Ketoacyl ACP synthase genes and uses thereof

• US 9,969,990 B2
• Filed: 07/10/2015
• Issued: 05/15/2018
• 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, Somanchi, Aravind, Casolari, Jason, Franklin, Scott, Ewing, Aren
• Primary Examiner(s): Pak, Yong D
• Application Number:
  US14/796,406
• Publication Number:
  US 20160010066A1
• Time in Patent Office: 1,040 Days
• 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 having at least 95% sequence identity or equivalent sequence by virtue of the degeneracy of the genetic code to SEQ ID NO:

31 encoding a β

-keto-acyl ACP synthase IV (KASIV) protein, or the complement of the polynucleotide, wherein said protein has 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 host cell comprising

a recombinant polynucleotide having at least 95% sequence identity or equivalent sequence by virtue of the degeneracy of the genetic code to SEQ ID NO:

31, encoding a β

-keto-acyl ACP synthase IV (KASIV) protein or the complement of the polynucleotide, wherein said protein has KASIV activity.

10. The host cell of claim 9 further comprising a polynucleotide encoding a fatty acyl-ACP thioesterase B (FATB) wherein the thioesterase has at least 90% amino acid sequence identity to SEQ ID NO:

1 or SEQ ID NO;

57.

11. The host cell of claim 9, wherein the host cell produces a cell oil characterized by a fatty acid profile with (i) at least 7, 8, 9, 10, 11, 12, 13, or 14 area % 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.

12. The host cell of claim 9, wherein the host cell is a plastidic oleaginous cell having a type II fatty acid biosynthesis pathway.

13. The host cell of claim 12, wherein the host cell is a microalga.

14. The host cell of claim 13, wherein the host cell is of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca.

15. The host cell of claim 14, wherein the microalga is of the species Prototheca moriformis.

16. The host cell of claim 9, 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.

17. The host cell of claim 16, wherein the coding sequence contains the most preferred codon for at least 80% of the codons of the coding sequence.

18. A recombinant polynucleotide having at least 85% sequence identity or equivalent sequence by virtue of the degeneracy of the genetic code to SEQ ID NO:

28 encoding a 3-keto-acyl ACP synthase IV (KASIV) protein, or the complement of the polynucleotide, wherein said protein has KASIV activity.

19. A transformation vector comprising the polynucleotide of claim 18.

20. The vector of claim 19, comprising promoter and 3′

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

21. A host cell comprising the vector of claim 19.

22. The host cell of claim 21, wherein the host cell is a plastidic oleaginous cell having a type II fatty acid biosynthesis pathway.

23. The host cell of claim 22, wherein the host cell is a microalga.

24. The host cell of claim 23, wherein the host cell is of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca.

25. The host cell of claim 24, wherein the microalga is of the species Prototheca moriformis.

26. A host cell comprising

a recombinant polynucleotide having at least 85% sequence identity or equivalent sequence by virtue of the degeneracy of the genetic code to SEQ ID NO:

28, encoding a β

-keto-acyl ACP synthase IV (KASIV) protein or the complement of the polynucleotide, wherein said protein has KASIV activity.

27. The host cell of claim 26 further comprising a polynucleotide encoding a fatty acyl-ACP thioesterase B (FATB), wherein the thioesterase has at least 90% amino acid sequence identity to SEQ ID NO:

1 or SEQ ID NO;

57.

28. The host cell of claim 26, wherein the host cell produces a cell oil characterized by a fatty acid profile with (i) at least 7, 8, 9, 10, 11, 12, 13, or 14 area % 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.

29. The host cell of claim 26, wherein the host cell is a plastidic oleaginous cell having a type II fatty acid biosynthesis pathway.

30. The host cell of claim 29, wherein the host cell is a microalga.

31. The host cell of claim 30, wherein the host cell is of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca.

32. The host cell of claim 31, wherein the microalga is of the species Prototheca moriformis.

33. The host cell of claim 26, 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.

34. The host cell of claim 33, wherein the coding sequence contains the most preferred codon for at least 80% of the codons of the coding sequence.