Recombinant microalgae cells producing novel oils

• US 8,951,777 B2
• Filed: 03/28/2013
• Issued: 02/10/2015
• Est. Priority Date: 11/28/2008
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Franklin, Scott, Somanchi, Aravind, Espina, Karen, Rudenko, George, Chua, Penelope
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US13/852,116
• Publication Number:
  US 20130273621A1
• Time in Patent Office: 684 Days
• US Class Current: 435/257.1
• 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

Disclosed herein are obligate heterotrophic microalgae cells containing an exogenous gene. In some embodiments the gene is a sucrose utilization gene, and further disclosed are methods of manufacturing triglyceride oils using sugar cane or sugar beets as a feedstock in a heterotrophic fermentation. In other embodiments the feedstock is depolymerized cellulosic material. Also disclosed are cells that produce medium chain fatty acids at levels not produced in non-recombinant cells of the same species and genus.

Claims

1. A method for preparing recombinant microalgal cells of the genus Prototheca, the method comprising:

heterotrophically cultivating recombinant microalgal cells of the genus Prototheca in a fermentation broth, the cells comprising a chromosomal knockout of a stearoyl ACP (acyl carrier protein) desaturase allele so that the cells produce an oil having an altered fatty acid profile comprising a reduction in unsaturated fatty acids; and

isolating the cells from the broth.

2. The method of claim 1, wherein the cells comprise a recombination event in the stearoyl-ACP desaturase allele.

3. The method of claim 2, wherein the recombination event is a double reciprocal recombination event.

4. The method of claim 2, wherein a selectable marker gene is incorporated into the chromosome so as to knock out the stearoyl-ACP desaturase allele.

5. The method of claim 1, wherein the cells are cultivated on glucose, fructose or depolymerized cellulosic material as a carbon source.

6. The method of claim 1, wherein the microalgal cells are of the species Prototheca moriformis.

7. The method of claim 1, further comprising isolating the oil from the microalgal cells.