US 11,859,225 B2
Methods of producing epimerases and benzylisoquinoline alkaloids
Christina D. Smolke, Menlo Park, CA (US); Stephanie Galanie, Palo Alto, CA (US); Isis Trenchard, Redwood City, CA (US); Catherine Thodey, Mountain View, CA (US); and Yanran Li, Mountain View, CA (US)
Assigned to The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
Appl. No. 15/567,358
Filed by The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
PCT Filed May 9, 2016, PCT No. PCT/US2016/031506
§ 371(c)(1), (2) Date Oct. 17, 2017,
PCT Pub. No. WO2016/183023, PCT Pub. Date Nov. 17, 2016.
Claims priority of provisional application 62/174,475, filed on Jun. 11, 2015.
Claims priority of provisional application 62/159,122, filed on May 8, 2015.
Prior Publication US 2018/0163241 A1, Jun. 14, 2018
Int. Cl. C12P 17/12 (2006.01); C12N 15/52 (2006.01); C12P 41/00 (2006.01); C12N 9/04 (2006.01); C12N 9/02 (2006.01); C12N 15/62 (2006.01)
CPC C12P 17/12 (2013.01) [C12N 9/0006 (2013.01); C12N 9/0071 (2013.01); C12N 15/52 (2013.01); C12N 15/62 (2013.01); C12P 41/002 (2013.01); C12Y 101/01247 (2013.01); C12N 2800/22 (2013.01)] 24 Claims
 
1. A method of producing an (R)-1-benzylisoquinoline alkaloid, the method comprising:
(a) culturing an engineered non-plant cell in a cell culture comprising a feedstock, wherein the engineered non-plant cell stably expresses a plurality of heterologous enzymes within the same engineered non-plant-cell for the production of an (S)-1-benzylisoquinoline alkaloid wherein the plurality of heterologous enzymes comprise tyrosine hydroxylase (TyrH), L-DOPA decarboxylase (DODC), norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), cytochrome P450 80B1 (CYP80B1), and 4′-O-methyltransferase (4′OMT),
(b) producing an (S)-1-benzylisoquinoline alkaloid within the same engineered non-plant cell,
(c) contacting, within the same engineered non-plant cell, the (S)-1-benzylisoquinoline alkaloid that is produced within the engineered non-plant cell with at least one heterologous, fused enzyme expressed within the same engineered non-plant cell that converts the (S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinoline alkaloid within the engineered non-plant cell, and
(d) producing the (R)-1-benzylisoquinoline alkaloid within the engineered non-plant cell.