(19) TZZ __ T (11) EP 2 617 817 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: C12N 9/16 (2006.01) C07H 21/02 (2006.01) 24.07.2013 Bulletin 2013/30 C07H 21/04 (2006.01) A01N 63/00 (2006.01) A61K 48/00 (2006.01) C12N 5/00 (2006.01) (2006.01) (21) Application number: 13164137.5 C12N 5/02 (22) Date of filing: 21.09.2007 (84) Designated Contracting States: • Trefzer, Axel AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 2264 KR Leidschendam (NL) HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE • Todaro, Tom SI SK TR San Diego, CA 91962 (US) • Solbak, Arne (30) Priority: 21.09.2006 US 846831 P San Diego, CA 92126 (US) •El-Farrah, Fatima (62) Document number(s) of the earlier application(s) in San Diego, CA 91962 (US) accordance with Art. 76 EPC: • Alvarado, Alberto 07842987.5 / 2 057 178 San Diego, CA 92110 (US) • Frey, Gerhard (71) Applicant: Verenium Corporation San Diego, CA 91129 (US) San Diego CA 92121 (US) (74) Representative: Wakerley, Helen Rachael (72) Inventors: Reddie & Grose LLP • Steer, Brian 16 Theobalds Road San Diego, CA 92109 (US) London WC1X 8PL (GB) • Dycaico, Mark San Diego, CA 92129 (US) Remarks: • Kline, Katie This application was filed on 17-04-2013 as a San Diego, CA 92111 (US) divisional application to the application mentioned under INID code 62. (54) Phytases, nucleic acids encoding them and methods for making and using them (57) This invention relates to phytases, polynucle- otides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high tem- peratures. The phytases of the invention can be thermo- tolerant and/or thermostable at low temperatures, in ad- dition to higher temperatures. The phytases of the inven- tion can be used in foodstuffs to improve the feeding val- ue of phytate rich ingredients. The phytases of the inven- tion can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pel- lets, liquids, powders and the like. In one aspect, phytas- es of the invention are stabile against thermal denatura- tion during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed. EP 2 617 817 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 617 817 A2 Description FIELD OF THE INVENTION 5 [0001] This invention relates to phytases, polynucleotides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be thermotolerant and/or thermostable at low temperatures, in addition to higher temperatures. The phytases of the invention can be used in foodstuffs to 10 improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, liquids, powders and the like. In one aspect, phytases of the invention are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed. 15 BACKGROUND [0002] Minerals are essential elements for the growth of all organisms. Dietary minerals can be derived from many source materials, including plants. For example, plant seeds are a rich source of minerals since they contain ions that 20 are complexed with the phosphate groups of phytic acid molecules. These phytate-associated minerals may, in some cases, meet the dietary needs of some species of farmed organisms, such as multi- stomached ruminants. Accordingly, in some cases ruminants require less dietary supplementation with inorganic phosphate and minerals because micro- organisms in the rumen produce enzymes that catalyze conversion of phytate (myo- inositol-hexaphosphate) to inositol and inorganic phosphate. In the process, minerals that have been complexed with phytate are released. The majority 25 of species of farmed organisms, however, are unable to efficiently utilize phytate- associated minerals. Thus, for example, in the livestock production of monogastric animals e.g.,( pigs, birds, and fish), feed is commonly supplemented with minerals and/or with antibiotic substances that alter the digestive flora environment of the consuming organism to enhance growth rates. [0003] Assuch, there aremany problematic burdens - related tonutrition, ex vivo processing steps, health and medicine, 30 environmental conservation, and resource management - that are associated with an insufficient hydrolysis of phytate in many applications. The following are non-limiting examples of these problems: 1) The supplementation of diets with inorganic minerals is a costly expense. 2) The presence of unhydrolyzed phytate is undesirable and problematic in manyex vivo applications (e.g. by 35 causing the presence of unwanted sludge). 3) The supplementation of diets with antibiotics poses a medical threat to humans and animals alike by increasing the abundance of antibiotic-tolerant pathogens. 4) The discharge of unabsorbed fecal minerals into the environment disrupts and damages the ecosystems of surrounding soils, fish farm waters, and surface waters at large. 40 5) The valuable nutritional offerings of many potential foodstuffs remain significantly untapped and squandered. [0004] Consequently, phytate-containing foodstuffs require supplementation with exogenous nutrients and/or with a source of phytase activity in order to amend their deficient nutritional offerings upon consumption by a very large number of species of organisms. 45 [0005] Consequently, there is a need for means to achieve efficient and cost effective hydrolysis of phytate in various applications. Particularly, there is a need for means to optimize the hydrolysis of phytate in commercial applications. In a particular aspect, there is a need to optimize commercial treatment methods that improve the nutritional offerings of phytate-containing foodstuffs for consumption by humans and farmed animals. 50 SUMMARY OF THE INVENTION [0006] This invention provides polypeptides having phytase activity, polynucleotides encoding them, uses of the poly- nucleotides and polypeptides of the invention, and methods for the production and isolation of such polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having phytase activity under high temperature condi- 55 tions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, tablets, pills, liquids, powders, sprays and the like. In one aspect, phytases of the invention 2 EP 2 617 817 A2 are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed. SUMMARY: 5 [0007] The invention provides isolated, synthetic or recombinant nucleic acids comprising (a) (i) a nucleic acid sequence having at least 95%, 96% 97%, 98% or 99% sequence identity to SEQ ID NO: 1, and comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all 10 fourteen of the nucleotide base pair sequence modifications to SEQ ID NO: 1, selected from the group consisting of: the nucleotides at positions 139 to 141 are TTT or TTC; the nucleotides at positions 289 to 291 are GTT, GTC, GTA or GTG; the nucleotides at positions 289 to 291 are GAA or GAG; the nucleotides at positions 406 to 408 are CAT or CAC; the nucleotides at positions 475 to 477 are GTT, GTC, GTA or GTG; the nucleotides at positions 475 to 477 are GAA or GAG; the nucleotides at positions 487 to 489 are CGT, CGC, CGA, CGG, 15 AGA or AGG; the nucleotides at positions 490 to 492 are CGT, CGC, CGA, CGG, AGA or AGG; the nucleotides at positions 502 to 504 are CGT, CGC, CGA, CGG, AGA or AGG; the nucleotides at positions 535 to 537 are CGT, CGC, CGA, CGG, AGA or AGG; the nucleotides at positions 676 to 678 are GAT or GAC; the nucleotides at positions 697 to 699 are TGG; the nucleotides at positions 823 to 825 are GTT, GTC, GTA or GTG; the nucleotides at positions 865 to 867 are GCT, GCC, GCA or GCG; the nucleotides at positions 1045 to 1047 20 are TAT or TAC; and the nucleotides at positions 1087 to 1089 are CCA, CCC, CCG or CCT; or, (ii) a nucleic acid sequence having at least 95%, 96% 97%, 98% or 99% sequence identity to SEQ ID NO:1, and comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all fourteen of the nucleotide base pair sequence modifications to SEQ ID NO: 1, selected from the group consisting of: the nucleotides at the equivalent of positions 139 to 141 are TTT or TTC; the nucleotides at