Arginine supplementation to improve efficiency in gas fermenting acetogens

Patent metadata

Field	Value
Publication number	US-2017159083-A1
Application number	US-201615368521-A
Country	US
Kind code	A1
Filing date	Dec 2, 2016
Priority date	Dec 3, 2015
Publication date	Jun 8, 2017
Grant date	—

Abstract

The invention provides methods for improving efficiency of fermentation by arginine supplementation, and genetically modified bacterium for use therefor. More particularly the invention provides methods for (i) increasing the production ATP intensive products with arginine supplementation, (ii) increasing utilization of arginine by a C1-fixing bacterium; and (iii) providing C1-fixing bacterium with optimized arginine de-aminase pathways.

First claim

1 . A method for increasing the production of at least one ATP-intensive product, the method comprising;
a. flowing a gaseous C1-containing substrate to a bioreactor containing a culture of a C1-fixing microorganism in a liquid nutrient media; and b. fermenting the culture to produce at least one product; wherein arginine is provided to the culture in excess of the cellular requirement of the culture; and wherein the C1-fixing microorganism comprises an arginine metabolism pathway.

2 . The method of claim 1 wherein the arginine metabolism pathway comprises at least one of an arginine deaminase pathway and an arginine decarboxylase pathway, wherein the arginine deaminase pathway comprises one or more enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6), ornithine carbomyltransferase (putrescine carbomyltransferase) (EC 2.1.3.3) and a carbamate kinase (EC 2.7.2.2) and the arginine decarboxylase pathway comprises one or more enzymes selected from the group consisting arginine decarboxylase (EC 4.1.1.19), putative arginine deaminase (EC 3.5.3.12), putrescine carbamoyl transferase (EC 2.1.3.6) and carbamate kinase (EC 2.7.2.2).

3 . The method of claim 1 , wherein arginine is provided to the culture in an amount ranging from the cellular requirement of the culture to about 1000 times the cellular requirement of the culture.

4 . The method of claim 1 , wherein arginine is provided to the culture in an amount ranging from 2 times the cellular requirement of the culture to 1000 times the cellular requirement of the culture.

5 . The method of claim 1 , wherein the concentration of arginine in the bioreactor is at least 20 mg/L.

6 . The method of claim 1 , wherein the cellular requirement of the culture is about 0.012 g of arginine per gram of cellular biomass.

7 . The method of claim 1 , wherein the doubling time of the culture is decreased by at least 10% when compared to a culture where arginine is not provided in excess of the cellular requirement of the culture.

8 . The method of claim 7 , wherein the doubling time of the culture is at decreased by at least 50% when compared to a culture where arginine is not provided in excess of the cellular requirement of the culture.

9 . The method of claim 1 , wherein selectivity to the ATP-intensive product is increased when compared to a culture where arginine is not provided in excess of the cellular requirement of the culture.

10 . The method of claim 1 , wherein productivity of the ATP-intensive product is at least 10% greater when compared to a culture where arginine is not provided in excess of the cellular requirement of the culture.

11 . The method of claim 1 , wherein the culture produces a reduced amount of acetate compared to a culture where arginine is not provided in excess of the cellular requirement of the culture.

12 . The method of claim 11 , wherein the culture produces no acetate.

13 . The method of claim 1 , wherein the C1-fixing microorganism is a Clostridium bacterium.

14 . The method of claim 1 wherein the product is selected from the group consisting of ethanol, ethanol, 2,3-butanediol, 1,3-butanediol, lactate, succinate, methyl ethyl ketone (MEK), butyrate, 2-butanol, 1,2-propanediol (1,2-PDO), 1-propanol, isopropanol (IPA), acetoin, iso-butanol, isoprene, farnesene, bisabolene, pinine, limonene, acetone, 3-hydroxybutyrate, 2-hydroxyisobutyric acid (2-HIBA), citramalate, butadiene, poly lactic acid, 1-butanol, 3-hydroxy propionate (3-HP), benzoate, fatty acid ethyl ester, and fatty acids, and isobutylene.

15 . A method for improving the sustainability of a fermentation process, the method comprising:
a. flowing a gaseous C1-containing substrate to a bioreactor containing a culture of at least one C1-fixing bacterium comprising at least one of an arginine deiminase pathway or an arginine decarboxylase pathway in a liquid nutrient media; b. fermenting the culture to produce at least one product; and c. providing arginine to the culture in excess of the cellular requirement of the culture, such that arginine is catabolized by the arginine deiminase pathway to produce ammonium, wherein the bacterium utilizes ammonium as a nitrogen source.

16 . A method for improving the efficiency of a fermentation process, the method comprising providing arginine as the sole nitrogen source for a C1-fixing microorganism.

17 . A genetically engineered C1-fixing microorganism comprising an optimized arginine deiminase pathway.

18 . The bacterium of claim 17 , wherein the C1-fixing microorganism comprises one or more enzymes selected from the group consisting of: arginine deiminase (EC 3.5.3.6), carbomyltransferase (ornithine carbomyltransferase, putrescine carbomyltransferase) (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.2) wherein each enzyme is an overexpressed endogenous enzyme, a mutated endogenous enzyme or an exogenous enzyme

19 . The bacterium of claim 18 , further comprising one or more enzymes selected from the group consisting of omithine racemase (EC 5.1.1.12), omithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12), and 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10), wherein each enzyme is an overexpressed endogenous enzyme, a mutated endogenous enzyme or an exogenous enzyme

20 . The bacterium of claim 18 wherein the bacterium further comprises a disruptive mutation in an arginine: ornithine transporter.

21 . A method for producing at least one product from a substrate, the method comprising culturing the bacterium of claim 19 in the presence of a gaseous C1-containing substrate.

22 . A method for improving the efficiency of arginine incorporation into metabolism, the method comprising culturing a genetically engineered C1-fixing microorganism comprising one or more genetic modifications selected from the group consisting of:
i. a disruptive mutation in an arginine transporter; ii. overexpression of one or more endogenous enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6), ornithine carbomyltransferase (putrescine carbomyltransferase) (EC 2.1.3.3), carbamate kinase (EC 2.7.2.2), omithine racemase (EC 5.1.1.12), omithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12), and 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10); iii. expression of one or more mutated endogenous enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6), ornithine carbomyltransferase (putrescine carbomyltransferase) (EC 2.1.3.3), carbamate kinase (EC 2.7.2.2), ornithine racemase (EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12), and 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10); and iv. expression of one or more exogenous enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6), ornithine carbomyltransferase (putrescine carbomyltransferase) (EC 2.1.3.3), carbamate kinase (EC 2.7.2.2), ornithine racemase (EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12), and 2-amino-4-oxopentanoate thiolase (EC 2.3.1.B10)

23 . A method for improving efficiency of arginine co-utilization with one or more gaseous substrates selected from the group consisting of CO, H 2 and CO 2 , the method comprising culturing a genetically engineered C1-fixing bacterium comprising one or more genetic modifications, wherein the one or more genetic modifications are selected from the group con

Assignees

• Lanzatech New Zealand Ltd

Inventors

• Valgepea Kaspar
• Koepke Michael
• Behrendorff James Bruce Yarnton Haycock
• Marcellin Esteban
• Nielsen Lars K
• De S P Lemgruber Renato

Classifications

• C12P7/54

  Acetic acid (vinegar C12J) · CPC title

• C12N1/38 Primary

  Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound (C12N1/34 takes precedence) · CPC title

• C12Y501/01012

  Ornithine racemase (5.1.1.12) · CPC title

• C12N9/1018

  Carboxy- and carbamoyl transferases (2.1.3) · CPC title

• C12P7/04

  acyclic · CPC title