US 12,264,354 B2
Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1,4-butanediol related thereto
Anthony P. Burgard, Bellefonte, PA (US); Robin E. Osterhout, San Diego, CA (US); Stephen J. Van Dien, Encinitas, CA (US); Cara Ann Tracewell, Solana Beach, CA (US); Priti Pharkya, San Diego, CA (US); and Stefan Andrae, San Diego, CA (US)
Assigned to Genomatica, Inc., San Diego, CA (US)
Filed by Genomatica, Inc., San Diego, CA (US)
Filed on Apr. 14, 2017, as Appl. No. 15/488,320.
Application 15/488,320 is a continuation of application No. 13/975,678, filed on Aug. 26, 2013, granted, now 9,657,316.
Claims priority of provisional application 61/766,609, filed on Feb. 19, 2013.
Claims priority of provisional application 61/693,683, filed on Aug. 27, 2012.
Prior Publication US 2018/0030484 A1, Feb. 1, 2018
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 7/18 (2006.01); C07D 207/12 (2006.01); C08G 18/32 (2006.01); C08G 63/06 (2006.01); C08G 63/12 (2006.01); C08G 63/66 (2006.01); C08G 69/26 (2006.01); C12N 9/04 (2006.01); C12N 9/06 (2006.01); C12N 9/10 (2006.01); C12N 9/88 (2006.01); C12P 7/24 (2006.01); C12P 19/02 (2006.01)
CPC C12P 7/18 (2013.01) [C07D 207/12 (2013.01); C08G 18/3206 (2013.01); C08G 63/06 (2013.01); C08G 63/12 (2013.01); C08G 63/66 (2013.01); C08G 69/26 (2013.01); C12N 9/0006 (2013.01); C12N 9/0028 (2013.01); C12N 9/1007 (2013.01); C12N 9/88 (2013.01); C12P 7/24 (2013.01); C12P 19/02 (2013.01); C12Y 101/01244 (2013.01); C12Y 101/02007 (2013.01); C12Y 105/0102 (2013.01); C12Y 201/01 (2013.01); C12Y 101/01224 (2013.01); C12Y 201/0109 (2013.01); C12Y 402/01 (2013.01); Y02P 20/52 (2015.11)] 15 Claims
 
1. A method of producing an intermediate of a metabolic pathway that can be used in the formation of biomass, comprising culturing a non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce the intermediate,
wherein the non-naturally occurring microbial organism has a methanol metabolic pathway and comprises at least three exogenous nucleic acids each encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, or metabolize methanol as a carbon source for biosynthesis of formaldehyde, wherein said methanol metabolic pathway comprises:
(i) a methanol methyltransferase, which catalyzes the conversion of methanol to methyltetrahydrofolate (methyl-THF),
(ii) a methylenetetrahydrofolate reductase, which catalyzes the conversion of methyl-THF to methylene-THF, and
(iii) a methylenetetrahydrofolate dehydrogenase, which catalyzes the conversion of methylene-THF to methenyl-THF; and
wherein the non-naturally occurring microbial organism has a formaldehyde assimilation pathway and comprises at least two exogenous nucleic acids each encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of a metabolic pathway that can be used in the formation of biomass, wherein the formaldehyde assimilation pathway comprises:
(i) a hexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase; or
(ii) a dihydroxyacetone synthase and a dihydroxyacetone kinase.