynthetic pathway from farnesyl pyrophosphate and isopentyl pyrophosphate to various carotenoids, especially .beta.-carotene, lycopene, zeaxanthin and cantaxanthin. BRIEF DESCRIPTION OF THE FIGURES FIG. 1: The biosynthesis pathway for the formation or carotenoids of Flavobacterium sp. R1534 is illustrated explaining the enzymatic activities which are encoded by DNA sequences of the present invention. FIG. 2: Southern blot of genomic Flavobacterium sp. R1534 DNA digested with the restriction enzymes shown on top of each lane and hybridized with Probe 46F. The arrow indicated the isolated 2.4 kb Xhol/PstI fragment. FIG. 3 (FIGS. 3A and 3B): Southern blot of genomic Flavobacterium sp. R1534 DNA digested with ClaI or double digested with ClaI and HindIII. Blots shown in FIGS. 3A and 3B were hybridized to probe A or probe B, respectively (see examples). Both ClaI/HindIII fragments of 1.8 kb and 9.2 kb are indicated. FIG. 4: Southern blot of genomic Flavobacterium sp. R1534 DNA digested with the restriction enzymes shown on top of each lane and hybridized to probe C. The isolated 2.8 kb SalI/HindIII fragment is shown by the arrow. FIG. 5: Southern blot of genomic Flavobacterium sp. R1534 DNA digested with the restriction enzymes shown on top of each lane and hybridized to probe D. The isolated BclI/SphI fragment of approx. 3 kb is shown by the arrow. FIG. 6: Physical map of the organization of the carotenoid biosynthesis cluster in Flavobacterium sp. R1534, deduced from the genomic clones obtained. The location of the probes used for the screening are shown as bars on the respective clones. FIG. 7 (FIGS. 7A-7C1): Nucleotide sequence of the Flavobacterium sp. R1534 carotenoid biosynthesis cluster and its flanking regions. The nucleotide sequence is numbered from the first nucleotide shown (see BamHI site of FIG. 6). The deduced amino acid sequence of the ORFs orf-5, orf-1, crtE (SEQ ID NO: 1), crtB (SEQ ID NO: 3), crtI (SEQ ID NO: 5), crtY (SEQ ID NO: 7), crtZ (SEQ ID NO: 9)and orf-16) are shown with the single-letter amino acid code. Arrow (.fwdarw.) indicate the direction of the transcription; asterisks, stop codons. FIG. 8: Amino acid sequence of the GGPP synthase (crtE) of Flavobacterium sp. R1534 (SEQ ID NO: 1) with a MW of 31331 Da. FIG. 9: Amino acid sequence of the prephytoene synthetase (crtB) of Flavobacterium sp. R134 (SEQ ID NO: 3) with a MW of 32615 Da. FIG. 10: Amino acid sequence of the phytoene desaturase (crtI) of Flavobacterium sp. R1534 (SEQ ID NO: 5) with a MW of 54411 Da. FIG. 11: Amino acid sequence of the lycopene cyclase (crtY) of Flavobacterium sp. R1534 (SEQ ID NO: 7) with a MW of 42368 Da. FIG. 12: Amino acid sequence of the .beta.-carotene -hydroxylase (crtZ) of Flavobacterium sp. R1534 (SEQ ID NO: 9) with a MW of 19282 Da. FIG. 13: Recombinant plasmids containing deletions of the Flavobacterium sp. R1534 carotenoid biosynthesis gene cluster. FIG. 14: Primers used for PCR reactions (SEQ ID NO: 28: through 39). The underlined sequence is the recognition site of the indicated restriction enzyme. Small caps indicate nucleotides introduced by mutagenesis. Boxes show the artificial RBS which is recognized in B. subtilis. Small caps in bold show the location of the original adenine creating the translation start site (ATG) of the following gene (see original operon). All the ATG's of the original Flavobacter carotenoid biosynthetic genes had to be destroyed to not interfere with the rebuild transcription start site. Arrows indicate start and ends of the indicated Flavobacterium R1534 WT carotenoid genes. FIG. 15: Linkers used for the different constructions (SEQ ID NO: 40 through 47). The underlined sequence is the recognition site of the indicated restriction enzyme. Small caps indicate nucleotides introduced by synthetic primers. Boxes show the artificial RBS which is recognized in B. subtilis. Arrow indicate start and ends of the indicated Flavobacterium carotenoid genes. FIG. 16 (FIGS. 16A and 16B): Construction of plasmids pBIIKS(+)-clone59-2, pLyco and pZea4. FIG. 17 (FIGS. 17A and 17B): Construction of plasmid p602CAR. FIG. 18 (FIGS. 18A and 18B): Construction of plasmids pBIIKS(+)-CARVEG-E and p602 CARVEG-E. FIG. 19 (FIGS. 19A and 19B): Construction of plasmids pHP13-2CARZYIB-EINV and pHP13-2PN25ZYIB-EINV. FIG. 20 (FIGS. 20A-20G): Construction of plasmid pXI12-ZYIB-EINVMUTRBS2C. FIG. 21 (FIGS. 21A-21B): Norhern blot analysis of B. subtilis strain BS1012::ZYIB-EINV4. Panel A: Schematic representation of a reciprocal integration of plasmid pXI12-ZYIB-EINV4 into the levan-sucrase gene of B-subtilis. Panel B: Northern blot obtained with probe A (PCR fragment which was obtained with CAR 51 and CAR 76 and hybridizes to the 3' end of crtZ and the 5' end or crtY). Panel C: Northern blot obtained with probe B (BamHI-Xhol fragment isolated from plasmid pBIIKS(+)-crtE/2 and hybridizing to the 5' part of the crtE gene). FIG. 22: Schematic representation of the integration sites of three transformed Bacillus subtilis strains: BS1012::SFCO, BS1012::SFCOCAT1 and BA1012::SFCONEO1. Amplification of the synthetic Flavobacterium carotenoid operon (SFCO) can only be obtained in those strains having amplifiable structures. Probe A was used to determine the copy number of the integrated SFCO. Erythromycine resistance gene (ermAM), chloramphenicol resistance gene (cat), neomycine resistance gene (neo), terminator of the cryT gene of B. subtilis (cryT), levan-sucrase gene (sac-B 5' and sac-B 3'), plasmid sequences of pXI12 (pXI12), promoter originating from site I of the veg promoter complex (Pvegl). FIG. 23 (FIGS. 23A and 23B): Construction of plasmids pXI12-ZYIB-EINV4MUTRBS2CNEO and pXI12-ZYIB-EINV4MUTRBS2CCAT. FIG. 24 (FIGS. 24A-24Y): Complete nucleotide sequence of plasmid pZea4. FIG. 25: Synthetic crtW gene of Alcaligenes PC-1 (SEQ ID NO: 11). The translated protein sequence is shown above the double stranded DNA sequence. The twelve oligonucleotides (crtW1-crtW12) used for the PCR synthesis are underlined. FIG. 26:Construction of plasmid pBIIKS-crtEBIYZW. The HindIII-Pm1I fragment of pALTER-Ex2-crtW, carrying the synthetic crtW gene, was cloned into the HindIII and MluI (blunt) sites. PvegI and Ptac are the promoters used for the transcription of the two opera. The ColE1 replication origin of this plasmid is compatible with the p15A origin present in the pALTER-Ex2 constructs. FIG. 27: Relevant inserts of all plasmids constructed in Example 7. Disrupted genes are shown by //. Restriction sites: S=SacI, X=Xbal, H=HindIII, N=NsiI, Hp=HpaI, Nd=Ndel. FIG. 28: Reaction products (carotenoids) obtained from .beta.-carotene by the process of the present invention. DETAILED DESCRIPTION OF THE INVENTION Novel proteins of Flavobacterium sp. R1534 and the DNA sequences which encode these proteins have been discovered which provide an improved biosynthetic pathway from farnesyl pyrophosphate and isopentyl pyrophosphate to various carotenoid precursors and carotenoids, especially .beta.-carotene, lycopene, zeaxanthin and cantaxanthin. One aspect of the invention is the geranylgeranyl pyrophosphate (GGPP) synthase of Flavobacterium sp. R1534 and a polynucleotide comprising a DNA sequence which encodes said GGPP synthase (crtE), said synthase and polynucleotide being substantially free of other proteins and polynucleotides, respectively, of Flavobacterium sp. R1534. Also encompassed by this aspect of the present invention is a polynucleotide comprising a DNA sequence which is substantially homologous to said DNA sequence. Said GGPP synthase catalyzes the condensation of farnesyl pyrophosphate and isopentyl pyrophosphate to obtain geranylgeranyl pyrophosphate, a carotenoid precursor. The preferred GGPP synthase has the amino acid sequence of FIG. 8 (SEQ ID NO: 1), and the preferred DNA sequence encodes said amino acid sequence. The especially preferred DNA sequence is bases 2521-3408 shown in FIG. 7 (SEQ ID NO: 2). This aspect of the present invention also includes a vector comprising the aforesaid polynucleotide, preferably in the form of an expression vector. Furthermore this aspect of the present invention also includes a recombinant cell comprising a host cell which is transformed by the aforesaid polynucleotide or vector which contains such a polynucleotide. Preferably said host cell is a prokaryotic cell and more preferably said host cell is E. coli or a Bacillus strain. However, said host cell may also be a eukaryotic cell, preferably a yeast cell or a fungal cell. Finally this aspect of the present invention also comprises a process for the preparation of geranylgeranyl pyrophosphate by culturing said recombinant cell of the invention in the presence of farnesyl pyrophosphate and isopentyl pyrophosphate in a culture medium under suitable culture conditions whereby said GGPP synthase is expressed by said cell and catalyzes the condensation of farnesyl pyrophosphate and isopentyl pyrophosphate to geranylgeranyl pyrophosphate, and isolating the geranylgeranyl pyrophosphate from such cells or the culture medium. A further aspect of the present invention is the prephytoene synthase of Flavobacterium sp. R1534 and a polynucleotide comprising a DNA sequence which encodes said prephytoene synthase of Flavobacterium sp. R1534 (crtB), said synthase and polynucleotide being substantially free of other proteins and polynucleotides, respectively, of Flavobacterium sp. R1534. Also encompassed by this aspect of the present invention is a polynucleotide comprising a DNA sequence which is substantially homologous to said DNA sequence. Said prephytoene synthase catalyzes the condensation of two geranylgeranyl pyrophosphates to the carotenoid, prephytoene, and then catalyzes the rearrangement of the cyclopropyl ring of prephytoene to produce phytoene. The preferred prephytoene synthase has the amino acid sequence of FIG. 9 (SEQ ID NO: 3), and the preferred DNA sequence is one which encodes said amino acid sequence. The especially preferred DNA sequence is bases 4316-3405 shown in FIG. 7 (SEQ ID NO: 4). This aspect of the present invention also includes a vector comprising the aforesaid polynucleotide, preferably in the form of an expression vector. Furthermore this aspect of the present invention also includes a recombinant cell comprising a host cell which is transformed by the aforesaid polynucleotide or vector which contains such a polynucleotide. Preferably said host cell is a prokaryotic cell and more preferably said host cell is E. coli or a Bacillus strain. However, said host cell may also be a eukaryotic cell, preferably a yeast cell or a fungal cell. Finally this aspect of the present invention also comprises a process for the preparation of phytoene by culturing said recombinant cell of the invention in the presence of geranylgeranyl pyrophosphate in a culture medium under suitable culture conditions whereby said prephytoene synthase is expressed by said cell and catalyzes the condensation of two geranylgeranyl pyrophosphates to the carotenoid, prephytoene, and then catalyzes the rearrangement of the cyclopropyl ring of said prephytoene to produce phytoene, and isolating the phytoene from such cells or the culture medium. A further aspect of the present invention is the phytoene desaturase of Flavobacterium sp. R1534 and a polynucleotide comprising a DNA sequence which encodes said phytoene desaturase of Flavobacterium sp. R1534 (crtI), said desaturase and polynucleotide being substantially free of other proteins and polynucleotides, respectively, of Flavobacterium sp. R1534. Also encompassed by this aspect of the present invention is a polynucleotide comprising a DNA sequence which is substantially homologous to said DNA sequence. Said phytoene desaturase catalyzes the desatuation of phytoene in four steps to obtain lycopene. The preferred prephytoene desaturase has the amino acid sequence of FIG. 10 (SEQ ID NO: 5), and the preferred DNA sequence is one which encodes said amino acid sequence. The especially preferred DNA sequence is bases 4313-5797 shown in FIG. 7 (SEQ ID NO: 6). This aspect of the present invention also includes a vector comprising the aforesaid polynucleotide, preferably in the form of an expression vector. Furthermore this aspect of the present invention also includes a recombinant cell comprising a host cell which is transformed by the aforesaid polynucleotide or vector which contains such a polynucleotide. Preferably said host cell is a prokaryotic cell and more preferably said host cell is E. coli or a Bacillus strain. However, said host cell may also be a eukaryotic cell, preferably a yeast cell or a fungal cell. Finally this aspect of the present invention also comprises a process for the preparation of lycopene by culturing said cell of the invention in the presence of phytoene in a culture medium under suitable culture conditions whereby said phytoene desaturase is expressed by said cell and catalyzes the desatuation of phytoene in four steps to obtain lycopene, and isolating the lycopene from such cells or the culture medium. A still further aspect of the present invention is the lycopene cyclase of Flavobacterium sp. R1534 and a polynucleotide comprising a DNA sequence which encodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY), said cyclase and polynucleotide being substantially free of other proteins and polynucleotides, respectively, of Flavobacterium sp. R1534. Also encompassed by this aspect of the present invention is a polynucleotide comprising a DNA sequence which is substantially homologous to said DNA sequence. Said lycopene cyclase catalyzes the closure of rings at both ends of lycopene to produce .beta.-carotene. The preferred lycopene cyclase has the amino acid sequence of FIG. 11 (SEQ ID NO: 7), and the preferred DNA sequence is one which encodes said amino acid sequence. The especially preferred DNA sequence is bases 5794-6942 shown in FIG. 7 (SEQ ID NO: 8). This aspect of the present invention also includes a vector comprising the aforesaid polynucleotide, preferably in the form of an expression vector. Furthermore this aspect of the present invention also includes a recombinant cell comprising a host cell which is transformed by the aforesaid polynucleotide or vector which contains such a polynucleotide. Preferably said host cell is a prokaryotic cell and more preferably said host cell is E. coli or a Bacillus strain. However, said host cell may also be a eukaryotic cell, preferably a yeast cell or a fungal cell. Finally this aspect of the present invention also comprises a process for the preparation of .beta.-carotene by culturing said recombinant cell of the invention in the presence of lycopene in a culture medium under suitable culture conditions whereby said lycopene cyclase is expressed by said cell and catalyzes the closure of rings at both ends of lycopene to produce .beta.-carotene, and isolating the .beta.-carotene from such cells or the culture medium. A still further aspect of the present invention is the .beta.-carotene hydroxylase of Flavobacterium sp. R1534 and a polynucleotide comprising a DNA sequence which encodes said .beta.-carotene hydroxylase of Flavobacterium sp. R1534 (crtZ), said hydroxylase and polynucleotide being substantially free of other proteins and polynucleotides, respectively, of Flavobacterium sp. R1534. Also encompassed by this aspect of the present invention is a polynucleotide comprising a DNA sequence which is substantially homologous to said DNA sequence. Said .beta.-carotene hydroxylase catalyzes the hydroxylation of .beta.-carotene to produce the xanthophyll, zeaxanthin. The preferred .beta.-carotene hydroxylase has the amino acid sequence of FIG. 12 (SEQ ID NO: 9), and the preferred DNA sequence is one which encodes said amino acid sequence. The especially preferred DNA sequence is a DNA sequence comprising bases 6939-7448 shown in FIG. 7 (SEQ ID NO: 10). This aspect of the present invention also includes a vector comprising the aforesaid polynucleotide, preferably in the form of a n expression vector. Furthermore this aspect of the present invention also includes a recombinant cell comprising a host cell which is transformed by the aforesaid polynucleotide or vector which contains such a DNA sequence. Preferably said host cell is a prokaryotic cell and more preferably said host cell is E. coli or a Bacillus strain. However, said host cell may also be a eukaryotic cell, preferably a yeast cell or a fungal cell. Finally this aspect of the present invention also comprises a process for the preparation of zeaxanthin by culturing said recombinant cell of the invention in the presence of .beta.-carotene in a culture medium under suitable culture conditions whereby said .beta.-carotene hydroxylase is expressed by said cell and catalyzes the hydroxylation of .beta.-carotene to produce the xanthophyll, zeaxanthin, and isolating the zeaxanthin from such cells or the culture medium. It is contemplated, and in fact preferred, that the aforementioned DNA sequences, crtE, crtB, crtI crtY and crtZ, which terms refer to the above-described genes of Flavobacterium sp. R1534 encompassed by the invention herein described, are incorporated into a polynucleotide of the invention whereby two or more of said DNA sequences which encode enzymes catalyzing contiguious steps in the process shown in FIG. 1 are contained in said polynucleotide, said polynucleotide being substantially free of other polynucleotides of Flavobacterium sp. R1534. Examples of preferred polynucleotides which encode enzymes which catalyze such contiguous steps are polynucleotides which comprise crtE and crtB (conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to phytoene), crtE, crtB, and crtI (conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to lycopene), crtB and crtI (conversion of geranylgeranyl pyrophosphate to lycopene) and the like. The present invention also comprises a vector comprising a polynucleotide of the invention which contains said DNA sequences, preferably in the form of an expression vector. Furthermore the present invention also comprises a recombinant cell comprising a host cell which is transformed by a polynucleotide of the invention or vector which contains such a polynucleotide. Preferably said host cell is a prokaryotic cell and more preferably said host cell is E. coli or a Bacillus strain. However, said host cell may also be a eukaryotic cell, preferably a yeast cell or a fungal cell. Finally the present invention also comprises a process for the preparation of a desired carotenoid by culturing a recombinant cell of the invention in the presence of a starting material in a culture medium under suitable culture conditions and isolating the desired carotenoid from such cells or the culture medium wherein the cell utilizes the polynucleotide of the invention which contains said DNA sequences to express the enzymes which catalyze the reactions necessary to produce the desired carotenoid from the starting material. Where an enzyme catalyzes two sequential steps and it is preferred to produce the product of the second step, a higher copy number of the DNA sequence encoding the enzyme may be used to further production of the product of the second of the two steps in comparison to the first product. The present invention further comprises a process for the preparation of a food or feed composition which process comprises mixing a nutritionally effective amount of the carotenoid isolated from the aforementioned recombinant cells or culture medium with said food or feed. One preferred embodiment of the present invention is a polynucleotide which comprises the following DNA sequences: crtE or a DNA sequence which is substantially homologous thereto, crtB or a DNA sequence which is substantially homologous thereto, and crtI or a sequence which is substantially homologous thereto, said polynucleotide being substantially free of other polynucleotides of Flavobacterium sp. R1534. This polynucleotide encodes enzymes which catalyze the conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to lycopene. It is particularly preferred that this embodiment of the invention is a polynucleotide which contains crtE, crtB, and crtI. It is especially preferred that this polynucleotide of the invention comprises DNA sequences which encode the amino acid sequences of FIGS. 8, 9 and 10, and it is most preferred that this polynucleotide of the invention contain as the three DNA sequences crtE, crtB and crtI, bases 2521-3408, 4316-3405 and 4313-5797, respectively, shown in FIG. 7. This embodiment of the present invention also comprises a vector, recombinant cell, process of making a carotenoid and process of making a food or feed stuff containing a carotenoid, as described hereinbefore, wherein the polynucleotide is a polynucleotide of this embodiment and the carotenoid is lycopene. Another preferred embodiment of the present invention is a polynucleotide comprising the following DNA sequences: crtE or a DNA sequence which is substantially homologous thereto, crtB or a DNA sequence which is substantially homologous thereto, crtI or a DNA sequence which is substantially homologous thereto, and crtY or a DNA sequence which is substantially homologous thereto, said polynucleotide being substantially free of other polynucleotides of Flavobacterium sp. R1534. This polynucleotide encodes enzymes which catalyze the conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to .beta.-carotene. It is especially preferred that this embodiment of the invention is a polynucleotide which contains the following DNA sequences: crtE, crtB, crtI, and crtY. It is especially preferred that this polynucleotide of the invention comprises DNA sequences which encode the amino acid sequences of FIGS. 8, 9, 10 and 11, and it is most preferred that this polynucleotide of the invention contain as the four DNA sequences crtE, crtB, crtI and crtY, bases 2521-3408, 4316-3405, 4313-5797 and 5794-6942, respectively, shown in FIG. 7. This embodiment of the present invention also comprises a vector, recombinant cell, process of making a carotenoid and process of making a food or feed stuff containing the carotenoid, as described hereinbefore, wherein the polynucleotide is a polynucleotide of this embodiment and the carotenoid is .beta.-carotene. Further, the polynucleotide of the present embodiment which contains crtE, crtB, crtI and crtY, or corresponding DNA sequences which are substantially homologous, may additionally contain a DNA sequence which encodes the .beta.-carotene .beta.4-oxygenase of Alcaligenes strain PC-1 (crt W) [Misawa, supra] or a DNA sequence which is substantially homologous to said .beta.-carotene .beta.4-oxygenase. Because the crtW subsequence encodes an enzyme which catalyzes the conversion of .beta.-carotene to echinenone, this DNA sequence encodes enzymes which catalyze the conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to echinenone, and with the further catalysis of echinenone by the enzyme encoded by crtW, to cantaxanthin. Such a polynucleotide preferably contains crtE, crtB, crtI, crtY and crt W. It is most preferred that this polynucleotide of the invention contain as the four subsequences crtE, crtB, crtI and crtY, bases 2521-3408, 4316-3405, 4313-5797 and 5794-6942, respectively, shown in FIG. 7. This embodiment of the present invention in which the polynucleotide contains crtE, crtB, crtI, crtY and crt W, or DNA sequences which are substantially homologous thereto, also comprises a vector, recombinant cell, process of making a carotenoid and process of making a food or feed stuff containing the carotenoid, as described hereinbefore, wherein the polynucleotide is the polynucleotide of this embodiment and the carotenoid is echinenone and also cantaxanthin. It is also contemplated that, instead of being transformed by one expression vector comprising crtE, crtB, crtI, crtY and crt W, a recombinant cell of the invention which expresses the aforesaid enzymes which catalyze the conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to echinenone, and with the further catalysis of echinenone by the enzyme encoded by crtW, to cantaxanthin, may be obtained by transforming a host cell with two expression vectors, one of which comprises crtE, crtB, crtI, crtY, but not crtW, and the second of which comprises crtW. The preferred expression vector which contains a polynucleotide comprising crtE, crtB, crtI and crtY is as described above. Another preferred embodiment of the present invention is a polynucleotide comprising the following DNA sequences: crtE or a DNA sequence which is substantially homologous thereto, crtB or a DNA sequence which is substantially homologous thereto, crtI or a DNA sequence which is substantially homologous thereto, crtY or a DNA sequence which is substantially homologous thereto, and crtZ or a DNA sequence which is substantially homologous thereto, said polynucleotide being substantially free of other polynucleotides of Flavobacterium sp. R1534. This polynucleotide encodes enzymes which catalyze the conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to cryptoxanthin, and with further catalysis of cryptoxanthin by the enzyme encoded by crtZ, to zeaxanthin. Such a polynucleotide preferably contains crtE, crtB, crtI, crtY and crt Z. It is most preferred that this polynucleotide of the invention contain as the five subsequences crtE, crtB, crtI, crtY and crtZ, bases 2521-3408, 4316-3405, 4313-5797, 5794-6942 and 6939-7448, respectively, shown in FIG. 7. This embodiment of the present invention in which the polynucleotide contains crtE, crtB, crtI, crtY and crt Z, or DNA sequences which are substantially homologous thereto, also comprises a vector, recombinant cell, process of making a carotenoid and process of making a food or feed stuff containing the carotenoid, as described hereinbefore, wherein the polynucleotide is the polynucleotide of this embodiment and the carotenoid is cryptoxanthin and also zeaxanthin. Further, the polynucleotide of the present embodiment which contains crtE, crtB, crtI, crtY and crtZ, or corresponding DNA sequences which are substantially homologous, may additionally contain a DNA sequence which encodes the .beta.-carotene .beta.4-oxygenase of Alcaligenes strain PC-1 (crt W) [Misawa, supra] or a DNA sequence which is substantially homologous to said .beta.-carotene .beta.4-oxygenase. Because the crtW DNA sequence encodes a enzyme which catalyzes the conversion of zeaxanthin to adonixanthin, this polynucleotide encodes enzymes which catalyze the conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to adonixanthin, and with the further catalysis of adonixanthin by the enzyme encoded by crtW, to astaxanthin. Such a polynucleotide preferably contains crtE, crtB, crtI, crtY, crtZ and crt W. It is most preferred that this polynucleotide of the invention contain as the five subsequences crtE, crtB, crtI, crtY and crtZ, bases 2521-3408, 4316-3405, 4313-5797, 5794-6942 and 6939-7448, respectively, shown in FIG. 7. This embodiment of the present invention in which the polynucleotide contains crtE, crtB, crtI, crtY, crtZ and crt W, or DNA sequences which are substantially homologous thereto, also comprises a vector, recombinant cell, process of making a carotenoid-and process of making a food or feed stuff containing the carotenoid, as described hereinbefore, wherein the polynucleotide is the polynucleotide of this embodiment and the carotenoid is adonixanthin and also astaxanthin. It is also contemplated that, instead of being transformed by one expression vector comprising crtE, crtB, crtI, crtY, crtZ and crt W, a recombinant cell of the invention which expresses the aforesaid enzymes which catalyze the conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to adonixanthin, and with the further catalysis of adonixanthin by the enzyme encoded by crtW, to astaxanthin, may be obtained by transforming a host cell with two expression vectors, one of which comprises crtE, crtB, crtI, crtY, and crtZ, but not crtW, and the second of which comprises crtW. The preferred expression vector which contains a polynucleotide comprising crtE, crtB, crtI, crtY, and crtZ is as described above. The expression "a DNA sequence which is substantially homologous" refers with respect to the crtE encoding DNA sequence to a DNA sequence which encodes an amino acid sequence which shows more than 45%, preferably more than 60% and more preferably more than 75% and most preferably more than 90% identical amino acids when compared to the amino acid sequence of crtE of Flavobacterium sp. 1534 and is the amino acid sequence of a polypeptide which shows the same type of enzymatic activity as the enzyme encoded by crtE of Flavobacterium sp. 1534. In analogy with respect to crtB this means more than 60%, preferably more than 70%, more preferably more than 80% and most preferably more than 90%; with respect to crtI this means more than 70%, preferably more than 80% and most preferably more than 90%; with respect to crtY this means 55%, preferably 70%, more preferably 80% and most preferably 90%; with respect to crtZ this means more than 60%, preferably 70%, more preferably 80% and most preferably 90%; with respect to crt W this also means more than 60%, preferably 70%, more preferably 80% and most preferably 90%. Sequences which are substantially homologous to crtW are known, e.g., in form of the 0-carotene P4-oxygenase of Agrobacterium aurantiacum or the green algae Haematococous pluvialis (bkt). The expression "said polynucleotide being substantially free of other polynucleotides of Flavobacterium sp. R1534" is meant to preclude the present invention from encompassing the polynucleotides as they exist in Flavobacterium sp. R1534, itself. The polynucleotides herein described which are combinations of two or more DNA sequences of Flavobacterium sp. R1534 are also "substantially free of other polynucleotides of Flavobacterium sp. R1534" in any circumstance where a polynucleotide containing only a single such DNA sequence would be "substantially free of other polynucleotides of Flavobacterium sp. R1534." DNA sequences in the form of genomic DNA, cDNA or synthetic DNA can be prepared as known in the art [see, e.g., Sambrook et al., Molecular Cloning, Cold Spring Habor Laboratory Press 1989] or, e.g., as specifically described in Examples 1, 2 or 7. The cloning of the DNA sequences of the present invention from such genomic DNA can than be effected, e.g., by using the well known polymerase chain reaction (PCR) method. The principles of this method are outlined, e.g., in PCR Protocols: A guide to Methods and Applications, Academic Press, Inc. (1990). PCR is an in vitro method for producing large amounts of a specific DNA of defined length and sequence from a mixture of different DNA sequences. Thereby, PCR is based on the enzymatic amplification of the specific DNA fragment of interest which is flanked by two oligonucleotide primers which are specific for this sequence and which hybridize to the opposite strand of the target sequence. The primers are oriented with their 3' ends pointing toward each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences and extension of the annealed primers with a DNA polymerase result in the amplification of the segment between the PCR primers. Since the extension product of each primer can serve as a template for the other, each cycle essentially doubles the amount of the DNA fragment produced in the previous cycle. By utilizing the thermostable Taq DNA polymerase, isolated from the thermophilic bacteria Thermus aquaticus, it has been possible to avoid denaturation of the polymerase which had necessitated the addition of enzyme after each heat denaturation step. This development has led to the automation of PCR by a variety of simple temperature-cycling devices. In addition, the specificity of the amplification reaction is increased by allowing the use of higher temperatures for primer annealing and extension. The increased specificity improves the overall yield of amplified products by minimizing the competition by non-target fragments for enzyme and primers. In this way the specific sequence of interest is highly amplified and can be easily separated from the non-specific sequences by methods known in the art, e.g., by separation on an agarose gel, and cloned by methods known in the art using vectors as described, e.g., by Holten and Graham in Nucleic Acid Res. 19, 1156 (1991), Kovalic et. al. in Nucleic Acid Res. 19, 4560 (1991), Marchuk et al. in Nucleic Acid Res. 19, 1154 (1991) or Mead et al. in BiolTechnology 9, 657-663 (1991). The oligonucleotide primers used in the PCR procedure can be prepared as known in the art and described, e.g., in Sambrook et al., supra. Amplified DNA sequences can then be used to screen DNA libraries by methods known in the art (Sambrook et al., supra) or as specifically described in Examples 1 and 2. Once complete DNA sequences of the present invention have been obtained, they can be used as a guideline to define new PCR primers for the cloning of substantially homologous DNA sequences from other sources. In addition, the DNA sequences of the invention and such homologous DNA sequences can be integrated into expression vectors by methods known in the art and described, e.g., in Sambrook et al., supra to express or overexpress the encoded polypeptide(s) in appropriate host systems. The expression vector into which the polynucleotides of the invention are integrated is not critical. Conventional expression vectors may be selected based upon the size of the polynucleotide of the invention to be inserted into the vector and the host cell to be transformed by the vector. Such conventional expression vectors contain a regulatory sequence for the synthesis of mRNA derived from the polynucleotide of the invention being expressed and possible marker genes. Conventional regulatory sequences generally contain, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Suitable vectors which can be used for expression in E. coli are known, e.g., the vectors described by Sambrook et al., supra or by Fiers et al. in "Procd. 8th Int. Biotechnology Symposium" [Soc. Franc. de Microbiol., Paris (Durand et al., eds.), pp. 680-697 (1988)] or by Bujard et al. in Methods in Enzymology, eds. Wu and Grossmann, Academic Press, Inc. Vol. 155, 416-433 (1987) and Stuber et al. in Immunological Methods, eds. Lefkovits and Pernis, Academic Press, Inc., Vol. IV, 121-152 (1990). Vectors which could be used for expression in Bacilli are known in the art and described, e.g., in EP 405 370, EP 635 572, Proc. Nat. Acad. Sci. USA 81, 439 (1984) by Yansura and Henner, Meth. Enzym. 185, 199-228 (1990) or EP 207 459. Vectors which can be used for expression in fungi are known in the art and described, e.g., in EP 420 358. Vectors which can be used for expression in yeast are known in the art and are described, e.g., in EP 183 070, EP 183 071, EP 248 227 and EP 263 311. The polynucleotides of the invention themselves, or expression vectors containing them, can be used to transform suitable host cells to get overexpression of the encoded enzyme or enzymes. The transformation of host cells to obtain a cell of the invention may be performed by any conventional means. Appropriate host cells are for example bacteria, e.g., E. coli, Bacilli as, e.g., Bacillus subtilis or Flavobacter strains. E. coli which could be used are E. coli K12 strains, e.g., M15 [described as DZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 (1974)], HB 101 [ATCC No. 33694] or E. coli SG13009 [Gottesman et al., J. Bacteriol. 148, 265-273 (1981)]. Suitable eukaryotic host cells are, for example, fungi, like Aspergilli, e.g., Aspergillus niger [ATCC 9142] or yeasts, like Saccharomyces, e.g., Saccharomyces cerevisiae or Pichia, like pastoris, all available from ATCC. Once the polynucleotides of the invention have been expressed in an appropriate host cell in a suitable medium, thereby causing the catalysis of the starting materials to the desired carotenoids, the carotenoids can be isolated either from the medium, in the case they are secreted into the medium, or from the host organism and, if necessary separated from other carotenoids that may be present in case one specific carotenoid is desired, by methods known in the art (see, e.g., Carotenoids Vol IA: Isolation and Analysis, G. Britton, S. Liaaen-Jensen, H. Pfander; 1995, Birkhauser Verlag, Basel). The carotenoids produced in accordance with the present invention can be used in a process for the preparation of food or feeds. A man skilled in the art is familiar with such processes. Such compound foods or feeds can further comprise additives or components generally used for such purpose and known in the state of the art. After the invention has been described in general hereinbefore, the following examples are intended to illustrate details of the invention, without thereby limiting it in any matter. |
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