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(12) United States Patent (10) Patent No.: US 7,252,985 B2 Cheng Et Al

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(12) United States Patent (10) Patent No.: US 7,252,985 B2 Cheng Et Al US007252985B2 (12) United States Patent (10) Patent No.: US 7,252,985 B2 Cheng et al. (45) Date of Patent: Aug. 7, 2007 (54) CAROTENOID KETOLASES Echinenone in the Cyanobacterium Synechocystis sp. PCC 6803*, J. Biol. Chem., vol. 272(15):9728-9733, 1997. (75) Inventors: Qiong Cheng, Wilmington, DE (US); Norihiko Misawa et al., Metabolic engineering for the production of Luan Tao, Claymont, DE (US); Henry carotenoids in non-carotenogenic bacteria and yeasts, J. of Biotech., Yao, Boothwyn, PA (US) vol. 59:169-181, 1998. National Center for Biotechnology Information General Identifier (73) Assignee: E. I. du Pont de Nemours and No. 5912291, Accession No. Y 15112, Sep. 15, 1999, M. Harker et Company, Wilmington, DE (US) al., Carotenoid biosynthesis genes in the bacterium Paracoccus narcusii MH1. National Center for Biotechnology Information General Identifier (*) Notice: Subject to any disclaimer, the term of this No. 2654317. Accession No. X86782, Sep. 9, 2004, M. Harker et patent is extended or adjusted under 35 al., Biosynthesis of ketocarotenoids in transgenic cyanobacteria U.S.C. 154(b) by 326 days. expressing the algal gene for beta-C-4-oxygenase, crtO. National Center for Biotechnology Information General Identifier (21) Appl. No.: 11/015,433 No. 903298, Accession No. D58422, N. Misawa et al., Canthaxanthin biosynthesis by the conversion of methylene to keto (22) Filed: Dec. 17, 2004 groups in a hydrocarbon beta-carotene by a single gene Biochem. Biophys. Res. Commun. 209(3), 867-876 (1995). (65) Prior Publication Data National Center for Biotechnology Information General Identifier No. 61629280, Accession No. D58420, N. Misawa et al., US 2005/0227311 A1 Oct. 13, 2005 Canthaxanthin biosynthesis by the conversion of methylene to keto groups in a hydrocarbon beta-carotene by a single gene Biochem. Related U.S. Application Data Biophys. Res. Commun. 209(3), 867-876 (1995). (60) Provisional application No. 60/531,310, filed on Dec. National Center for Biotechnology Information General Identifier 19, 2003. No. 1136838, Accession No. D45881, Feb. 10, 1999, S. Kajiwara et al., Isolation and functional identification of a novel cDNA for (51) Int. Cl. astaxanthin biosynthesis from Haematococcus pluvialis, and CI2N L/20 (2006.01) astaxanthin synthesis in Escherichia coli. CI2N 9/02 (2006.01) National Center for Biotechnology Information General Identifier No. 8650414, Accession No. AF2 18415, L. Hannibal et al., Isolation C07H 2L/04 (2006.01) and characterization of canthaxanthin biosynthesis genes from the (52) U.S. Cl. ................ 435/252.3; 435/189: 435/320.1; photosynthetic bacterium Bradyrhizobium sp. strain ORS278 J. 435/148; 435/410; 435/254.3:435/255.2: Bacteriol. 182(13), 3850-3853 (2000). 435/255.5; 536/23.2 H. J. Nelis et al., Principal carotenoids permitted as additives in (58) Field of Classification Search ............. 435/252.3, foods and feeds, J. of Applied Bacteriology, vol.70:181-191, 1991. 435/189,148,410, 254.3, 255.2, 255.5; 536/23.2 Laure Hannibal et al., Isolation and Characterization of See application file for complete search history. Canthaxanthin Biosynthesis Genes from the Photosynthetic Bacte rium Bradyrhizobium sp. Strain ORS278, J. of Bacteriology, vol. (56) References Cited 182(13):3850-3853, Jul. 2000. U.S. PATENT DOCUMENTS Primary Examiner Tekchand Saidha 6,150,130 A 11/2000 Misawa et al. 6,551,807 B1 4/2003 Cunningham (57) ABSTRACT 2003/0O87337 A1 5, 2003 Giraud et al. Novel CrtW carotenoid ketolase are provided that are useful FOREIGN PATENT DOCUMENTS for the production of ketocarotenoids. The ketolases genes WO WO O2/O79395 A2 10, 2002 of the present invention exhibit low homology in compari son to other CrtW ketolases previously reported. Expression OTHER PUBLICATIONS of the carotenoid ketolases in heterologous hosts enabled production of canthaxanthin and astaxanthin. Coexpression Takakazu Kaneko et al., Complete Genomic Sequence of the experiments using divergent crtW genes resulted in Filamentous Nitrogen-fixing Cyanobacterium Anabaena sp. Strain PCC 7120, DNA Research, vol. 8:205-213, 2001. increased production of the desired ketocarotenoids. Blanca Fernandez-Gonzalez et al., A New Type of Asymmetrically Acting beta-Carotene Ketolase Is Required for the Synthesis of 10 Claims, 7 Drawing Sheets U.S. Patent US 7,252,985 B2 IQun??Ht—ausdoosK U.S. Patent Aug. 7, 2007 Sheet 2 of 7 US 7,252,985 B2 E so e AZS ---so st t s 3 3 3 GEO st 2 re s 5 3 S 3 U.S. Patent Aug. 7 9 2007 Sheet 3 of 7 US 7,252,985 B2 8| qZ9.InãIH 627 -s 09 U.S. Patent Aug. 7, 2007 Sheet S of 7 US 7,252,985 B2 Figure 3 Dao D, Sigelos Reiss50.5 (112403217-0701.D. 2000 - DC18 CritW 1soo! co so fait d, sig-arts ref=6505 i2a32G2ai04.D) DC263 CritW 2.5 s 5 12.5 s 7.5 Ap1d, sig=4705 Ref=650,5112403205-soid) So K1-202C CtW s O 15 s S. U.S. Patent Aug. 7, 2007 Sheet 6 of 7 US 7,252,985 B2 Figure 4 dio, Sig=4705 Ref=850,5(11 1703LT001-0101 o A. : 500 - 10G(pDCQ335) 10G(pDCQ335) -- DC263 CritW 10G(pDCQ335) +K1-202C crtW U.S. Patent Aug. 7, 2007 Sheet 7 of 7 US 7,252,985 B2 Figure 5 MWM1200(pDCQ340) Alu A, Sigel-ou,s resolub Lutuq Nstuz-uzu.Lu MWM1200(pDCQ341) DAD B, Sig=47 U.5 Ref=8505 (030204 SSG TUC04.D) --- mAU 700 600 500 400 300 200 100 US 7,252,985 B2 1. 2 CAROTENOID KETOLASES the CrtOketolase enzyme from Synechocystis sp. PCC6803 adds a keto-group asymmetrically to only one of the two This application claims the benefit of U.S. Provisional B-ionone rings of B-carotene. Application No. 60/531,310, filed Dec. 19, 2003. Several examples of CrtW ketolases have been reported in variety of bacteria including Agrobacterium aurantiacum FIELD OF THE INVENTION (U.S. Pat. No. 6,150,130), Bradyrhizobium sp. (U.S. Patent Publication No. 20030087337), and Brevundimonas auran This invention is in the field of microbiology and molecu tiacum (WO 02/079395). However, there is a need to lar biology. More specifically, this invention pertains to identify additional novel CrtW ketolase genes useful for nucleic acid fragments encoding enzymes useful for micro 10 genetically engineering industrially suitable microorganisms bial production of cyclic ketocarotenoid compounds. for the production of valuable ketocarotenoids, such as canthaxanthin and astaxanthin. Additionally, there is a par BACKGROUND OF THE INVENTION ticularly important need to identify CrtW type ketolases having relatively low to moderate sequence homology (i.e. Carotenoids are pigments that are ubiquitous throughout 15 <65% nucleotide sequence identity) as coexpression of nature and synthesized by all photosynthetic organisms, and highly homologous genes tends to result genetic instability in some heterotrophic growing bacteria and fungi. Caro (i.e. undesirable homologous recombination). Expressing tenoids provide color for flowers, vegetables, insects, fish crtW genes having relatively low to moderate sequence and birds. Colors of carotenoid range from yellow to red homology should decrease the probability of genetic insta with variations of brown and purple. As precursors of bility normally associated with expression of highly vitamin A, carotenoids are fundamental components in our homologous genes. This is particularly important when diet and they play additional important role in human health. developing genetically-stable commercial strains for opti Because animals are unable to synthesize carotenoid de mal production of the desired product (i.e. ketocarotenoids). novo, they must obtain them by dietary means. Thus, CrtW genes having divergent nucleotide sequences are manipulation of carotenoid production and composition in 25 most suitable for expressing multiple ketolases in a single plants or bacteria can provide new or improved source for recombinant host cell. This is especially important when carotenoids. Industrial uses of carotenoids include pharma ketolase activity becomes the rate-limiting step in the keto ceuticals, food supplements, animal feed additives, and carotenoid biosynthesis pathway. Increasing the number of colorants in cosmetics, to mention a few. crtW genes that can be simultaneously expressed in the Industrially, only a few carotenoids are used for food 30 production host is expected to increase ketocarotenoid pro colors, animal feeds, pharmaceuticals, and cosmetics, duction, assuming that the pool of available substrates is not despite the existence of more than 600 different carotenoids limiting. identified in nature. This is largely due to difficulties in Additionally, CrtW ketolases tend to exhibit substrate production. Presently, most of the carotenoids used for flexibility. However, it can be envisioned that different CrtW industrial purposes are produced by chemical synthesis: 35 ketolases may exhibit preferential activity towards one or however, these compounds are very difficult to make chemi more possible substrates (i.e. f6-caroteine versus zeaxanthin). cally (Nelis and Leenheer, Appl. Bacteriol. 70:181-191 Simultaneous expression of multiple CrtW ketolases, each (1991)). Natural carotenoids can either be obtained by selected based on their preferred substrate, may be used for extraction of plant material or by microbial synthesis; but, optimal production of a desired ketocarotenoid. One of skill only a few plants are widely used for commercial carotenoid 40 in the art may optimize production of the desired ketocaro production and the productivity of carotenoid synthesis in tenoid end product by analyzing the available substrate pool these plants is relatively low. As a result, carotenoids pro within the desired host cell, selectively expressing an appro duced from these plants are very expensive. One way to priate combination of ketolases for optimal production of the increase the productive capacity of biosynthesis would be to desired ketocarotenoid. apply recombinant DNA technology (reviewed in Misawa 45 The problem to be solved therefore is to identify and and Shimada, J. Biotech., 59:169-181 (1998)).
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