Methods for producing a triglyceride composition from algae

• US 9,464,304 B2
• Filed: 11/30/2009
• Issued: 10/11/2016
• Est. Priority Date: 11/28/2008
• Status: Expired due to Fees

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Terravia Holdings, Inc. (Corbion NV)
• Inventors: Franklin, Scott, Somanchi, Aravind, Espina, Karen, Rudenko, George, Chua, Penelope
• Primary Examiner(s): Steadman, David J
• Application Number:
  US12/628,144
• Publication Number:
  US 20100151538A1
• Time in Patent Office: 2,507 Days
• Field of Search: 435/134, 435/105, 435/196, 435/257.2, 435/320.1
• US Class Current: 1/1
• CPC Class Codes: C07C 1/2078 by a transformation in which at least one -C(=O)-O- moiety is eliminated
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C10L 1/02 essentially based on components consisting of carbon, hydrogen, and oxygen only
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12P 7/20 Glycerol
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• CPC Class Codes: C12P 7/6409 Fatty acids
• CPC Class Codes: C12P 7/6418 by hydrolysis of fatty acid esters
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6458 by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
• CPC Class Codes: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: C12P 7/649 Biodiesel, i.e. fatty acid alkyl esters
• CPC Class Codes: C12Y 301/02014 Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase
• CPC Class Codes: C12Y 302/01026 Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts
• CPC Class Codes: Y02P 60/87 Re-use of by-products of food processing for fodder production
• CPC Class Codes: Y02T 50/678 : Aviation using fuels of non-fossil origin

Abstract

Methods are disclosed for cultivation of oleaginous microorganisms on feedstocks including depolymerized cellulosic material such as corn stover, Miscanthus, forage sorghum, sugar beet pulp and sugar cane bagasse. Also disclosed are methods of manufacturing novel triglyceride oil compositions using cellulosic feedstocks, including oil compositions comprising fatty acids such as those having chain lengths of 8, 10, 12, and 14 carbon atoms.

Claims

1. A method of producing a lipid composition comprising:

(a) providing a culture medium with a population of Prototheca cells, the cells comprising an exogenous gene operably linked to a promoter sequence selected from the group consisting of SEQ ID NO;

96, SEQ ID NO;

97, and SEQ ID NO;

98, wherein the exogenous gene is heterologous to the promoter sequence;

(b) adding depolymerized cellulosic material to the culture medium, wherein the added depolymerized cellulosic material has a glucose concentration of at least 600 g/L and a salt concentration of less than 300 mM;

(c) cultivating the population of cells heterotrophically in the dark thereby accumulating lipid in the cells; and

(d) isolating lipid from the cells to provide the lipid composition.

2. The method of claim 1, wherein the depolymerized cellulosic material is selected from the group consisting of corn stover, Miscanthus, forage sorghum, sugar beet pulp and sugar cane bagasse.

3. The method of claim 1, wherein the depolymerized cellulosic material is washed with water or deionized prior to addition to the culture medium.

4. The method of claim 1, wherein the exogenous gene encodes a fatty acyl-ACP thioesterase that has hydrolysis activity towards one or more fatty acyl-ACP substrates of chain length C8, C10, C12 or C14.

5. The method of claim 4, wherein the fatty acyl-ACP thioesterase has activity towards fatty acyl-ACP substrates of chain length C8 or C10.

6. The method of claim 4, wherein the fatty acyl-ACP thioesterase has activity towards fatty acyl-ACP substrates of chain length C12 or C14.

7. The method of claim 4, wherein the lipid composition has a profile of:

a. at least 4% C8-C14 fatty acids; and

b. one or more of the following attributes;

i. less than 0.4 micrograms/ml total carotenoids;

ii. less than 0.001 micrograms/ml lycopene;

iii. less than 0.02 micrograms/ml beta carotene;

iv. less than 0.02 milligrams of chlorophyll per kilogram of oil;

v. 0.40-0.60 milligrams of gamma tocopherol per 100 grams of oil;

vi. 3-9 mg campesterol per 100 grams of oil; and

vii. less than 0.5 milligrams of total tocotrienols per gram of oil.

8. The method of claim 7, wherein the fatty acyl-ACP thioesterase has activity towards fatty acyl-ACP substrates of chain length C14 and the percent C8-C14 fatty acids is more than 30%.

9. The method of claim 1, wherein the Prototheca cells are Prototheca moriformis.

10. The method of claim 9, wherein the exogenous gene encodes a fatty acyl-ACP thioesterase that has hydrolysis activity towards one or more fatty acyl-ACP substrates of chain length C8, C10, C12 or C14.

11. The method of claim 1, wherein the population of cells is cultivated in fed-batch cultivation mode.

12. The method of claim 1 wherein the depolymerized cellulosic material is depolymerized by an enzyme.

13. The method of claim 1 wherein the depolymerized cellulosic material is depolymerized by using acid, heat and pressure.

14. The method of claim 1, wherein the depolymerized cellulosic material comprises glucose and xylose.

15. The method of claim 1, wherein the promoter sequence is selected from the group consisting of SEQ ID NO:

96 and 97.