Production of oil in microorganisms

• US 8,512,999 B2
• Filed: 04/30/2010
• Issued: 08/20/2013
• Est. Priority Date: 06/01/2007
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Trimbur, Donald E., Im, Chung-Soon, Dillon, Harrison F., Day, Anthony G., Franklin, Scott, Coragliotti, Anna
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US12/772,173
• Publication Number:
  US 20110015417A1
• Time in Patent Office: 1,208 Days
• Field of Search: 435/257.1
• US Class Current: 435/257.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C10L 1/026 for compression ignition
• CPC Class Codes: C10L 1/06 for spark ignition
• CPC Class Codes: C10L 2200/0484 Vegetable or animal oils
• CPC Class Codes: C10L 2270/026 for diesel engines, e.g. automobiles, stationary, marine
• CPC Class Codes: C10L 2270/04 for turbines, planes, power generation
• CPC Class Codes: C11C 3/00 Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom sulfonated fats or oils C07C309/62; factice C08H; drying oils C09F
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 1/32 Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2431 Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12P 5/02 : acyclic C12P5/007 takes precedence
• CPC Class Codes: C12P 7/6409 : Fatty acids
• 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 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: Y02E 50/30 : Fuel from waste, e.g. synthetic alcohol or diesel
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts
• CPC Class Codes: Y02T 50/678 : Aviation using fuels of non-fossil origin

Abstract

The invention provides methods of cultivating oil-bearing microbes using xylose alone or in combination with other depolymerized cellulosic material. Also provided are microorganisms comprising an exogenous gene encoding a polysaccharide degrading enzyme, such as a cellulase, a hemicellulase, a pectinase, or a driselase. Some methods of microbial fermentation are provided that comprise the use of xylose and depolymerized cellulosic materials for the production of oil-bearing microorgansims.

Claims

1. A microalgae comprising an exogenous gene encoding a sucrose invertase, which is expressed in the microalgae and hydrolyzes sucrose to fructose and glucose, and wherein the microalgae comprises at least 20% lipid by dry cell weight.

2. The microalgae of claim 1, wherein the microalgae is selected from Table 1.

3. The microalgae of claim 1 that is an obligate heterotroph.

4. The microalgae of claim 1 that is a species of the genus Prototheca.

5. The microalgae of claim 4, wherein the microalgae is selected from the group consisting of Prototheca stagnora, Prototheca portoricensis, Prototheca wickerhamii, Prototheca moriformis, and Prototheca zopfii.

6. The microalgae of claim 1 that further comprises a second exogenous gene that encodes an acyl-ACP thioesterase, which is expressed in the microalgae and thereby alters the proportions of fatty acids produced in the microalgae.

7. The microalgae of claim 6, wherein the thioesterase is selected from the group consisting of Umbellularia californica fatty acyl-ACP thioesterase, Cinnamomum camphora fatty acyl-ACP thioesterase, Myristica fragrans fatty acyl-ACP thioesterase, Elaeis guineensis fatty acyl-ACP thioesterase, Populus tomentosa fatty acyl-ACP thioesterase, Arabidopsis thaliana fatty acyl-ACP thioesterase, Gossypium hirsutum fatty acyl-ACP thioesterase, Cuphea lanceolata fatty acyl-ACP thioesterase, Cuphea hookeriana fatty acyl-ACP thioesterase, Cuphea calophylla subsp. mesostemon fatty acyl-ACP thioesterase, Cuphea lanceolata fatty acyl-ACP thioesterase, Vitis vinifera fatty acyl-ACP thioesterase, Garcinia mangostana fatty acyl-ACP thioesterase, Brassica juncea fatty acyl-ACP thioesterase, Madhuca longifolia fatty acyl-ACP thioesterase, Brassica napus fatty acyl-ACP thioesterase, Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase, and Oryza sativa (japonica cultivar-group) fatty acyl-ACP thioesterase.

8. The microalgae of claim 1, wherein the gene encoding the sucrose invertase includes a secretion signal so that sucrose invertase is secreted from the microalgae thereby converting available extracellular sucrose to fructose and glucose, and wherein the microalgae is capable of transporting and utilizing the glucose and fructose so produced.

9. The microalgae of claim 8, wherein the microalgae further comprises a second exogenous gene that encodes an acyl-ACP thioesterase, which is expressed in the microalgae.

10. The microalgae of claim 9, wherein the proportions of fatty acids of the microalgae are altered relative to the microalgae lacking the exogenous acyl-ACP thioesterase.