(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date PCT (10) International Publication Number 19 June 2008 (19.06.2008) WO 2008/073367 Al (51) International Patent Classification: Quinn, Qun [US/US], 544 Revere Road, West Chester, C12N 1/16 (2006 01) C07C 403/24 (2006 01) Pennsylvania 19381 (US) C12N 1/15 (2006 01) (74) Agent: FELTHAM, S., NeU, E I du Pont de Nemours and (21) International Application Number: Company, Legal Patent Records Center, 4417 Lancaster PCT/US2007/025222 Pike, Wilmington, Delaware 19805 (US) (22) International Filing Date: (81) Designated States (unless otherwise indicated for every 10 December 2007 (10 12 2007) kind of national protection available): AE, AG, AL, AM, (25) Filing Language: English AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY,BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, (26) Publication Language: English ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, (30) Priority Data: IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, 60/869,576 12 December 2006 (12 12 2006) US LR, LS, LT, LU, LY,MA, MD, ME, MG, MK, MN, MW, 60/869,574 12 December 2006 (12 12 2006) US MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, 60/869,591 12 December 2006 (12 12 2006) US PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, 60/869,582 12 December 2006 (12 12 2006) US TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, 60/869,580 12 December 2006 (12 12 2006) US ZM, ZW (71) Applicant (for all designated States except US): E. I. DU (84) Designated States (unless otherwise indicated for every PONT DE NEMOURS AND COMPANY [US/US] , 1007 kind of regionalprotection available): ARIPO (BW, GH, Market Street, Wilmington, Delaware 19898 (US) GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (72) Inventors; and ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), (75) Inventors/Applicants (for US only): SHARPE, Pamela, European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, L. [US/US], 19 King Circle, Apartment C, Newark, FR, GB, GR, HU, IE, IS, IT, LT,LU, LV,MC, MT, NL, PL, Delaware 19702 (US) YE, Rick, W. [US/US], 27 Pier PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, sons Ridge, Hockessin, Delaware 19707 (US) ZHU, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG) [Continued on next page] (54) Title: CAROTENOID PRODUCTION IN A RECOMBINANT OLEAGINOUS YEAST CrtB Crt! LuH GGPP — ► Phytoene — ► Lycopene • α-Carote πe - Zeinoxanthin — ► Lutein β Carotene Canthaxanthin / 0 3 -Hydroxyechinenone \ CrtZ / L ' ' Crtw\ C X OH O Adonixanthin \crt W HO Tl Astaxanthin O (57) Abstract: Engineered strains of the oleaginous yeast Yarrowia lipolytica capable of producing carotenoids (e g , β carotene, lycopene, lutein, zeaxanthin, canthaxanthin, astaxanthin) are provided The strains may also be engineered to co produce at least one ω 3/ω 6 polyunsaturated fatty acid and/or at least one additional antioxidant Methods of using the carotenoid products obtained (e g , biomass and/or pigmented oils) in food and feed applications are also provided WO 2008/073367 Al Published: with sequence listing part of description published sep a — with international search report rately in electronic form and available upon request from — before the expiration of the time limit for amending the the International Bureau claims and to be republished in the event of receipt of amendments — with (an) indications) in relation to deposited biological material furnished under Rule 13bis separately from the description WO 2008/073367 PCT7US2007/025222 TITLE CAROTENOID PRODUCTION IN A RECOMBINANT OLEAGINOUS YEAST This application claims the benefit of U.S. Provisional Applications 5 No. 60/869,576, No. 60/869,591 , No. 60/869,574, No. 60/869,582 and No. 60/869,580, each filed December 12, 2006. FIELD OF THE INVENTION This invention is in the field of biotechnology. More specifically, this invention pertains to a process of producing carotenoids in oleaginous 10 yeast, pigmented yeast biomass produced by the process, and pigmented oil obtained from the yeast biomass. The carotenoid-producing oleaginous yeast may also be engineered to produce one or more ω- lω polyunsaturated fatty acids and/or antioxidants (e.g., coenzyme Q and/or resveratrol). 15 BACKGROUND OF THE INVENTION Carotenoids (e.g., lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin) represent one of the most widely distributed and structurally diverse classes of natural pigments, producing pigment colors of light yellow to orange to deep red color. Eye-catching examples of 20 carotenogenic tissues include carrots, tomatoes, red peppers, and the petals of daffodils and marigolds. Carotenoids are synthesized by all photosynthetic organisms, as well as some bacteria and fungi. These pigments have important functions in photosynthesis, nutrition, and protection against photooxidative damage; as such, they are used today in 25 e.g., food ingredients/colors, animal feed ingredients, pharmaceuticals, cosmetics and as nutritional supplements. Animals do not have the ability to synthesize carotenoids but must obtain these nutritionally important compounds through their dietary sources. Many animals exhibit an increase in tissue pigmentation when 30 carotenoids are included in their diets, a characteristic often valued by consumers. For example, canthaxanthin and astaxanthin are commonly used in commercial aquaculture industries to pigment shrimp and salmonid fish. It has also been reported that astaxanthin may be a dietary 1 WO 2008/073367 PCT7US2007/025222 requirement for the growth and survival of some salmonid species (Christiansen et al., Aquaculture Nutrition, 1:189-198 (1995)). Similarly, lutein, canthaxanthin and astaxanthin are commonly used as pigments in poultry feeds to increase the pigmentation of chicken skin and egg yolks. 5 Industrially, only a few carotenoids are used, despite the existence of more than 600 different carotenoids identified in nature. This is largely due to difficulties in production and high associated costs. For example, the predominant source of aquaculture pigments used in the market today are produced synthetically and are sold under such trade names as 10 Carophyll® Red (canthaxanthin; DSM Nutritional Products, Heerlen, NL) and Carophyll® Pink (astaxanthin; DSM Nutritional Products); however, the cost of utilizing the synthetically produced pigments is quite high even though the amount of pigment incorporated into the fishmeal is typically less than 100 ppm. 15 A further concern associated with the use of synthetically produced carotenoids for feed formulations is the common addition of synthetic antioxidants to act as a preservative and to help protect the pigments from oxidation. Many of these synthetic antioxidants are facing significant questions concerning their safety; for example, many adverse health 20 effects (e.g., carcinogenesis, cytotoxicity, etc.) have been reported with the use of ethoxyquin [6-ethoxy-2,2,4-trimethyl-1 H-quinoline] (Blaszczyk et. al., Cell MoI. Biol. Lett., 10(1 ):1 5-21 (2005); Blaszczyk et al., Mutat. Res., 542:1 17-128 (2003); Little, A.D., Chemical Evaluation Committee Draft Report, Ethoxvquin. CAS Number 91-53-2 , submitted to National 25 Toxicology Program, Executive Summary of Safety and Toxicity Information, U.S. Department of Health and Human Services, (1990)). As such, there is a need for a source of natural carotenoids that optionally comprise natural antioxidants suitable for their stabilization. Natural carotenoids can either be obtained by extraction of plant 30 material or by microbial synthesis; but, only a few plants are widely used for commercial carotenoid production and the productivity of carotenoid synthesis in these plants is relatively low. Microbial production of carotenoids is a more attractive production route. Examples of carotenoid- 2 WO 2008/073367 PCT7US2007/025222 producing microorganisms include: algae (Haematococcus pluvialis, sold under the tradename NatuRose™ (Cyanotech Corp., Kailua-Kona, HI; Dunaliella sp.), yeast (Phaffia rhodozyma, recently renamed as Xanthophyllomyces dendrorhous; Thraustochytrium sp.; Labyrinthula sp.; 5 and Saccharomyces cerevisiae), and bacteria (Paracoccus marcusii, Bradyrhizobium, Rhodobactersp., Brevibacterium, Escherichia coli and Methylomonas sp.). Additionally, recombinant production of carotenoids is also possible, since the genes involved in carotenoid biosynthesis are well-known and have been heterologously expressed in a variety of host 10 cells (e.g., E. coli, Candida utilis, Saccharomyces cerevisiae, Methylomonas sp.). Thus far, few of these demonstrations are suitable to produce a carotenoid product in significant quantities in a cost-effective manner for industrial use. Many commercial products are formulated to contain a mixture of 15 pigments and fats/lipids and/or natural antioxidants. For example, animal feeds, dietary supplements, and personal care products are often formulated to contain carotenoids, polyunsaturated fatty acids (PUFAs) and antioxidants (e.g., CoQi 0). For fish feed formulations, fish oil is often supplemented to the feed to provide the necessary caloric intake and to 20 provide essential fatty acids such as the ω-3/ω-6 PUFAs. Typically, for example, a commercial product formulator will obtain these compounds from a variety of sources and formulate them into a final product that contains an effective amount of each ingredient. The composition, purity and source of each ingredient may vary, resulting in a final product 25 formulation that may require significant monitoring and/or processing to obtain the desired product specifications. Engineering a microorganism to simultaneously produce carotenoids and fats/lipids and/or an additional antioxidant could create a higher value product and prove advantageous for commercial production 30 economics (and therefore availability) to consumers. One class of organisms that are especially suitable as a production platform for synthesis of pigmented microbial oils (optionally comprising PUFAs and/or additional antioxidants) are the oleaginous yeast.