US 2011 O165635A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0165635 A1 Copenhaver et al. (43) Pub. Date: Jul. 7, 2011 (54) METHODS AND MATERALS FOR Publication Classification PROCESSINGA FEEDSTOCK (51) Int. Cl. CI2P I 7/04 (2006.01) (75) Inventors: Gregory P. Copenhaver, Chapel CI2P I/00 (2006.01) Hill, NC (US); Daphne Preuss, CI2P 7/04 (2006.01) Chicago, IL (US); Jennifer Mach, CI2P 7/16 (2006.01) Chicago, IL (US) CI2P 7/06 (2006.01) CI2P 5/00 (2006.01) CI2P 5/02 (2006.01) (73) Assignee: CHROMATIN, INC., Chicago, IL CI2P3/00 (2006.01) (US) CI2P I/02 (2006.01) CI2N 5/10 (2006.01) (21) Appl. No.: 12/989,038 CI2N L/15 (2006.01) CI2N I/3 (2006.01) (52) U.S. Cl. ........... 435/126; 435/41; 435/157; 435/160; (22) PCT Fled: Apr. 21, 2009 435/161; 435/166; 435/167; 435/168; 435/171; 435/419,435/254.11: 435/257.2 (86) PCT NO.: PCT/US2O09/041260 (57) ABSTRACT S371 (c)(1), The present disclosure relates generally to methods for pro (2), (4) Date: Mar. 11, 2011 cessing a feedstock. Specifically, methods are provided for processing a feedstock by mixing the feedstock with an addi tive organism that comprises one or more transgenes coding Related U.S. Application Data for one or more enzymes. The expressed enzymes may be (60) Provisional application No. 61/046,705, filed on Apr. capable of breaking down cellulosic and lignocellulosic 21, 2008. materials and converting them to a biofuel. US 2011/01 65635 A1 Jul. 7, 2011 METHODS AND MATERALS FOR enzymes (e.g., gene Stack), mixing the feedstock with the PROCESSINGA FEEDSTOCK additive organism, and incubating the mixture under condi tions wherein the feedstock is processed by the activity of the FIELD one or more enzymes on the feedstock. In some embodi ments, the additive organism can secrete one or more 0001. The present disclosure relates generally to methods enzymes encoded by one or more transgenes (along with for processing a feedstock. Specifically, methods are pro optionally other enzymes produced by the additive organism) vided for processing a feedstock by mixing the feedstock with into its environment. an additive organism that comprises one or more transgenes 0008. The present disclosure also provides methods for coding for one or more enzymes. Enzymes released from the converting a feedstock to a biofuel by mixing the feedstock additive organism may be used to manufacture a biofuel or with an additive organism that comprises one or more trans another hydrocarbon or co-product by converting the feed genes coding for one or more enzymes (e.g., gene Stack). stock into Sugars that can be fermented or chemically con converting the feedstock into Sugars, and fermenting or verted or by extracting oils that may be processed into biodie chemically converting the Sugars to produce a biofuel or other sel. hydrocarbon. BACKGROUND 0009. The present disclosure provides methods for con verting a feedstock to a biofuel by mixing the feedstock with 0002. The use of biofuels are considered a means for an additive organism that comprises one or more transgenes reducing greenhouse gas emissions and increasing energy coding for one or more enzymes (e.g., gene Stack), extracting security by providing an alternative to fossil fuels. Biofuels one or more oils from the feedstock, and converting the oils to may be produced from the conversion of a biomass (e.g., a biofuel. trees, grasses, agricultural crops or other biological material) 0010. The present disclosure provides method for gener into liquid or gaseous fuels (e.g., ethanol, propanol, butanol, ating revenue from a biofuel manufacturing process by mix methanol, methane, 2,5-dimethylfuran, dimethyl ether, ing the feedstock with an additive organism that comprises biodiesel, biogasoline, paraffins, other hydrocarbons or co one or more transgenes coding for one or more enzymes (e.g., products or hydrogen) by converting the biomass into Sugars gene stack), converting the feedstock into Sugars and selling that can be fermented or chemically converted to form a the Sugars to a buyer (e.g., a Supplier, a distributor, a manu biofuel, or otherwise extracting oils from the biomass. facturer, a dealer, a reseller, a wholesaler, a retailer and/or a 0003. The manufacture of a biofuel from a biomass consumer), or fermenting or chemically converting the Sugars requires accessibility to plant constituents (e.g., cellulosic to produce a biofuel, and selling the biofuel to a buyer (e.g., a materials need to be broken-down). AS Such, biomass may Supplier, a distributor, a manufacturer, a dealer, a reseller, a require a pre-treatment step that uses heat, chemicals and/or wholesaler, a retailer and/or a consumer). purified enzyme additives. These treatments are often expen 0011. The present disclosure also provides methods for sive, inefficient, and produce by-products that are inhibitory generating revenue from a biofuel manufacturing process by of downstream processing or are toxic. mixing the feedstock with an additive organism that com prises one or more transgenes coding for one or more SUMMARY enzymes (e.g., gene Stack), extracting one or more oils from 0004. The present disclosure relates generally to methods the feedstock, converting the oils to a biofuel, and selling the for processing a feedstock (e.g., a biomass) by mixing the biofuel to a buyer (e.g., a Supplier, a distributor, a manufac feedstock with an additive organism (e.g., a transgenic organ turer, a dealer, a reseller, a wholesaler, a retailer and/or a ism including but not limited to a plant, alga, or fungus) that consumer). comprises one or more transgenes coding for one or more 0012. The present disclosure provides an additive organ enzymes (e.g., gene Stack), including methods for processing ism for processing a feedstock, that comprises one or more a feedstock by preparing an additive organism that comprises transgenes coding for one or more enzymes (e.g., gene stack), one or more transgenes coding for one or more enzymes and wherein the enzymes include, for example, those enzymes mixing the feedstock with the additive organism. An additive listed in Table 1. organism may produce one or more enzymes from one or 0013 The present disclosure also provides methods of more transgenes and may optionally produce one or more preparing an additive organism by introducing one or more enzymes not from transgenes. transgenes (e.g., gene stack) into the additive organism, 0005 Such methods may additionally include the step of wherein the enzymes include, for example, those enzymes incubating the mixture under conditions wherein the feed listed in Table 1. stock is processed by the activity of one or more enzymes on 0014. The present disclosure also provides an additive the feedstock. organism for processing a feedstock, that comprises one or 0006. The present disclosure provides methods for pro more transgenes (e.g., gene Stack) that when transcribed pro cessing a feedstock by preparing an additive organism that duce an RNA product that is capable of inhibiting (RNAi) the comprises one or more transgenes coding for one or more production of one or more enzymes, wherein the enzymes enzymes (e.g., gene Stack), disrupting cells of the additive includebut are not limited to those listed in Table 1. Inhibitory organism, mixing the feedstock with the disrupted cells, and RNA products include, for example, antisense RNA and incubating the mixture under conditions wherein the feed microRNAs. stock is processed by the activity of the one or more enzymes 0015 The present disclosure also provides methods of on the feedstock. preparing an additive organism by introducing one or more 0007. The present disclosure provides methods for pro transgenes (e.g., gene stack) into the additive organism that cessing a feedstock by preparing an additive organism that when transcribed produce an RNA product that is capable of comprises one or more transgenes coding for one or more inhibiting (RNAi) the production of one or more enzymes, US 2011/01 65635 A1 Jul. 7, 2011 wherein the enzymes include but are not limited to those forth by SEQ ID NOs: 1-40. In further embodiments, the listed in Table 1. Inhibitory RNA products include, for transgenes each separately comprise one of the polynucle example, antisense RNA and microRNAs. otide sequences set forth by SEQID NOs: 7, 16, 18 and 28. In 0016. The present disclosure also provides methods for further embodiments, the transgenes each separately com processing a feedstock by expressing one or more transgenes prise one of the polynucleotide sequences set forth by SEQID (e.g., gene stack) coding for one or more enzymes in the NOS: 2, 16, 18 and 25. additive organism, and mixing a feedstock with the additive 0022. In some embodiments, the additive organism com organism. prises five transgenes (e.g., gene stack) each of which sepa 0017. In some embodiments, the one or more transgenes rately comprises one of the polynucleotide sequences set are present in a nucleic acid construct that integrates into a forth by SEQ ID NOs: 1-40. In further embodiments, the chromosome in the additive organism. Integrative constructs transgenes each separately comprise one of the polynucle include, for example, those that integrate into nuclear chro otide sequences set forth by SEQID NOs: 3, 5, 16, 18 and 25. mosomes, mitochondrial chromosomes, chloroplast chromo In further embodiments, the transgenes each separately com Somes or any other non-nuclear portion of the genome. In prise one of the polynucleotide sequences set forth by SEQID Some embodiments, the one or more transgenes are present in NOS: 6, 16, 18, 26 and 27. a nucleic acid construct that does not integrate into the chro 0023.