US 12,480,146 B2
Microbial production of mogrol and mogrosides
Michelle N. Goettge, Cambridge, MA (US); Ryan N. Philippe, Orleans (CA); Ajikumar Parayil Kumaran, Cambridge, MA (US); Christine Nicole S. Santos, Cambridge, MA (US); Jason Eric Donald, Cambridge, MA (US); Christopher Frei, Cambridge, MA (US); Aaron Love, Cambridge, MA (US); Kaitlin Chambers, Cambridge, MA (US); Joyce Samson, Cambridge, MA (US); and Christopher Toomey, Cambridge, MA (US)
Assigned to Manus Bio Inc., Cambridge, MA (US)
Appl. No. 17/785,488
Filed by Manus Bio Inc., Cambridge, MA (US)
PCT Filed Dec. 16, 2020, PCT No. PCT/US2020/065285
§ 371(c)(1), (2) Date Jun. 15, 2022,
PCT Pub. No. WO2021/126960, PCT Pub. Date Jun. 24, 2021.
Claims priority of provisional application 63/075,631, filed on Sep. 8, 2020.
Claims priority of provisional application 63/085,557, filed on Sep. 30, 2020.
Claims priority of provisional application 62/948,657, filed on Dec. 16, 2019.
Prior Publication US 2023/0042171 A1, Feb. 9, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 17/02 (2006.01); C12N 9/02 (2006.01); C12N 9/14 (2006.01); C12P 5/00 (2006.01); C12P 19/18 (2006.01); C12P 19/56 (2006.01)
CPC C12P 17/02 (2013.01) [C12N 9/0077 (2013.01); C12N 9/14 (2013.01); C12P 5/007 (2013.01); C12P 19/18 (2013.01); C12P 19/56 (2013.01); C12Y 303/0201 (2013.01); C12Y 504/99008 (2013.01)] 20 Claims
 
1. A method for making mogrol or mogroside, comprising:
providing a recombinant microbial host cell expressing a heterologous enzyme pathway catalyzing the conversion of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to mogrol or mogroside, the heterologous enzyme pathway comprising:
a farnesyl diphosphate synthase (FPPS),
a squalene synthase (SQS),
a squalene epoxidase (SQE) catalyzing the formation of 2,3;22,23-dioxidosqualene from squalene,
a triterpene cyclase (TTC) catalyzing the formation of 24,25-epoxycucurbitadienol from 2,3;22,23-dioxidosqualene,
an epoxide hydrolase (EPH) catalyzing the formation of 24,25-dihydroxycucurbitadienol from 24,25-epoxycucurbitadienol, and
a cytochrome P450 enzyme catalyzing the formation of mogrol from 24,25-dihydroxycucurbitadienol;
wherein the cytochrome P450 enzyme comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 171; and
culturing the host cell under conditions for producing the mogrol or mogroside.