Gene disruptants producing fatty acyl-CoA derivatives

• US 9,157,103 B2
• Filed: 12/19/2011
• Issued: 10/13/2015
• Est. Priority Date: 12/23/2010
• Status: Active Grant

• Current Assignee: Shell Oil Company (Shell Plc)
• Original Assignee: Shell Oil Company (Shell Plc)
• Inventors: Hattendorf, Douglas A., Shock, Jennifer L., Clark, Louis
• Primary Examiner(s): RAMIREZ, DELIA M
• Application Number:
  US13/330,650
• Publication Number:
  US 20120165562A1
• Time in Patent Office: 1,394 Days
• Field of Search: 435/252.3, 435/254.11, 435/252.2, 435/257.2, 435/69.1, 435/440, 435/189, 435/191, 530/350
• US Class Current: 1/1
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12P 7/62 Carboxylic acid esters
• 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: C12Y 102/01042 Hexadecanal dehydrogenase (acylating) (1.2.1.42), i.e. fatty acyl-CoA reductase
• CPC Class Codes: C12Y 102/0105 Long-chain-fatty-acyl-CoA reductase (1.2.1.50)
• CPC Class Codes: C12Y 102/01084 Alcohol-forming fatty acyl-CoA reductase (1.2.1.84)

Abstract

This invention provides microbial organisms, particularly yeasts such as Yarrowia lipolytica, that have one or more disrupted genes. The gene disruption(s) may yield improved production of fatty acyl-CoA derivatives.

Claims

1. A Y. lipolytica recombinant yeast cell comprising:

(a) at least one genetic modification in the YALI0C17545 endogenous gene and at least one genetic modification in the YALI0E28534 endogenous gene; and

(b) an exogenous gene encoding a fatty acyl reductase (FAR) protein, wherein the exogenous gene is operably linked to a promoter, wherein the FAR protein has at least 80% amino sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO;

2, 4, 6, and 65-73, wherein the at least one genetic modification comprises;

(i) a deletion of all or a portion of the coding region of the endogenous gene;

(ii) a mutation in the endogenous gene such that the gene encodes a polypeptide having reduced activity;

(iii) a modified regulatory sequence that reduces expression of the endogenous gene;

or (iv) any combination of (i)-(iii).

2. The Y. lipolytica recombinant yeast cell of claim 1 further comprising at least one genetic modification in the YALI0E28336 endogenous gene and/or the YALI0E11099 endogenous gene, wherein the at least one genetic modification comprises:

(i) a deletion of all or a portion of the coding region of the endogenous gene;

(ii) a mutation in the endogenous gene such that the gene encodes a polypeptide having reduced activity;

(iii) a modified regulatory sequence that reduces expression of the endogenous gene;

or (iv) any combination of (i)-(iii).

3. The Y. lipolytica recombinant yeast cell of claim 2 comprising at least one genetic modification in each of the YALI0E28336 endogenous gene and the YALI0E11099 endogenous gene, wherein the at least one genetic modification comprises:

(i) a deletion of all or a portion of the coding region of the endogenous gene;

(ii) a mutation in the endogenous gene such that the gene encodes a polypeptide having reduced activity;

(iii) a modified regulatory sequence that reduces expression of the endogenous gene;

or (iv) any combination of (i)-(iii).

4. The Y. lipolytica recombinant yeast cell of claim 1 further comprising at least one genetic modification in at least one endogenous gene selected from the group consisting of YALI0B10406, YALI0A19536, YALI0E32769, YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212, YALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, and YALI0B17512, wherein the at least one genetic modification comprises:

(i) a deletion of all or a portion of the coding region of the endogenous gene;

(ii) a mutation in the endogenous gene such that the gene encodes a polypeptide having reduced activity;

(iii) a modified regulatory sequence that reduces expression of the endogenous gene;

or (iv) any combination of (i)-(iii).

5. The Y. lipolytica recombinant yeast cell of claim 4 comprising at least one genetic modification in the YALI0B17512 endogenous gene, wherein the at least one genetic modification comprises:

(i) a deletion of all or a portion of the coding region of the endogenous gene;

(ii) a mutation in the endogenous gene such that the gene encodes a polypeptide having reduced activity;

(iii) a modified regulatory sequence that reduces expression of the endogenous gene;

or (iv) any combination of (i)-(iii).

6. The Y. lipolytica recombinant yeast cell of claim 1, wherein multiple copies of the exogenous gene are expressed.

7. The Y. lipolytica recombinant yeast cell of claim 1, further comprising a second exogenous gene that encodes a fatty acid synthase (FAS), an ester synthase, an acyl-ACP thioesterase (TE), a fatty acyl-CoA synthase (FACS), an acetyl-CoA carboxylase (ACC), a xylose isomerase, or an invertase.

8. The Y. lipolytica recombinant yeast cell of claim 1, wherein said Y. lipolytica recombinant yeast cell exhibits at least one-fold increase in the production of a fatty acyl-CoA derivative relative to the corresponding Y. lipolytica yeast cell lacking the at least one modification in the YALI0C17545 endogenous gene and the at least one genetic modification in the YALI0E28534 endogenous gene.

10. A method of producing a fatty acyl-CoA derivative, the method comprising culturing the Y. lipolytica recombinant yeast cell of claim 1 under conditions in which the fatty acyl-CoA derivative is produced.

11. The method of claim 10, wherein the fatty acyl-CoA derivative is a fatty alcohol, fatty acid, fatty aldehyde, fatty ester, fatty acetate, wax ester, alkane, or alkene.

12. The method of claim 10, comprising:

contacting a cellulose-containing biomass with one or more cellulases to yield fermentable sugars; and

contacting said Y. lipolytica recombinant yeast cell with the fermentable sugars under conditions in which the fatty acyl-CoA derivative is produced.

13. The method of claim 10, wherein at least 5 g/L of fatty acyl-CoA derivatives per liter of culture medium is produced.

9. An isolated Y. lipolytica recombinant yeast cell comprising (i) an exogenous gene encoding a fatty acyl reductase (FAR) protein, wherein the exogenous gene is operably linked to a promoter, wherein the FAR protein has at least 80% amino sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

2, 4, 6, and 65-73, and (ii) at least one genetic modification in each of the two or more endogenous genes selected from;

(a) the YALI0C17545 and YALI0E28534 endogenous genes;

(b) the YALI0C17545, YALI0E28336, and YALI0E28534 endogenous genes;

(c) the YALI0C17545, YALI0E28534, and YALI0B17512 endogenous genes; and

(d) the YALI0C17545, YALI0E28336, YALI0E11099, and YALI0E28534 endogenous genes,

wherein the at least one genetic modification comprises;

(A) a deletion of all or a portion of the coding region of the endogenous gene;

(B) a mutation in the endogenous gene such that the gene encodes a polypeptide having reduced activity;

(C) a modified regulatory sequence that reduces expression of the endogenous gene;

or (D) any combination of (A)-(C).

14. An isolated Y. lipolytica recombinant yeast cell comprising at least one genetic modification in the YALI0C17545 endogenous gene and at least one genetic modification in the YALI0E28534 endogenous gene;

wherein the at least one genetic modification comprises;

(i) a deletion of all or a portion of the coding region of the endogenous gene;

(ii) a mutation in the endogenous gene such that the gene encodes a polypeptide having reduced activity;

(iii) a modified regulatory sequence that reduces expression of the endogenous gene;

or (iv) any combination of (i)-(iii).