MICROORGANISMS AND METHODS FOR PRODUCTION OF 4-HYDROXYBUTYRATE, 1,4-BUTANEDIOL AND RELATED COMPOUNDS

• US 20140030779A1
• Filed: 06/03/2013
• Published: 01/30/2014
• Est. Priority Date: 06/04/2012
• Status: Active Grant

• Current Assignee: Genomatica Incorporated
• Original Assignee: Genomatica Incorporated
• Inventors: PHARKYA, Priti, BURGARD, Anthony P., OSTERHOUT, Robin E., BURK, Mark J., TRAWICK, John D., STEER, Brian, VAN DIEN, Stephen J., KUCHINSKAS, Michael P.
• Granted Patent: US 9,677,045 B2
• Time in Patent Office: Days
• US Class Current: 435/146
• CPC Class Codes: C07C 31/207 1,4-Butanediol; 1,3-Butanediol; 1,2-Butanediol; 2,3-Butanediol
• CPC Class Codes: C07C 59/01 Saturated compounds having only one carboxyl group and containing hydroxy or O-metal groups
• CPC Class Codes: C08G 18/284 Compounds containing ester groups, e.g. oxyalkylated monocarboxylic acids
• CPC Class Codes: C08G 18/3206 aliphatic
• CPC Class Codes: C08G 63/06 derived from hydroxycarboxylic acids
• CPC Class Codes: C08G 63/16 Dicarboxylic acids and dihydroxy compounds
• CPC Class Codes: C08G 63/181 Acids containing aromatic rings
• CPC Class Codes: C08G 69/02 Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
• CPC Class Codes: C12N 1/14 Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• CPC Class Codes: C12N 1/16 Yeasts; Culture media therefor
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12Y 301/01 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention provides non-naturally occurring microbial organisms having a 4-hydroxybutyrate pathway and being capable of producing 4-hydroxybutyrate, wherein the microbial organism comprises one or more genetic modifications. The invention additionally provides methods of producing 4-hydroxybutyrate or related products using the microbial organisms.

Claims

1. A non-naturally occurring microbial organism, said microbial organism having a 4-hydroxybutyrate pathway and being capable of producing 4-hydroxybutyrate, wherein said microbial organism comprises a genetic modification, said genetic modification selected from:

(A) a genetic modification that increases expression of phosphoenolpyruvate carboxylase;

(B) a genetic modification that increases expression of alpha-ketoglutarate dehydrogenase;

(C) a genetic modification that increases expression of a non-phosphotransferase (PTS) glucose uptake system;

(D) a genetic modification that increases expression of a gamma-butyrolactone esterase;

(E) a genetic modification that decreases expression of succinyl-CoA synthetase;

(F) a genetic modification that decreases expression of an acyl coenzyme A thioesterase;

(G) a genetic modification that decreases expression of an alcohol dehydrogenase

(H) a genetic modification that decreases expression of a non-energy-producing NADH dehydrogenase;

(I) a genetic modification that decreases expression of a cytochrome oxidase; and

(J) a combination of two or more of the genetic modifications of parts (A)-(I).

2. The microbial organism of claim 1, wherein:

(K) the microbial organism of part (A), (B) or (C) has decreased production of ethanol, acetate, pyruvate or alanine, or a combination thereof, relative to a parent microbial organism in the absence of said genetic modification;

(L) the microbial organism of part (B) has decreased production of glutamate relative to a parent microbial organism in the absence of said genetic modification;

(M) the microbial organism of part (C) has a genetic modification comprising increased expression of a permease, glucokinase, or a glucose facilitator, or a combination thereof;

(N) the microbial organism of part (D) has decreased production of gamma-butyrolactone relative to a parent microbial organism in the absence of said genetic modification;

(O) the microbial organism of part (E) has increased production of 4-hydroxybutyrate relative to a parent microbial organism in the absence of said genetic modification;

(P) the microbial organism of part (F) has decreased production of gamma-butyrolactone relative to a parent microbial organism in the absence of said genetic modification;

(Q) the microbial organism of part (F) has a genetic modification comprising at least two genetic modifications that decrease expression of at least two acyl coenzyme A thioesterases;

(R) the microbial organism of part (G) has decreased backflux from a downstream product of the 4-hydroxybutyrate pathway relative to a parent microbial organism in the absence of said genetic modification;

(S) the microbial organism of part (H) has suppressed depletion of the NADH pool or increased energy efficiency in the microbial organism, or a combination thereof, relative to a parent microbial organism in the absence of said genetic modification;

(T) the microbial organism of part (I) has increased energy efficiency relative to a parent microbial organism in the absence of said genetic modification;

or

(U) the microbial organism of part (I) has increased tolerance to a range of oxygen concentrations relative to a parent microbial organism in the absence of said genetic modification.

3. The microbial organism of claim 1, wherein said microbial organism of parts (D) or (F) further comprises a 4-hydroxybutyryl-CoA pathway.

4. A method for producing 4-hydroxybutyrate, comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce 4-hydroxybutyrate.

5. The method of claim 4, wherein:

(K) the microbial organism of part (A), (B) or (C) has decreased production of ethanol, acetate, pyruvate or alanine, or a combination thereof, relative to a parent microbial organism in the absence of said genetic modification;

(L) the microbial organism of part (B) has decreased production of glutamate relative to a parent microbial organism in the absence of said genetic modification;

(M) the microbial organism of part (C) has a genetic modification comprising increased expression of a permease, glucokinase, or a glucose facilitator, or a combination thereof;

(N) the microbial organism of part (D) has decreased production of gamma-butyrolactone relative to a parent microbial organism in the absence of said genetic modification;

(O) the microbial organism of part (E) has increased production of 4-hydroxybutyrate relative to a parent microbial organism in the absence of said genetic modification;

(P) the microbial organism of part (F) has decreased production of gamma-butyrolactone relative to a parent microbial organism in the absence of said genetic modification;

(Q) the microbial organism of part (F) has a genetic modification comprising at least two genetic modifications that decrease expression of at least two acyl coenzyme A thioesterases;

(R) the microbial organism of part (G) has decreased backflux from a downstream product of the 4-hydroxybutyrate pathway relative to a parent microbial organism in the absence of said genetic modification;

(S) the microbial organism of part (H) has suppressed depletion of the NADH pool or increased energy efficiency in the microbial organism, or a combination thereof, relative to a parent microbial organism in the absence of said genetic modification;

(T) the microbial organism of part (I) has increased energy efficiency relative to a parent microbial organism in the absence of said genetic modification;

or

(U) the microbial organism of part (I) has increased tolerance to a range of oxygen concentrations relative to a parent microbial organism in the absence of said genetic modification.

6. The method of claim 4, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.