	US 11,987,824 B2
	Additional endoglucanase variants and methods
Jie Yang, Foster City, CA (US); Xiyun Zhang, San Ramon, CA (US); William Buchanan Porterfield, Oakland, CA (US); Wenhua Lu, Dublin, CA (US); Khin Oo, Daly City, CA (US); Goutami Banerjee, Daly City, CA (US); Tatsuya Fukushima, Fremont, CA (US); and Eric Lin Hu, Millbrae, CA (US)
Assigned to FORNIA BIOSOLUTIONS, INC., Hayward, CA (US)
Filed by Fornia BioSolutions, Inc., Hayward, CA (US)
Filed on Dec. 20, 2021, as Appl. No. 17/556,504.
Claims priority of provisional application 63/129,143, filed on Dec. 22, 2020.
Prior Publication US 2022/0204956 A1, Jun. 30, 2022
Int. Cl. C12N 9/42 (2006.01); C12N 15/63 (2006.01); D21H 17/00 (2006.01)

CPC C12N 9/2437 (2013.01) [C12N 15/63 (2013.01); C12Y 302/01004 (2013.01); D21H 17/005 (2013.01)]

19 Claims

1. A composition comprising a variant endoglucanase enzyme comprising one or more amino acid modifications as compared to SEQ ID NO:1, wherein said one or more amino acid modifications occur at positions corresponding to positions selected from the group consisting of positions 2, 17, 23, 25, 29, 33, 36, 38, 39, 42, 43, 44, 48, 51, 54, 66, 67, 68, 75, 77, 80, 82, 90, 102, 116, 121, 122, 123, 132, 135, 136, 141, 144, 145, 153, 160, 161, 164, 167, 174, 180, 184, 185, 190, 192, 194, 195, 196, 204, 205, 206, 215, 216, 217, 218, 219, 220, 221, 222, 223, 228, 229, 230, 231, 232, 233, 234, 235, 237, 238, 239, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 258, 259, 267, 268, 269, 270, 271, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, and 293, wherein said variant endoglucanase enzyme exhibits at least 80% sequence identity to SEQ ID NO:1; wherein said variant endoglucanase enzyme has endoglucanase activity; and

wherein said variant endoglucanase enzyme comprises said amino acid modification(s) selected from the group consisting of D215*,

A23D/I38L/Y39S/G42N/A43E/K44E/E48N/S54T/A77Q/N116D/N122S/L141S/L281G, A23D/I38L/Y39S/G42N/A43E/K44Q/E48N/S54T/N116E/W258S/A259D/G275E/T277D, A23D/I38L/Y39S/G42N/A43S/K44E/E48N/A77E/N116S/N122A/N180T/W258S/A259D/G275E/T277D/I29 1S/L293T, A23D/I38L/Y39S/G42N/A43S/K44E/E48N/S54T/A77E/N122S/P164T/N180T/A259D/S286A/L293T, A23D/I38L/Y39S/G42N/A43S/K44Q/E48N/A77Q/N116E/L141S/G275E/T277D/W284R, A23D/I38L/Y39S/G42N/A43S/K44Q/E48N/S54T/N116D/W258S/G275T/L281E/1291Q, A23D/I38L/Y39S/G42N/A43S/K44Q/E48N/V102Q/W258S/L281E/N282S/I291A, A23D/I38L/Y39S/G42N/A43S/K44Q/E48N/V102R/W258R/A259D/L281E/N282S, A23D/I38L/Y39S/G42N/A43T/K44E/E48N/P164T/N180T/A194T/W258S/A259D/G275T/L281G, A23D/I38L/Y39S/G42N/A43T/K44E/E48N/V102Q/N116D/L281G/N282S, A23D/I38L/Y39S/G42N/A43T/K44Q/E48N/A77E/V102Q/N116D/W258S/G275T/L281E/1291A, A23D/I38L/Y39S/G42N/A43T/K44Q/E48N/A77Q/N180T/W258S/G275E/1291Q, A23D/I38L/Y39S/G42N/A43V/K44E/E48N/A77E/N116D/G275T/L281E, A23D/I38L/Y39S/G42N/A43V/K44E/E48N/N122A/N180T/T277D/L281E/N282S, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/A161K/P164T/N180T, A23D/ILK/Y39S/G42N/A43V/K44Q/E48N/A77E/A161K/P164T/N180T, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E/N116D/W258S/L281E, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/A77Q/N116S/N180T/G275E/T277E, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/A77Q/N180T, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/F67Y/A77Q/N116S/N180T/W258S/A259D/G275T/T277E/L28 1K/N282S/1291S/L293N, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/F67Y/A77Q/N122S/L141S/N180T/A194T/W258R/L281K, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/F67Y/N180T, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/L141S, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/N116D/G275T/L281E/N282S/1291A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/N116D/L281K/N282S/1291S/N292S/L293N, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/N116D/W258S/L281G, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/N180T, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/N180T/A259D/G275T/N282S, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/Q66T/A77Q/N122A/L141S/G275T/W284G/S286A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/A77E/A161P/W258S/W284M/1291Q, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/A77E/T277E/L281E/S286A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/A77E/V102Q/G275T/T277D/W284M/1291Q, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/A77E/W258S/T277D/W284M/1291A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/1291A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/L281E, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/A161P/L281E/N282S, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/A161P/W258S/G275T/L281E/1291A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/A161P/W258S/W284M/1291A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/D215G/S216N/P217G/S218G/S219T/S220G/S22 1-/A222-/A223T/S228A/T229P/S230G/Q231S/Q232G/P233Q/Q234T/Q235S/T237G/S238G/S239G/S241-/Q242-/A243-/S244-/V245-/P246-/T247-/S248-/N249-/P250-/G251-/T267S/T279Q/W284Y/T289-/M290-/1291-/N292-, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/L141S/A161K/N180T/W258R/A259D/G275E/T2 77D/W284R/1291Q/N292S/L293G, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/L141S/N180T/G275T/W284R/1291Q/L293N, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/W258S/G275T/N282S/1291A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/W284M/1291Q, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116E/L141S/N180T/L281G/N282S, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116S/G275E/Y285*, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/Q66T/F67Y/N122S/N180T/L281G/1291S/N292S/L293G, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/V102Q/N116D/A161P/T277D/W284M/1291Q, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/V102Q/N116D/W258S/G275T/1291Q, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/V102Q/N116D/W258S/G275T/T277E, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/V102Q/N116D/W258S/G275T/T277E/L281G, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/V102Q/N116D/W258S/L281G/1291A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/V102Q/W258S/G275T/T277D/N282S, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/V102Q/W258S/W284M/S286A, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/W258S, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/V102Q/A161P/W258S, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/V102Q/W258S/1291Q, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/V102R/N180T, A23D/I38L/Y39S/G42N/A43V/K44Q/E48N/W258S/1291A, A23D/I38L/Y39S/G42N/K44Q/E48N/A77E/N116D/L281E/1291A, A23D/S25N/I38L/Y39S/G42N/A43E/K44E/E48N/V102R/N116S/N122A/L141S/A161K/P164T/A194T/W25 8R/A259D/L281E, A23D/S25N/I38L/Y39S/G42N/A43E/K44Q/E48N/Q66T/F67Y/L141S/N180T, A23D/S25N/I38L/Y39S/G42N/A43S/K44Q/E48N/S54T/Q66T/L141S/N180T/W258S/A259D/T277D/W284 G/Y285*, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A161K/N180T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A161P, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E/N180T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E/N180T/A259D/L281K/L293T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E/V102Q/N180T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77Q/N180T/A194T/W258S/W284G/S286G, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77Q/V102Q/N180T/A194T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77Q/W258S/G275T/T277D/W284M/1291S/L293T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/F67Y/A77E/A161P/P164T/N180T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/F67Y/N180T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/L281E/N282S/N292S/L293G, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/N116D/A194T/W258S/A259D/G275T/W284G/S286G/L 293T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/N116E/N122S/A194T/T277L/C278A/T279P/K280S/L281*, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/N180T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/Q66T/F67Y/A77Q/N180T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/Q66T/N122S/L141S/W284R/C288*, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/A77Q/N116D/L141S/W258S/A259D/G275T/L281E/N292S/L293T, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/L281E/N282S/C288A/T289Q/M290*, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/A161P/N180T/W258R/G275E/T277E/1291 S/L293G, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N180T/L281E/N282S/C288A/T289Q/M290*, A23D/S25N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/Q66T/F67Y/A77Q/N122S/L141S/W258R/N282S, A23D/S25N/I38L/Y39S/G42N/K44E/E48N/A161P/A194T/G275E/T277D, A23D/S25N/I38L/Y39S/G42N/K44Q/E48N/S54T/A77E/V102R/N122S/L141S/A259D/W284G/1291S/L293T, A23N/I38L/Y39S/G42N/A43E/K44E/E48N/S54T/A77E/V102Q/N116D/W258S/W284M/1291Q, A23N/I38L/Y39S/G42N/A43E/K44Q/E48N/S54T/A77E/A161P/G275T/N282S/1291Q, A23N/I38L/Y39S/G42N/A43E/K44Q/E48N/S54T/A77E/N116D/W284M/S286A/1291Q, A23N/I38L/Y39S/G42N/A43E/K44Q/E48N/S54T/V102Q/N116D/W258S/G275T/L281E/N282S/1291A, A23N/I38L/Y39S/G42N/A43L/K44Q/E48N/Q66T/F67Y/A77E/V102Q/N116D/L141S/N180T/W258R/1291S, A23N/I38L/Y39S/G42N/A43S/K44E/E48N/S54T/A77E/L141S/W258S/G275T, A23N/I38L/Y39S/G42N/A43S/K44Q/E48N/S54T/A77E/1291Q, A23N/I38L/Y39S/G42N/A43S/K44Q/E48N/S54T/A77E/N116E/N180T/W258S/W284R/S286G, A23N/I38L/Y39S/G42N/A43S/K44Q/E48N/S54T/N116D/S286A/1291A, A23N/I38L/Y39S/G42N/A43S/K44Q/E48N/S54T/Q66T/F67Y/N116D/L141S, A23N/I38L/Y39S/G42N/A43V/K44E/E48N/A77Q/N122S/L141S/N180T/W284M/S286A, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/A161P/G275T/L281E/N282S/1291A, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/A161P/N180T, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E/N116D/R195S/W258S/T277E/N282S/1291A, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E/N180T, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E/N180T/A194T, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/A77E/V102Q/W284M/S286A/1291A, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/F67Y/A77Q/N116D/N180T/G275E/T277D/W284R/1291S/L293 N, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/N180T, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/A161P/G275T/L281G/1291Q, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/A77E/V102Q/A161P/W258S/W284M, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/A161P/G275T/L281E, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/N116D/A161P/P164T/G275E/L281E, A23N/I38L/Y39S/G42N/A43V/K44Q/E48N/S54T/Q66T/F67Y/A161P/W284M/1291Q, 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