USOO696.9595 B2 (12) United States Patent (10) Patent No.: US 6,969,595 B2 Brzostowicz et al. (45) Date of Patent: Nov. 29, 2005 (54) CAROTENOID PRODUCTION FROM A WO WO 02079395 A2 10/2002 SINGLE CARBON SUBSTRATE OTHER PUBLICATIONS (75) Inventors: Patricia C. Brzostowicz, West Chester, Hundle et al., Functional Assignment of Erwinia herbicola PA (US); Qiong Cheng, Wilmington, Eh010 Carotenoid Genes Expressed in Escherichia coli, DE (US); Deana DiCosimo, Rockland, Molecular and General Genetics, Springer Verlag, Berlin, DE (US); Mattheos Kofas, DE, vol. 245, 1994, pp. 406-416, XP002947 192. Wilmington, DE (US); Edward S. Pasamontes et al., Isolation and characterization of the Miller, Wilmington, DE (US); James carotenoid biosynthesis genes of Flavobacterium sp. Strain M. Odom, Kennett Square, PA (US); R1534, Gene: An International Journal on Genes and Stephen K. Picataggio, Landenberg, PA Genomes, Elsevier Science Publishers, Braking, GB, vol. (US); Pierre E. Rouviere, Wilmington, 185, No. 1, Jan. 31, 1997, pp. 35–41, XP004.093151. DE (US) Harker et al., BioSynthesis of Ketocarotenoids in transgenic cyanobacteria expressing the algal gene for (73) ASSignee: E. I. du Pont de Nemours and beta-C-4-oxygenase, crt0, FEBS Letters, Elsevier Science Company, Wilmington, DE (US) Publishers, Amsterdan, NL. Vol. 404, Mar. 1, 1997, pp. 129-134, XP002087149. Notice: Subject to any disclaimer, the term of this Fernandez-Gonzalez Blanca et al., A new type of asym patent is extended or adjusted under 35 metrically acting beta-caroteine ketolase is required for the U.S.C. 154(b) by 728 days. Synthesis of echinenone in the cyanobacterium SynechocyS tissp. PCC 6803., Journal of Biological Chemistry vol. 272, (21) Appl. No.: 09/941,947 No. 15, 1997, pp. 9728-9733, XP002222602. Hirschberg, Production of high-value compounds: Caro (22) Filed: Aug. 29, 2001 tenoids and Vitamin E, Current Opinion in biotechnology, (65) Prior Publication Data London GB, vol. 10, No. 2, Apr. 1999, pp. 186-191, XPOO2162837. US 2003/0003528 A1 Jan. 2, 2003 Misawa et al., Expression of an Wrwinia Phytoene Desatu rase Gene not only confers Multiple Resistance to Herbi Related U.S. Application Data cides Interfering with Carotenoid Biosynthesis but also (60) Provisional application No. 60/229.907, filed on Sep. 1, 2000, and provisional application No. 60/229,858, filed on alters Xanthophyll Metabolism in Transgenic Plants, Plant Sep. 1, 2000. Journal, Blackwell Scientific Publications, Oxford, GB, vol. 6, No. 4, 1994, pp. 481–489, XP002012.919. (51) Int. Cl................................................. 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A 5/1998 Inomata et al. ............. 585/312 dopsis thaliana is localized in mitochondria, Plant Molecu 5,972,642 A 10/1999 Flen.o slashed. et al. ..... 435/67 lar Biology, Nijhoff Publishers, Dordrecht, NL, vol. 35, No. 6,015,684 A 1/2000 Jacobson et al. ............. 435/67 3, 1997, pp. 331-341, XP002153683. 6,124,113 A 9/2000 Hohmann et al. ............ 435/67 (Continued) FOREIGN PATENT DOCUMENTS Primary Examiner Nashaat T. Nashed EP O747483 A2 12/1996 EP O872554 A2 10/1998 (57) ABSTRACT WO WO 97 23633 A1 7/1997 A method for the production of carotenoid compounds is WO WO 9907867 A1 2/1999 WO WO996.1652 A1 12/1999 disclosed. The method relies on the use of microorganisms WO WO 2000007718 A1 2/2000 which metabolize Single carbon Substrates for the production WO WO 01/66703 A1 9/2001 of carotenoid compounds in high yields. WO WO O22O733 A2 3/2002 WO WO O2/41833 A2 5/2002 22 Claims, 14 Drawing Sheets US 6,969,595 B2 Page 2 OTHER PUBLICATIONS Miura, Y. et al., 1998. Appl. Environm. Microbiol. 64: 1226-1229. Misawa et al., “Elucidation of the Erwinia uredovora Carenoid Biosynthetic Pathway by Functional Analysis of Albrecht, M. et al., 1999, Biotechnol. Lett. 21: 791-795. Gene Products Expressed in Escherichia coli”, Journal of Lidstrom and Stirling (Annu. Rev. Microbiol. 44:27-58, Bacteriology, Washington, D.C., vol. 172, No. 12, Dec. 1990. 1990, pp. 6704-6712. Murrell et al., Arch. Microbiol., 2000, 173(5-6), 325–332. Armstrong, J. Bact. 176: pp. 4795-4802. Grigoryan, E. A., Kinet. Catal., 1999, 40(3), 350-363. Armstrong, Annu. Rev. Microbiol. 51: 629–659, 1997. Beschastnyi et al., Inst. Biochem. Physiol. Microor., Push Nelis and Leenheer, Appl. Bacteriol. 70:181-191, 1991. chino, Russia, Biokhimiya (Moscow) 1992, 57(8), pp. Farmer, W. R. and J. C. Liao, 2001, Biotechnol. Prog. 17: 1215-1221. 57-61. Wang, C. et al., 2000 Biotechnol. Prog. 16:922–926. Shishkina et al., Inst. Bikhim. Fiziol. Mikroorg., Pushchino, Misawa, N. and H. Shimada, 1998, J. Biotechnol. 59: Russia, Mikrobiologiya, 1990, 59(4), 533–8. 169-181. Trotsenko et al., Studies on Phosphate metabolism in obli Shimada, H. et al., 1998, Appl. Environm. Microbiol. gate methanotrophs, Fems Microbiology Reviews 87, 1990, 64:2676-2680. pp. 267–272. U.S. Patent US 6,969,595 B2 H0 was,† U.S. Patent US 6,969,595 B2 U.S. Patent Nov. 29, 2005 Sheet 5 of 14 US 6,969,595 B2 Cri Y CritB critz ECORI (5301 ) U.S. Patent Nov. 29, 2005 Sheet 6 of 14 US 6,969,595 B2 400 300 MOU 200 100 O 5 10 15 2O 25 450 Mies, nn FIG. 3B 250 200. 150 OU OO 50 Minutes 450 nm BOnd E 4 nm. FIG. 3C U.S. Patent Nov. 29, 2005 Sheet 7 of 14 US 6,969,595 B2 tro promoter region U.S. Patent Nov. 29, 2005 Sheet 8 of 14 US 6,969,595 B2 A. O 200 400 600 800 1 000 200 1400 SeCOndS FIG. 4B 35000 30000 25000 OU 20000 15000 H U.S. Patent Nov. 29, 2005 Sheet 9 of 14 US 6,969,595 B2 Nhe 14 ) ECOR ) Phips ECORI ( 257 ) tro promoter Crit region 5 10 15 20 25 Minutes 450 nm Bond = 4 nm. F G. 5B U.S. Patent Nov. 29, 2005 Sheet 10 of 14 US 6,969,595 B2 ECORI ( 1)- - Crit O Nne (1631) - Phmps ECOR 187 ) PCO region F.G. 6A U.S. Patent Nov. 29, 2005 Sheet 11 of 14 US 6,969,595 B2 10 11 12 13 14 15 16 17 18 9 20 Minutes 474 nm Bond E 4 nm F. G. 6B U.S. Patent Nov. 29, 2005 Sheet 12 of 14 US 6,969,595 B2 200 250 300 350 400 450 500 550 600 n FIG. 60 U.S. Patent Nov. 29, 2005 Sheet 13 of 14 US 6,969,595 B2 DXP-5-PReduct OSe dxS dxr OCZ Tn5Kn 14994 bp U.S. Patent Nov. 29, 2005 Sheet 14 of 14 US 6,969,595 B2 d NNNNNNNNNNNSNSSSSSSSSSSSNSSNSZZZZZZZ -g NSSSSSSS-gYZZY ? V SSNSSSNSSNSSSSSSSSSSSSSSSSSNSNSZZZZZZZZZ Sk ZZZZZZZZY e ?h NSNSN g ? N SSSSSSSNSNSSSSSSSSNSSNSSO k - ed . C-d 8. e-r at s ZZZZZZZZZ 9 ---a stby N-9. A CD A- f : / - ZZZZZZZZ - a gig R OO ll N w n is a 2ZYZZYZZ k - A nar Perror-rr A all - C-9 (C. O seas ZZZZYZZYA - 4 ZZZZZY O T sers an C O C C Cd O C C C O s N C O t s N s r - - "4M Ku bu IO 4M Kud 6/43 fin US 6,969,595 B2 1 2 CAROTENOID PRODUCTION FROM A Although more than 600 different carotenoids have been SINGLE CARBON SUBSTRATE identified in nature, only a few are used industrially for food colors, animal feeding, pharmaceuticals and cosmetics. This application claims the benefit of U.S. Provisional Presently, most of the carotenoids used for industrial pur poses are produced by chemical Synthesis, however, these Application No. 60/229,907, filed Sep. 1, 2000 and the compounds are very difficult to make chemically (Nelis and benefit of U.S. Provisional Application No. 60/229,858 filed Leenheer, Appl. Bacteriol. 70:181-191 (1991)). Natural Sep. 1, 2000. carotenoids can either be obtained by extraction of plant FIELD OF THE INVENTION material or by microbial Synthesis. At the present time, only a few plants are widely used for commercial carotenoid The invention relates to the field of molecular biology and production.