Sucrose feedstock utilization for oil-based fuel manufacturing

• US 8,476,059 B2
• Filed: 06/02/2008
• Issued: 07/02/2013
• 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:
  US12/131,793
• Publication Number:
  US 20090035842A1
• Time in Patent Office: 1,856 Days
• Field of Search: None
• 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 manufacturing oils and oil-based products such as transportation fuels, industrial chemicals, edible oils, lubricants and plastics using sugar cane, sugar beets, and cane/beet agricultural processing byproducts as a feedstock for bioproduction processes. The disclosed processes utilize oil-bearing microbes as a conversion technology to convert chemical energy produced by sugar cane and sugar beets into energy-containing oils and oil derivatives. Also provided herein are oil-bearing microbes containing one or more exogenous sucrose utilization genes.

Claims

1. A method of cultivating microalgae Chlorella or Prototheca in a culture medium, the microalgae comprising an exogenous gene encoding a secretable sucrose invertase, the method comprising the steps of:

(a) providing the microalgae;

(b) adding sucrose to the culture medium; and

(c) cultivating the microalgae in the culture medium, wherein the invertase enzyme allows the microalgae to grow on sucrose.

2. The method of claim 1, wherein the sucrose is provided from a source selected from the group consisting of sugar cane juice, sugar beet juice and molasses, wherein the sugar cane juice, sugar beet juice or molasses has a sucrose concentration of at least about 50 μ

M.

3. The method of claim 2, wherein the sugar cane juice, sugar beet juice or molasses has a sucrose concentration of at least about 500 μ

M.

4. The method of claim 3, wherein the sugar cane juice, sugar beet juice or molasses has a sucrose concentration of at least about 5 mM.

5. The method of claim 4, wherein the sugar cane juice, sugar beet juice or molasses has a sucrose concentration of at least about 50 mM.

6. The method of claim 5, wherein the sugar cane juice, sugar beet juice or molasses has a sucrose concentration of at least about 500 mM.

7. The method of claim 1, wherein said microalgae further comprises another exogenous gene selected from the group consisting of a sucrose transporter, a hexokinase, a glucokinase, and a fructokinase.

8. The method of claim 1, wherein the source is sugar cane juice.

9. The method of claim 1, wherein the sucrose invertase gene comprises SEQ ID NO:

8.

10. The method of claim 1, wherein the sucrose invertase enzyme comprises SEQ ID NO:

19.

11. The method of claim 1, wherein the microalgae is a species of the genus Chlorella.

12. The method of claim 11, wherein the microalgae are selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, and Chlorella zofingiensis.

13. The method of claim 1, wherein the culture medium further comprises at least one additional carbon source selected from the group consisting of glucose, glycerol, xylose, fructose, arabinose, mannose, galactose, acetate, depolymerized cellulosic material, sugarcane bagasse, rice hulls, corn stover, wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels, hardwood thinnings, softwood thinnings, wood chips, sawdust, pulp mill waste, urban paper waste, grass clippings, switchgrass, hybrid poplar wood, miscanthus, fiber cane and fiber sorghum.

14. The method of claim 13, wherein the additional carbon source is depolymerized cellulosic material.

15. The method of claim 13, wherein the additional carbon source is sugar cane bagasse.

16. The method of claim 13, wherein the additional carbon source is sugar beet pulp.

17. The method of claim 1, where sucrose is the sole carbon source.

18. The method of claim 1, where the culture medium comprises at least 1% sucrose.

19. The method of claim 1, wherein the microalgae are cultivated in the absence of light.

20. The method of claim 1, wherein said microalgae are obligate heterotrophs.

21. The method of claim 1, wherein said microalgae is Prototheca.

22. A method of cultivating microalgae Chlorella or Prototheca in a culture medium that comprises sucrose, the method comprising the steps of:

(a) providing the microalgae comprising an exogenous secretable sucrose invertase gene; and

(b) cultivating the microalgae in said culture medium, wherein the culture medium comprises limited nitrogen and wherein the invertase enzyme allows the microalgae to grow on sucrose.

23. The method of claim 22, wherein said sucrose is provided to the culture medium from a source selected from the group consisting of sugar cane juice, sugar beet juice and molasses.

24. The method of claim 22, wherein said recombinant microalgae is cultured without selecting for a selectable marker other than sucrose.

25. A method for producing triglycerides, the method comprising:

(a) heterotrophically cultivating a population of microalgae Chlorella or Prototheca on a sucrose medium, the microalgae expressing a transgene encoding a secreted invertase enzyme and a secretion signal such that the invertase enzyme hydrolyzes sucrose in the medium and allows the microalgae to grow on sucrose; and

(b) recovering the triglycerides.