Nucleic Acids Useful in the Manufacture of Oil

• US 20100151567A1
• 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 8,268,610 B2
• Time in Patent Office: Days
• US Class Current: 435/320.100
• 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

Novel gene sequences from microalgae are disclosed, as well as novel gene sequences useful in the manufacture of triglyceride oils. Also disclosed are sequences and vectors that allow microalgae to be cultivated on sugar cane and sugar beets as a feedstock. In some embodiments, the vectors are useful for the purpose of performing targeted modifications to the nuclear genome of heterotrophic microalgae.

Claims

1. A recombinant nucleic acid molecule comprising a promoter that is upregulated at least 3-fold in a cell of the genus Prototheca when the extracellular environment changes from containing at least 5 mM nitrogen to containing no nitrogen.

2. The recombinant nucleic acid of claim 1, wherein the nucleic acid comprises a segment of 50 or more nucleotides of one of SEQ ID NOs:

91-102.

3. A nucleic acid vector comprising an expression cassette comprising (a) a promoter that is active in a cell of the genus Prototheca;

and (b) a coding sequence in operable linkage with the promoter wherein the coding sequence contains the most or second most preferred codons of Table 1 for at least 20% of the codons of the coding sequence.

4. The vector of claim 3, wherein the coding sequence comprises a plastid targeting sequence in-frame with a fatty acyl-ACP thioesterase.

5. The vector of claim 4, wherein the fatty acyl-ACP thioesterase has hydrolysis activity towards one or more fatty acyl-ACP substrates of chain length C8, C10, C12 or C14.

6. The vector of claim 3, wherein the promoter is upregulated at least 3-fold in a cell of the genus Prototheca when the extracellular environment changes from containing at least 5 mM nitrogen to containing no nitrogen.

7. The vector of claim 4, wherein the plastid targeting sequence encodes a peptide that is capable of targeting a protein to the plastid of a cell of the genus Prototheca.

8. The vector of claim 4, wherein the plastid targeting sequence is from microalgae.

9. The vector of claim 8, wherein the plastid targeting sequence has at least 25% amino acid sequence identity to one or more of SEQ ID NOs:

127-133 and is capable of targeting a protein to the plastid of a cell of the genus Prototheca.

10. The nucleic acid vector of claim 3, further comprising a nucleic acid sequence endogenous to the nuclear genome of a cell of the genus Prototheca, wherein the sequence is at least 200 nucleotides long.

11. The nucleic acid vector of claim 10, further comprising first and second nucleic acid sequences endogenous to the nuclear genome of a cell of the genus Prototheca, wherein the first and second sequences (a) are each at least 200 nucleotides long;

(b) flank the expression cassette; and

(c) are located on the same Prototheca chromosome no more than 20 kB apart.

12. (canceled)

13. A recombinant nucleic acid encoding a protein with at least 90% amino acid identity to one or both of SEQ ID NOs:

136-137.