(19) TZZ¥_¥ZZ_T (11) EP 3 168 300 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 17.05.2017 Bulletin 2017/20 C12N 9/42 (2006.01) C12P 7/06 (2006.01) (21) Application number: 16192843.7 (22) Date of filing: 03.06.2011 (84) Designated Contracting States: • WARNER, Anne, K. AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Lebanon, NH 03766 (US) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • MELLON, Mark PL PT RO RS SE SI SK SM TR Grantham, NH 03753 (US) • SKINNER, Ryan (30) Priority: 03.06.2010 US 351165 P White River Junction, VT 05001 (US) 06.12.2010 US 420142 P • SHIKHARE, Indraneel Lebanon, NH 03766 (US) (62) Document number(s) of the earlier application(s) in • DEN HAAN, Riaan accordance with Art. 76 EPC: 7550 Durbanville (ZA) 11790526.5 / 2 576 762 • GANDHI, Chhayal, V. Nashua, NH 03062-2858 (US) (71) Applicants: • BELCHER, Alan • Lallemand Hungary Liquidity Management LLC Nashua, NH 03063 (US) Budapest 1077 (HU) • RAJGARHIA, Vineet, B. • Stellenbosch University Lebanon, NH 03766 (US) 7600 Stellenbosch (ZA) • FROEHLICH, Allan, C. Lebanon, NH 03766 (US) (72) Inventors: • DELEAULT, Kristen, M. • BREVNOVA, Elena Canaan, NH 03741 (US) Lebanon, NH 03766 (US) • STONEHOUSE, Emily • MCBRIDE, John, E. Lebanon, NH 03766 (US) Lyme, NH 03768 (US) • TRIPATHI, Shital, A. • WISWALL, Erin Emeryville California 94608 (US) Danbury, NH 03230 (US) • GOSSELIN, Jennifer • WENGER, Kevin, S. Lebanon, NH 03766 (US) Hanover, NH 03755 (US) • CHIU, Yin-Ying • CAIAZZA, Nicky West Lebanon, NH 09784 (US) Rancho Santa Fe, CA 92067 (US) • XU, Haowen • HAU, Heidi, H. Lebanon, NH 03766 (US) Lebanon, NH 03766 (US) • ARGYROS, Aaron (74) Representative: Cornish, Kristina Victoria Joy White River Junction, VT 05001 (US) Kilburn & Strode LLP • AGBOGBO, Frank 20 Red Lion Street Lebanon, NH 03766 (US) London WC1R 4PJ (GB) • RICE, Charles, F. Hopkinton, NH 03229 (US) Remarks: • BARRETT, Trisha •Thecomplete document including Reference Tables Bradford, VT 05033 (US) and the Sequence Listing can be downloaded from • BARDSLEY, John, S. the EPO website Newport, NH 03773 (US) •This application was filed on 07-10-2016 as a • FOSTER, Abigail, S. divisional application to the application mentioned South Strafford, VT 05070 (US) under INID code 62. EP 3 168 300 A1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 3 168 300 A1 (54) YEAST EXPRESSING SACCHAROLYTIC ENZYMES FOR CONSOLIDATED BIOPROCESSING USING STARCH AND CELLULOSE (57) The present invention is directed to a yeast to produce ethanol from granular starch without liquefac- strain, or strains, secreting a full suite, or any subset of tion. The resulting strain, or strains, can be further used that full suite, of enzymes to hydrolyze corn starch, corn to reduce the amount of external enzyme needed to hy- fiber, lignocellulose, (including enzymes that hydrolyze drolyze a biomass feedstock during an Simultaneous linkages in cellulose, hemicellulose, and between lignin Saccharification and Fermentation (SSF) process, or to and carbohydrates) and to utilize pentose sugars (xylose increase the yield of ethanol during SSF at current sac- and arabinose). The invention is also directed to the set charolytic enzyme loadings. In addition, multiple en- of proteins that are well expressed in yeast for each cat- zymes of the present invention can be co-expressed in egory of enzymatic activity. The resulting strain, or strains cells of the invention to provide synergistic digestive ac- can be used to hydrolyze starch and cellulose simulta- tion on biomass feedstock. In some aspects, host cells neously. The resulting strain, or strains can be also met- expressing different heterologous saccharolytic en- abolicallyengineered toproduce less glyceroland uptake zymes can also be co-cultured togetherand used to pro- acetate. The resulting strain, or strains can also be used duce ethanol from biomass feedstock. 2 EP 3 168 300 A1 Description BACKGROUND OF THE INVENTION 5 [0001] Biomass is biological material from living, or recently living organisms, such as wood, waste, (hydrogen) gas, and alcohol fuels. Biomass is carbon, hydrogen and oxygen based. Nitrogen and small quantities of other atoms, including alkali, alkaline earth and heavy metals can be found as well. Metals are often found in functional molecules such as the porphyrins which include chlorophyll which contains magnesium. Plants in particular combine water and carbon dioxide to sugar building blocks. The required energy is produced from light via photosynthesis based on chlorophyll. On average, 10 between 0.1 and 1 % of the available light is stored as chemical energy in plants. The sugar building blocks are the starting point for all of the major fractions found in terrestrial plants, lignin, hemicellulose and cellulose. Biomass is wide ly recognized as a promising source of raw material for production of renewable fuels and chemicals. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost tech- nology for overcoming the recalcitrance of these materials to conversion into useful fuels. Biomass contains carbohydrate 15 fractions (e.g., starch, cellulose, and hemicellulose) that can be converted into ethanol. In order to convert these fractions, the starch, cellulose, and, hemicellulose must ultimately be converted or hydrolyzed into monosaccharides; it is the hydrolysis that has historically proven to be problematic. [0002] Biologically mediated processes are promising for energy conversion, in particular for the conversion of biomass into fuels. Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically 20 mediated transformations: (1) the production of saccharolytic enzymes (amylases, cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process 25 step for cellulase and/or hemicellulase production. [0003] CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated saccharolytic enzyme production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with saccharolytic enzyme production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy 30 and the use of thermophilic organisms and/or complexed saccharolytic systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring saccharolytic microorganisms to improve product-related properties, such as yield and titer; and engineering non-sac- 35 charolytic organisms that exhibit high product yields and titers to express a heterologous saccharolytic enzyme system enabling starch, cellulose, and, hemicellulose utilization. [0004] The breakdown of starch down into sugar requires amylolytic enzymes. Amylase is an example of an amylolytic enzyme that is present in human saliva, where it begins the chemical process of digestion. The pancreas also makes amylase (alpha amylase) to hydrolyze dietary starch into disaccharides and trisaccharides which are converted by other 40 enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylases. Amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds. [0005] Several amylolytic enzymes are implicated in starch hydrolysis. Alpha-amylases (EC 3.2.1.1) (alternate names: 1,4-α-D-glucan glucanohydrolase; glycogenase) are calcium metalloenzymes, i.e., completely unable to function in the absence of calcium. By acting at random locations along the starch chain, alpha-amylase breaks down long-chain 45 carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. Because it can act anywhere on the substrate, alpha-amylase tends to be faster-acting than beta-amylase. Another form of amylase, beta-amylase (EC 3.2.1.2) (alternate names: 1,4-α-D-glucan maltohydrolase; glycogenase; saccharogen amylase) catalyzes the hydrolysis of the secondα -1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. The third amylase is gamma-amylase (EC 3.2.1.3) (alternate names: Glucan 1,4-α-glucosidase; 50 amyloglucosidase; Exo-1,4-α-glucosidase; glucoamylase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase). In addition to cleaving the last α(1-4)glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, gamma-amylase will cleave α(1-6) glycosidic linkages. [0006] A fourth enzyme, alpha-glucosidase, acts on maltose and other short maltooligosaccharides produced by alpha-, beta-, and gamma-amylases, converting them to glucose. 55 [0007] Three major types of enzymatic activities are required for native