Ketoacyl ACP synthase genes and uses thereof

• US 10,316,299 B2
• Filed: 04/10/2018
• Issued: 06/11/2019
• Est. Priority Date: 07/10/2014
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Corbion Biotech, Inc. (Corbion NV)
• Inventors: Davis, David, Rudenko, George N., Somanchi, Aravind, Casolari, Jason, Franklin, Scott, Ewing, Aren
• Primary Examiner(s): Pak, Yong D
• Application Number:
  US15/950,048
• Publication Number:
  US 20180230442A1
• Time in Patent Office: 427 Days
• Field of Search: None
• CPC Class Codes: C12N 15/82 for plant cells , e.g. plant artificial chromosomes (PACs)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 33/00 Preparation of steroids
• 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: C12Y 203/01041 Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41)

Abstract

The present invention relates to beta-ketoacyl ACP synthase genes of the KASI/KASIV type and proteins encoded by these genes. The genes can be included in nucleic acid constructs, vectors or host cells. Expression of the gene products can alter the fatty acid profile of host cells. The KAS genes can be combined with a FATA or FATB thioesterase gene to create a cell that produces an increased amount of C8-C16 fatty acids. Suitable host cells include plastidic cells of plants or microalgae. Oleaginous microalga host cells with the new genes are disclosed.

Claims

1. A recombinant polynucleotide or a complement thereof encoding a polypeptide having at least 94% sequence identity to amino acids 34 to 502 of SEQ ID NO:

8 or 92% sequence identity to amino acids 34 to 525 of SEQ ID NO;

14 wherein said polypeptide has β

-keto-acyl ACP synthase (KASIV) activity.

2. A transformation vector comprising the polynucleotide of claim 1.

3. The vector of claim 2, comprising promoter and 3′

UTR sequences in operable linkage to the polynucleotide, and optionally a flanking sequence for homologous recombination.

4. A host cell comprising the vector of claim 2.

5. The host cell of claim 4, wherein the host cell is a plastidic oleaginous cell having a type II fatty acid biosynthesis pathway.

6. The host cell of claim 5, wherein the host cell is a microalga.

7. The host cell of claim 6, wherein the host cell is of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca.

8. The host cell of claim 7, wherein the microalga is of the species Prototheca moriformis.

9. A method for making a cell-oil, the method comprising cultivating a host cell of any one of claim 4, so as produce the cell-oil, wherein the oil comprises triglcyerides and microalgal sterols.

10. The method of claim 9, wherein the cell oil comprises sterols characterized by a sterol profile and the sterol profile has an excess of ergosterol over β

-sitosterol and/or the presence of 22, 23-dihydrobrassicasterol, poriferasterol or clionasterol.

21. The recombinant polynucleotide of claim 1, wherein the polynucleotide is codon-optimized for expression in microalgae.

22. The recombinant polynucleotide of claim 21, wherein the polynucleotide'"'"'s coding sequence comprises the most or second most preferred codon for at least 60% of the codons.

23. The recombinant polynucleotide of claim 22, wherein the coding sequence comprises the most preferred codon for at least 80% of the codons.

24. The recombinant polynucleotide of claim 1, wherein the polynucleotide comprises nucleic acids 105 to 1515 of SEQ ID NO:

27 or nucleic acids 150 to 1629 of SEQ ID NO;

33.

25. The recombinant polynucleotide of claim 22, wherein the polynucleotide comprises nucleic acids 102 to 1542 of SEQ ID NO:

45 or nucleic acids 100 to 1611 of SEQ ID NO;

51.

11. A host cell comprising

a recombinant polynucleotide or a complement thereof encoding a polypeptide having at least 94% sequence identity to amino acids 34 to 502 of SEQ ID NO:

8 or 92% sequence identity to amino acids 34 to 525 of SEQ ID NO;

14 wherein said polypeptide has β

-keto-acyl ACP synthase (KASIV) activity.

12. The host cell of claim 11, further comprising a polynucleotide encoding a fatty acyl-ACP thioesterase (FATB) polypeptide, wherein the FATB polypeptide has at least 90% amino acid sequence identity to SEQ ID NO:

1 or SEQ ID NO;

57.

13. The host cell of claim 12, wherein the host cell produces a cell oil characterized by a fatty acid profile with (i) at least 30, 40, 50, or 55% C14:

0, (ii) at least 7, 8, 9, 10, 11, 12, 13, or 14% C8;

0, (ii) at least 10, 15, 20, 25, 30, or 35 area % for the sum of C8;

0 and C10;

0, or (iii) a C8/C10 ratio in the range of 2.2-2.5, 2.5-3.0, or 3.0-3.4.

14. The host cell of any one of claim 11, wherein the host cell is a plastidic oleaginous cell having a type II fatty acid biosynthesis pathway.

15. The host cell of claim 14, wherein the host cell is a microalga.

16. The host cell of claim 15, wherein the host cell is of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca.

17. The host cell of claim 16, wherein the microalga is of the species Prototheca moriformis.

18. The host cell of claim 11, wherein one or more of the polynucleotides is codon-optimized for expression in the host cell such that the polynucleotide'"'"'s coding sequence contains the most or second most preferred codon for at least 60% of the codons of the coding sequence such that the codon-optimized sequence is more efficiently translated in the host cell relative to a non-optimized sequence.

19. The host cell of claim 18, wherein the coding sequence comprises the most preferred codon for at least 80% of the codons of the coding sequence.

20. A method for making a cell-oil, the method comprising cultivating a host cell of claim 11, so as produce the cell-oil, wherein the oil comprises triglcyerides and microalgal sterols.