Cellulosic Cultivation of Oleaginous Microorganisms

• US 20100151538A1
• Filed: 11/30/2009
• Published: 06/17/2010
• Est. Priority Date: 11/28/2008
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Rudenko, George, Somanchi, Aravind, Chua, Penelope, Espina, Karen, Franklin, Scott
• Granted Patent: US 9,464,304 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• 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 triglyceride composition, comprising:

(a) culturing a population of cells of the genus Prototheca in the presence of a fixed carbon source, wherein;

(i) the cells contain an exogenous gene;

(ii) the cells accumulate at least 10% of their dry cell weight as lipid; and

(iii) the fixed carbon source is selected from the group consisting of sorghum and depolymerized cellulosic material; and

(b) isolating lipid components from the cultured microorganisms.

2. The method of claim 1, wherein the fixed carbon source is depolymerized cellulosic material 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 fixed carbon source is depolymerized cellulosic material that has been subjected to washing with water prior to the culturing step.

4. The method of claim 1, wherein the fixed carbon source is depolymerized cellulosic material and the glucose level of the depolymerized cellulosic material is concentrated to a level of at least 300 g/liter, at least 400 g/liter, at least 500 g/liter, or at least 600 g/liter of prior to the culturing step and is fed to the culture over time as the cells grow and accumulate lipid.

5. 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.

6. The method of claim 1, wherein the triglyceride composition has a profile of:

a. at least 4% C8-C14; 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.

7. A method of producing a triglyceride composition, comprising:

(a) culturing a population of microorganisms in the presence of depolymerized cellulosic material, wherein;

(i) the depolymerized cellulosic material is subjected to washing with water prior to the culturing step;

(ii) the cells accumulate at least 10% of their dry cell weight as lipid; and

(iii) the depolymerized cellulosic material is concentrated to at least 300 g/liter of glucose prior to the cultivation step;

(iv) the microorganisms are cultured in a fed-batch reaction in which depolymerized cellulosic material of at least 300 g/liter of glucose is fed to the microorganisms; and

(b) isolating lipid components from the cultured microorganisms.

8. The method of claim 7, wherein the depolymerized cellulosic material is selected from the group consisting of corn stover, Miscanthus, forage sorghum, sugar beet pulp and sugar cane bagasse.

9. The method of claim 7, wherein the microorganisms are a species of the genus Prototheca and contain an exogenous gene.

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. A method of manufacturing triglyceride oil comprising cultivating a cell that has a 23S rRNA sequence with at least 96% nucleotide identity to SEQ ID NO:

30 in the presence of sucrose as a carbon source.