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WO 2011/026008 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 3 March 2011 (03.03.2011) WO 2011/026008 Al (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12P 1/00 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/US20 10/047091 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, 30 August 2010 (30.08.2010) KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (25) Filing Language: English NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (26) Publication Language: English SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/238,072 28 August 2009 (28.08.2009) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant (for all designated States except US): PHY- GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, CAL LLC [US/US]; 51 Alpha Park, Highland Heights, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, OH 44143 (US). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors; and LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, (75) Inventors/Applicants (for US only): ALLNUTT, F.C., SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Thomas [US/US]; 14692 Mustang Path, Glenwood, MD GW, ML, MR, NE, SN, TD, TG). 21748 (US). POSTIER, Bradley, Lynn [US/US]; 26 Tu pelo Court, St. Charles, MO 63303 (US). SAYRE, Declarations under Rule 4.17: Richard, T. [US/US]; 140 N. Rock Hill Road, Webster — of inventorship (Rule 4Λ 7(iv)) Groves, MO 63 119 (US). COURY, Daniel, A. [US/US]; 2026 Midfield Lane, St. Louis, MO 63 146 (US). Published: (74) Agent: RUSSELL, Raymond, N.; Calfee, Halter & Gris- — with international search report (Art. 21(3)) wold LLP, 800 Superior Ave., Ste. 1400, Cleveland, OH 441 14 (US). (54) Title: BIOFUEL FROM RECOMBINANT OLEGINOUS ALGAE USING SUGAR CARBON SOURCES (57) Abstract: Recombinant oleaginous alga that include one or more heterologous genes that increase the ability of the alga to use one or more natural saccharides such as cellulosic or hemicellulosic sugars for algal growth are described. The recombinant oleaginous algae are transformed to include one or more genes expressing sugar metabolizing enzymes or sugar transporting pro- teins, along with suitable control elements. Use of natural saccharides as a carbon source can allow the algae to produce biofuel precursors in a relatively efficient manner. Processes for preparing the alga, growing the alga, and extracting the biofuel precur- sors from the alga are also described. BIOFUEL FROM RECOMBINANT OLEAGINOUS ALGAE USING SUGAR CARBON SOURCES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to US Provisional Patent Application No. 61/238,072, filed August 28, 2009, the entirety of which is incorporated herein by reference. BACKGROUND [0002] Recent research indicates that microalgal biofuel systems can contribute substantially to domestic energy production in a sustainable and potentially carbon neutral manner. Chisti Y, Biotech Adv 25: 294-306 (2007). Microalgae can utilize feedstock materials that do not directly compete with food production, are 10-30 times more productive per hectare than terrestrial biofuel systems, can be harvested daily due to rapid doubling times, and can capture industrial CO2 emissions. To be competitive with petroleum-based fuels, however, microalgal biomass production and oil harvesting costs must be substantially reduced from current estimates of about $30/gallon. [0003] Oils, including polar membrane lipids and triacylglycerols (TAGs), typically make up 5-15% of the total biomass of algae. Oils can accumulate, however, up to 60% (w/w) of the total biomass in many algae when they are grown under conditions where the capacity to produce or metabolize reduced carbon exceeds the energy and carbon demands for growth. One way to encourage the accumulation of high levels of oil is to use heterotrophic growth, i.e., the use of an organic carbon source rather than sunlight and CO2, to provide an energy source for the algae. Heterotrophic growth enhances both cell division and sequestration of reduced carbon as oil. [0004] As a result of its abundance and renewable nature, biomass represents an excellent potential carbon source for producing affordable and environmentally sound biofuels to replace fossil fuels. Many biomass feedstocks can be used for the production of biofuel, such as lignocellulosic biomass (e.g., jatropha, switchgrass, and poplar), algae (e.g., macroalgae and microalgae), and oil crops (e.g., soybean, palm oil and sunflower). These biomass feedstocks have the advantage of being relatively inexpensive, but are relatively complex chemically and typically require deconstruction to obtain the useful biomass components (e.g., monosaccharides, fatty acids, etc.) that can be readily converted into biofuels. Starch (e.g., derived from corn, wheat, barley, etc.) and sugar crops (e.g., derived from cane, beet, etc.) are feedstocks that are easier to process, but are more expensive and compete with food production. [0005] Lignocellulosic biomass includes a large amount of cellulose, which is a polymeric form of glucose, and hemicellulose, which includes sugars such as xylose, mannose, galactose, rhamnose, and arabinose. However, many algae do not include the transporter proteins and/or sugar metabolizing enzymes necessary to utilize these sugars derived from lignocellulosic biomass. Selective growth has been used to encourage Chlorella algae to grow using pentose sugars. See Hawkins, R., Curr. Microb., 38, p. 360-363 (1999). However, selective growth is an imprecise and unreliable method for modifying algae to be able to utilize alternative carbon sources. Accordingly, the need remains for algae that are able to convert natural saccharides, such as cellulosic and hemicellulosic sugars, into biofuel precursors, such as oils and lipids. SUMMARY OF THE INVENTION [0006] The need for algae able to convert natural saccharides into biofuel precursors and other unmet needs of the prior art is met by exemplary compositions and methods described herein. [0007] Cellulose and hemicellulose are estimated to be the most abundant organic molecules on earth. The efficient and complete conversion of cellulose or hemicellulose into biofuel precursors through the heterotrophic growth of algae provides significant advantages, even over fermentation of the same substrates to ethanol or butanol. Biofuel derived from the process has a higher energy density, is compatible with existing transportation engines and distribution systems, is non-toxic, and is not limited by final concentration of endproduct in the reactor vessel, such as occurs for ethanol and butanol. As such biofuel precursors, derived from algae produced phototrophically or heterotrophically are an excellent replacement fuel for petroleum based diesel and jet fuel. Since oleaginous algae are highly efficient (up to 90% energy conversion efficiency) at converting reduced carbon into oil, the recombinant oleaginous algae described herein can efficiently convert natural saccharides such as cellulose into a liquid, high-energy density fuel. [0008] In one aspect, the present invention provides a recombinant oleaginous alga that includes one or more heterologous genes that increase the ability of the alga to use one or more natural saccharides for algal growth. In some embodiments, the natural saccharide is a cellulosic or hemicellulosic sugar, or a monosaccharide derived therefrom. In further embodiments, the one or more heterologous genes express one or more sugar metabolizing enzymes that are lacking in a wild-type form of the oleaginous alga. In a further embodiment, the one or more heterologous genes express one or more sugar transporters that are lacking in a wild-type form of the oleaginous alga. In additional embodiments, the recombinant algae also includes a nucleotide sequence expressing an RNAi construct or an expression vector that alters the metabolism of the algae to produce a larger amount of biofuel precursors such as fatty acids. [0009] hi another aspect, the present invention provides a method of making biofuel precursors that includes growing an algal culture including a recombinant oleaginous alga of any one of the preceding claims using a natural saccharide as a primary carbon source, and extracting one or more biofuel precursors from the algal culture. In some embodiments, the natural saccharide is a cellulosic or hemicellulosic sugar, or a monosaccharide derived therefrom. In further embodiments, the biofuel precursors are extracted by mixing a portion of the algal culture with an oil-extracting solvent to obtain a solvent-algae mixture, partitioning the solvent-algae mixture to obtain a solvent- biofuel precursor fraction and viable extracted algae, and returning the viable extracted algae to the algal culture. BRIEF DESCRIPTION OF THE FIGURES [0010] The present invention may be more readily understood by reference to the following drawings wherein: [0011] Figure 1 provides a construct for containing ADP glucose pyrophosphorylase RNAi that allows one to block starch synthesis. [0012] Figure 2 provides two graphs showing the ability of various natural saccharides to support growth (Fig. 2A) or oil production (Fig. 2B) in Chlorella protothecoides. [0013] Figure 3 provides a scheme showing the enzymes involved in rhamnose metabolism. [0014] Figure 4 provides a schematic representation of a three gene operon from Clostridium that encodes the genes required for metabolism of rhamnose.
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