Nucleic acids useful in the manufacture of oil

• US 8,268,610 B2
• Filed: 11/30/2009
• Issued: 09/18/2012
• 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): Bui, Phuong T
• Application Number:
  US12/628,147
• Publication Number:
  US 20100151567A1
• Time in Patent Office: 1,023 Days
• Field of Search: 435/6, 435/69.1, 435/468, 435/419, 435/320.1, 435/6.1, 435/257.1, 435/196, 435/134, 435/183, 536/24.1, 536/23.1, 536/23.2, 800/278, 800/295, 530/370, 530/300
• 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

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 codon-optimized recombinant nucleic acid comprising a fatty acyl-ACP thioesterase coding sequence codon-optimized for expression in Prototheca, wherein the coding sequence contains the most or second most preferred codon of Table 1 for at least 60% of the codons of the coding sequence, such that the codon-optimized sequence is more efficiently translated in Prototheca than a native sequence encoding the fatty acyl-ACP thioesterase, the fatty acyl-ACP thioesterase having hydrolysis activity towards one or more fatty acyl-ACP substrates, the recombinant nucleic acid further comprising a plastid targeting sequence from microalgae that is in-frame with the codon-optimized fatty acyl-ACP thioesterase coding sequence.

2. The codon-optimized recombinant nucleic acid of claim 1, wherein the plastid targeting sequence is a sequence selected from the group consisting of SEQ ID NOs:

127-130.

9. A host cell of the genus Prototheca comprising the recombinant nucleic acid of claim 1.

10. A host cell of the genus Prototheca comprising the recombinant nucleic acid of claim 2.

3. A codon-optimized recombinant nucleic acid comprising a fatty acyl-ACP thioesterase coding sequence codon-optimized for expression in Prototheca, wherein the coding sequence contains the most or second most preferred codon of Table 1 for at least 60% of the codons of the coding sequence, such that the codon-optimized sequence is more efficiently translated in Prototheca than a native sequence encoding the fatty acyl-ACP thioesterase, the fatty acyl-ACP thioesterase having hydrolysis activity towards one or more fatty acyl-ACP substrates, the recombinant nucleic acid further comprising a plastid targeting sequence in-frame with the codon-optimized fatty acyl-ACP thioesterase coding sequence, the recombinant nucleic acid further comprising first and second flanking nucleic acid sequences endogenous to the nuclear genome of a cell of the genus Prototheca, wherein the first and second flanking sequences are each at least 200 nucleotides long and the first and second flanking sequences flank the codon-optimized recombinant nucleic acid for integration of the recombinant nucleic acid into the nuclear genome by homologous recombination with a corresponding genomic sequence.

4. The codon-optimized recombinant nucleic acid of claim 3, wherein the first and second flanking sequences are found on the same Prototheca chromosome and are located no more than 20 kB apart from each other.

11. A host cell of the genus Prototheca comprising the recombinant nucleic acid of claim 3.

12. A host cell of the genus Prototheca comprising the recombinant nucleic acid of claim 4.

5. A codon-optimized recombinant nucleic acid comprising a fatty acyl-ACP thioesterase coding sequence codon-optimized for expression in Prototheca, wherein the coding sequence contains the most or second most preferred codon of Table 1 for at least 60% of the codons of the coding sequence, such that the codon-optimized sequence is more efficiently translated in Prototheca than a native sequence encoding the fatty acyl-ACP thioesterase, the fatty acyl-ACP thioesterase having hydrolysis activity towards one or more fatty acyl-ACP substrates, the recombinant nucleic acid further comprising a plastid targeting sequence from microalgae that is in-frame with the codon-optimized fatty acyl-ACP thioesterase coding sequence, and wherein the amino acid sequence of the fatty acyl-ACP thioesterase comprises an amino acid sequence of a fatty acyl-ACP thioesterase from an organism selected from the group consisting of Cuphea hookeriana, Umbellularia californica, Cinnamomum camphora, Cuphea palustris and Ulmus americana.

13. A host cell of the genus Prototheca comprising the recombinant nucleic acid of claim 5.

6. A codon-optimized recombinant nucleic acid comprising a fatty acyl-ACP thioesterase coding sequence and a sucrose invertase coding sequence, each coding sequence codon-optimized for expression in Prototheca, wherein each coding sequence contains the most or second most preferred codon of Table 1 for at least 60% of the codons of the coding sequence, such that each codon-optimized sequence is more efficiently translated in Prototheca than a native sequence encoding the fatty acyl-ACP thioesterase or the native sequence encoding the sucrose invertase, the fatty acyl-ACP thioesterase having hydrolysis activity towards one or more fatty acyl-ACP substrates, the recombinant nucleic acid further comprising a plastid targeting sequence in-frame with the codon-optimized fatty acyl-ACP thioesterase coding sequence.

14. A host cell of the genus Prototheca comprising the recombinant nucleic acid of claim 6.

16. The codon-optimized recombinant nucleic acid of claim 6, wherein each coding sequence contains the most preferred codon of Table 1 for at least 60% of the codons of each coding sequence.

17. The codon-optimized recombinant nucleic acid of claim 6, wherein each coding sequence contains the most preferred codon of Table 1 for at least 80% of the codons of each coding sequence.

7. A host cell of the genus Prototheca comprising a recombinant nucleic acid comprising a fatty acyl-ACP thioesterase coding sequence codon-optimized for expression in Prototheca, wherein the coding sequence contains the most or second most preferred codon of Table 1 for at least 60% of the codons of the coding sequence, such that the codon-optimized sequence is more efficiently translated in Prototheca than a native sequence encoding the fatty acyl-ACP thioesterase, the fatty acyl-ACP thioesterase having hydrolysis activity towards one or more fatty acyl-ACP substrates.

8. A host cell of the genus Prototheca comprising a codon optimized recombinant nucleic acid comprising a fatty acyl-ACP thioesterase coding sequence codon-optimized for expression in Prototheca, wherein the coding sequence contains the most or second most preferred codon of Table 1 for at least 60% of the codons of the coding sequence, such that the codon-optimized sequence is more efficiently translated in Prototheca than a native sequence encoding the fatty acyl-ACP thioesterase, the fatty acyl-ACP thioesterase having hydrolysis activity towards a fatty acyl-ACP substrate of chain length C8, C10, C12 or C14, the recombinant nucleic acid further comprising a plastid targeting sequence that is in-frame with the codon-optimized fatty acyl-ACP thioesterase coding sequence.

15. A codon-optimized recombinant nucleic acid comprising a fatty acyl-ACP thioesterase coding sequence codon-optimized for expression in Prototheca, wherein the coding sequence contains the most or second most preferred codon of Table 1 for at least 60% of the codons of the coding sequence, such that the codon-optimized sequence is more efficiently translated in Prototheca than a native sequence encoding the fatty acyl-ACP thioesterase, the fatty acyl-ACP thioesterase having hydrolysis activity towards one or more fatty acyl-ACP substrates, further comprising a promoter comprising the nucleotide sequence of any of SEQ ID NOS:

91-102.