Genetically Engineered Microorganisms That Metabolize Xylose

• US 20120329109A1
• Filed: 05/04/2012
• Published: 12/27/2012
• Est. Priority Date: 05/06/2011
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Chua, Penelope, Somanchi, Aravind
• Granted Patent: US 8,846,352 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 1/22 Processes using, or culture media containing, cellulose or hydrolysates thereof
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8246 Non-starch polysaccharides, e.g. cellulose, fructans, levans
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12P 2203/00 Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source ethanol C12P7/10
• 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 7/6463 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: Y02E 50/10 Biofuels, e.g. bio-diesel
• 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 as a fixed carbon source. Also provided are microorganisms that have been genetically engineered to metabolize xylose as a fixed carbon source allowing them to convert xylose into oils. Particular advantages of the processes provided herein include production of oils rather than alcohols through the microbial fermentation processes utilizing xylose.

Claims

1. A microorganism of the kingdom Plantae, the microorganism comprising a recombinant nucleic acid operable to produce an active xylose metabolic pathway enzyme, wherein the microorganism is capable of being cultured on a carbon source consisting essentially of xylose.

4. The microorganism of claim 1, wherein the microorganism is capable of accumulating 10, 20, 30, 40, 50, 60, 70, or 80% triglyceride by dry cell weight.

9. The microorganism of claim 1, wherein the recombinant nucleic acid is operable to encode one or more of an active xylose transporter, a xylulose-5-phosphate translocator, a xylose isomerase, a xylulokinase, a xylitol dehydrogenase, or a xylose reductase.

10. The microorganism of claim 9, wherein the recombinant nucleic acid encodes an active xylulose-5-phosphate translocator in operable linkage with a plastid-targeting signal sequence and wherein the xylulose-5-phosphate translocator is operable to transport xylulose-5-phosphate into a plastid.

12. The microorganism of claim 1, wherein the microorganism is of the subkingdom Viridiplantae.

13. The microorganism of claim 12, wherein the microorganism is of the infrakingdom or phyla Chlorophytae, of the subphylum Tetraphytina, the class Trebouxiophyceae, or the order Chlorellales.

14. The microorganism of claim 13, wherein the microorganism is of the genus Chlorella or Prototheca.

15. The microorganism of claim 14, wherein the microorganism is of the species Prototheca moriformis or Chlorella protothecoides.

16. The microorganism of claim 1, further comprising a recombinant modification operable to alter the fatty acid profile of lipid produced by the microorganism.

17. The microorganism of claim 16, wherein the recombinant modification comprises an exogenous gene that encodes an active acyl-ACP thioesterase, ketoacyl-ACP synthase or a fatty acid desaturase, or is operable to reduce or ablate an acyl-ACP thioesterase, ketoacyl-ACP synthase or fatty acid desaturase.

24. A natural oil produced by the microorganism of claim 1.

25. An oleochemical, food, food oil, fuel, cosmetic ingredient, or other product produced from the natural oil of claim 24.

76. A microbial lipid composition produced by a process of cultivating the microorganism of claim 1, the process comprising the steps of:

(a) providing the microorganism in a culture medium;

(b) adding xylose to the culture medium;

(c) propagating or cultivating the microorganism until the microorganism accumulates at least about 20% of its dry weight as lipid; and

(d) lysing the microorganism and isolating the microbial lipid composition.

2-3. -3. (canceled)

5-8. -8. (canceled)

11. (canceled)

18. A method for producing microbial biomass or a product produced from the biomass, the method comprising cultivating a microorganism of the kingdom Plantae in a culture medium comprising xylose, the microorganism comprising a recombinant nucleic acid operable to produce an active xylose metabolic pathway enzyme, and wherein the microorganism is capable of being cultured on a carbon source consisting essentially of xylose.

19. The method of claim 18, wherein the microbial biomass comprises microbial lipid.

20. The method of claim 19, wherein the lipid is used to make a product that is a chemical, a lubricant, a detergent, a fuel, a food, a food oil, or a cosmetic ingredient.

21. The method of claim 20, wherein the product is a fuel selected from biodiesel or renewable diesel.

92. The method of claim 18, wherein the xylose is obtained from depolymerized cellulosic material.

22-23. -23. (canceled)

26-56. -56. (canceled)

57. A recombinant oleaginous microbe comprising an exogenous gene that encodes one or more xylose translocator proteins selected from GPT-A-XPT (SEQ ID NO:

45), GPT-F-XPT (SEQ ID NO;

     47) or S106SAD-XPT (SEQ ID NO;

     49).

91. The microbe of claim 57, further comprising one or more exogenous genes that encode a xylose transporter protein selected from SUT1 (SEQ ID NO:

37), GXS1 (SEQ ID NO;

     39), or XLT1 (SEQ ID NO;

     43), a symporter protein (SEQ ID NO;

     41), an oxidoreductase pathway protein selected from XylA (SEQ ID NO;

     15) or XYL3 (SEQ ID NO;

     51), or a xylose isomerase pathway protein selected from XYL1 (SEQ ID NO;

     35), XYL2 (SEQ ID NO;

     50) or XYL3 (SEQ ID NO;

     51).

58-75. -75. (canceled)

77-90. -90. (canceled)