	US 12,227,783 B2
	Metabolic engineering for microbial production of terpenoid products
Ajikumar Parayil Kumaran, Watertown, MA (US); Chin-Giaw Lim, Allston, MA (US); Souvik Ghosh, Walnut Creek, CA (US); Christopher Pirie, Seattle, WA (US); Jason Donald, Lexington, MA (US); Aaron Love, Boston, MA (US); Hong Nan, Daly City, CA (US); Hsien-Chung Tseng, Cambridge, MA (US); Christine Nicole S. Santos, Newton, MA (US); and Ryan Philippe, Somerville, MA (US)
Assigned to Manus Bio Inc., Waltham, MA (US)
Filed by MANUS BIO INC., Cambridge, MA (US)
Filed on Apr. 26, 2022, as Appl. No. 17/729,056.
Application 17/729,056 is a continuation of application No. 16/862,209, filed on Apr. 29, 2020, granted, now 11,339,412.
Application 16/862,209 is a continuation of application No. 15/888,573, filed on Feb. 5, 2018, granted, now 10,662,442, issued on May 26, 2020.
Claims priority of provisional application 62/454,121, filed on Feb. 3, 2017.
Prior Publication US 2022/0364127 A1, Nov. 17, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 5/00 (2006.01); C12N 9/02 (2006.01); C12N 9/04 (2006.01); C12N 9/10 (2006.01); C12P 7/02 (2006.01); C12P 7/26 (2006.01); C12P 7/40 (2006.01); C12P 17/02 (2006.01)

CPC C12P 5/007 (2013.01) [C12N 9/0006 (2013.01); C12N 9/0093 (2013.01); C12N 9/1085 (2013.01); C12Y 101/01267 (2013.01); C12Y 117/07001 (2013.01); C12Y 205/0109 (2013.01); C12P 7/02 (2013.01); C12P 7/26 (2013.01); C12P 7/40 (2013.01); C12P 17/02 (2013.01)]

31 Claims

1. A method for production of a terpene or terpenoid, comprising:

providing a bacterial strain that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an upstream methylerythritol pathway (MEP) and converts the IPP and DMAPP to a terpene or terpenoid through a downstream synthesis pathway; and

culturing the bacterial strain in culture media to produce the terpene or terpenoid, wherein greater than 15% of carbon entering glycolysis becomes MEP carbon;

wherein the bacterial strain overexpresses 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) and 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), such that expression levels of Dxr and IspE in the bacterial strain limits accumulation of each of 1-deoxy-D-xylulose (DOX) and 2-C-methyl-D-erythritol (ME) in the culture media to 1 g/L or less;

wherein the bacterial strain further overexpresses 1-hydroxy-2-methyl-2-E-butenyl 4-diphosphate synthase (IspG) and 1-hydroxy-2-methyl-2-(E) 4-diphosphate reductase (IspH), such that expression levels of IspG and IspH in the bacterial cell limits accumulation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) in the culture media.