US 12,312,627 B2
Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds
Priti Pharkya, San Diego, CA (US); Anthony P. Burgard, Bellefonte, PA (US); Stephen J. Van Dien, Encinitas, CA (US); Robin E. Osterhout, San Diego, CA (US); Mark J. Burk, San Diego, CA (US); John D. Trawick, La Mesa, CA (US); Michael P. Kuchinskas, Escondido, CA (US); Brian Steer, San Diego, CA (US); Stefan Andrae, San Diego, CA (US); and Amit Shah, San Diego, CA (US)
Assigned to Genomatica, Inc., San Diego, CA (US)
Filed by Genomatica, Inc., San Diego, CA (US)
Filed on Jun. 23, 2016, as Appl. No. 15/191,421.
Application 15/191,421 is a continuation of application No. 14/262,461, filed on Apr. 25, 2014, abandoned.
Claims priority of provisional application 61/854,611, filed on Apr. 26, 2013.
Prior Publication US 2017/0183694 A1, Jun. 29, 2017
Int. Cl. C12P 7/18 (2006.01); C07C 31/20 (2006.01); C07C 47/19 (2006.01); C07C 211/09 (2006.01); C07D 307/33 (2006.01); C12N 1/20 (2006.01); C12N 9/04 (2006.01); C12N 9/10 (2006.01); C12N 15/52 (2006.01); C12P 7/24 (2006.01); C12P 7/42 (2006.01); C12P 7/62 (2022.01); C12P 13/00 (2006.01); C12P 17/04 (2006.01); C12P 19/32 (2006.01)
CPC C12P 7/18 (2013.01) [C07C 31/207 (2013.01); C07C 47/19 (2013.01); C07C 211/09 (2013.01); C07D 307/33 (2013.01); C12N 1/20 (2013.01); C12N 9/0006 (2013.01); C12N 9/13 (2013.01); C12N 15/52 (2013.01); C12P 7/24 (2013.01); C12P 7/42 (2013.01); C12P 7/62 (2013.01); C12P 13/001 (2013.01); C12P 13/005 (2013.01); C12P 17/04 (2013.01); C12P 19/32 (2013.01); C12Y 208/03 (2013.01)] 17 Claims
 
1. An isolated nucleic acid molecule selected from:
(a) a nucleic acid molecule encoding a variant amino acid sequence of a parent polypeptide set forth in any one of:
SEQ ID NOS: 439-518, wherein said variant amino acid sequence comprises two or more variant amino acid positions selected from a group consisting of: 12L, 15P, 34K, 42D, 44L, 47T, 51R, 51K, 56G, 57A, 58C, 58L, 58H, 59G, 66F, 67S, 67A, 71A, 91R, 104R, 106T, 107V, 127I, 128N, 129A, 129G, 129Y, 129I, 131W, 132A, 133M, 133V, 135P, 136A, 139S, 139T, 142L, 156C, 158V, 159G, 164A, 167P, 172G, 178G, 184M, 187K, 188G, 200M, 204L, 204R, 208A, 208L, 211C, 215M, 215A, 217Q, 226R, 227L, 227I, 229A, 230V, 230R, 230Q, 231I, 233Q, 233V, 234G, 241S, 242S, 242D, 243R, 243N, 243E, 248T, 252S, 253F, 253L, 253V, 253A, 258R, 259T, 260S, 260T, 260C, 265I, 265C, 265W, 265V, 266S, 269L, 2731, 273T, 273S, 273M, 273N, 279T, 280S, 281L, 284P, 288L, 289K, 291V, 294V, 295F, 298L, 310L, 310P, 317A, 317T, 327K, 333A, 349W, 360E, 370L, 370H, 395P, 396H, 396K, 396W, 396V, 398F, 401M, 406H, 411W, 416G, 416S, 417A, 419S, 426G, 427L, 427F, 427V, 429L, 429M, 437P, 439H, 441S, 446V, 446I, 447P, 456A, 464T, 464S, 466N, 468N, 47V, 113N, 177Y, 193L, 260V, 294D, 353N, 355A, 383Y, 13L, 16P, 32K, 40D, 42L, 45T, 45V, 49R, 49K, 54G, 55A, 56C, 56L, 56H, 57G, 64F, 65S, 65A, 69A, 89R, 102R, 104T, 105V, 111N, 125I, 126N, 127A, 127G, 127Y, 129W, 130A, 131M, 131V, 133P, 134A, 137S, 137T, 140L, 154C, 156V, 157G, 162A, 165P, 170G, 175Y, 176G, 182M, 185K, 186G, 191L, 198M, 202L, 202R, 206A, 206L, 209C, 213M, 213A, 215Q, 224R, 225I, 227A, 228V, 228R, 228Q, 229I, 231Q, 231V, 232G, 239S, 240S, 240D, 241R, 241N, 241E, 246T, 250S, 251F, 251L, 251V, 251A, 256R, 257T, 258S, 258T, 263I, 263C, 263W, 263V, 264S, 267L, 271I, 271T, 271S, 271M, 271N, 277T, 278S, 279L, 282P, 286L, 287K, 289V, 292V, 292D, 293F, 296L, 308L, 308P, 316A, 316T, 326K, 332A, 348W, 352N, 354A, 359E, 369L, 369H, 382Y, 394P, 395H, 395K, 395W, 395V, 397F, 400M, 405H, 133R, 146R, 148T, 149V, 155N, 170N, 171A, 171G, 171Y, 171I, 173W, 174A, 175M, 175V, 177P, 178A, 181S, 181T, 184L, 198C, 200V, 201G, 209P, 214G, 219Y, 220G, 226M, 229K, 230G, 235L, 242M, 246R, 250A, 250L, 253C, 257M, 257A, 259Q, 268R, 269I, 271A, 272V, 272R, 272Q, 275Q, 275V, 276G, 283S, 284S, 284D, 285R, 285N, 285E, 290T, 294S, 295L, 295V, 295A, 300R, 301T, 302S, 302T, 302C, 302V, 307I, 307C, 307W, 307V, 308S, 311L, 315I, 315T, 315S, 315M, 315N, 321T, 322S, 323L, 326P, 330L, 331K, 333V, 336V, 336D, 337F, 340L, 352L, 352P, 359A, 359T, 379K, 385A, 401, 405N, 407A, 412E, 55C, 55H, 55L, 63F, 64S, 64A, 68A, 88R, 101R, 103T, 104V, 110N, 124I, 125N, 126A, 126G, 126Y, 126I, 128W, 130M, 130V, 132P, 133A, 136S, 136T, 139L, 153C, 155V, 156G, 160A, 169G, 174Y, 175G, 181M, 184K, 185G, 190L, 197M, 201L, 201R, 205A, 205L, 208C, 212M, 212A, 214Q, 223R, 224L, 224I, 226A, 227V, 227R, 227Q, 228I, 231G, 238S, 239D, 240R, 240N, 240E, 245T, 248S, 250F, 250V, 255R, 256T, 257S, 257C, 257V, 262I, 262C, 262W, 262V, 263S, 266L, 270I, 270T, 270S, 270M, 270N, 276T, 277S, 278L, 281P, 285L, 286K, 288V, 291D, 292F, 314A, 334K, 340A, 357W, 360N, 362A, 367E, 377L, 377H, 388Y, 402P, 403H, 403K, 403W, 403V, 405F, and 408M; wherein the variant amino acid sequence can convert 4-hydroxybutyryl-CoA (4HB-CoA) to 4-hydroxybutyraldehyde (4-HBAld); and
(b) a nucleic acid molecule that is complementary to the nucleic acid molecule of (a);
wherein other than the two or more variant amino acid positions, the variant amino acid sequence has at least 90% sequence identity to any one of the parent polypeptide amino acid sequences of SEQ ID NOS: 439-518; and
wherein the variant amino acid sequence is not the sequence of the parent polypeptide or a naturally occurring amino acid sequence.