IP Library Granted Patent US 9,005,940
Granted Patent B2
US 9,005,940 · App. 14/132,051 · Granted Apr 14, 2015

Down-regulation of a polynucleotide encoding a Sou2 sorbitol utilization protein to modify lipid production in microbial cells

Inventors: Quinn Qun Zhu (West Chester, PA); Seung-Pyo Hong (Hockessin, DE); Dongming Xie (Newark, DE); Zhixiong Xue (Chadds Ford, PA); Hyeryoung Yoon (Wilmington, DE); Michael Dauner (Wilmington, DE)
Assignee: E I du Pont de Nemours and Company
C12P7/6427C12P7/6472C12N9/0036
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Quick Facts
Patent No.
US 9,005,940
App. No.
14/132,051
Granted
Apr 14, 2015
Kind
B2
Abstract

Recombinant microbial cells are disclosed herein that comprise (i) a down-regulation of an endogenous polynucleotide sequence encoding Sou2 sorbitol utilization protein, and (ii) a polyunsaturated fatty acid (PUFA) biosynthetic pathway. The down-regulation of the polynucleotide sequence encoding Sou2 sorbitol utilization protein can increase the lipid content of the microbial cells and/or decrease the total amount of sugar alcohols produced by the microbial cells. Also disclosed are methods of using the recombinant microbial cells to produce oil containing omega-3 polyunsaturated fatty acids such as EPA.

Claims (22)

1. A recombinant microbial cell comprising:

(i) a down-regulation of an endogenous polynucleotide sequence encoding Sou2 sorbitol utilization protein comprising the amino acid sequence of an Sou2 sorbitol utilization protein selected from the group consisting of Asperqillus niger, Bifidobacterium dentium, Candida albicans, Candida dubliniensis, Candida orthopsilosis, Candida parapsilosis, Cordyceps militaris, Debaryomyces hansenii, Enterobacter hormaechei, Gaeumannomvces graminis, Glarea lozovensis, Lodderomyces elonqisporus, Maqnaporthe oryzae, Metarhizium acridum, Metarhizium anisopliae, Mycobacterium smeqmatis, Neurospora crassa, Neurospora tetrasperma, Paracoccidioides brasiliensis, Penicillium chrysoqenum, Scheffersomvces stipitis, Uncinocarpus reesii, Verticillium dahliae , and Yarrowia lipolytica , and

(ii) a polyunsaturated fatty acid (PUFA) biosynthetic pathway, wherein said down-regulation increases the lipid content of the microbial cell and/or decreases the total amount of sugar alcohols produced by the microbial cell, as compared to a suitable control cell.

2. The recombinant microbial cell of claim 1 , wherein said down-regulation is due to a mutation of said endogenous polynucleotide sequence.

3. The recombinant microbial cell of claim 2 , wherein said mutation is selected from the group consisting of a substitution, deletion and insertion.

4. The recombinant microbial cell of claim 3 , wherein said mutation is a deletion that removes

(i) one or more nucleotides from an open reading frame encoding said Sou2 sorbitol utilization protein, and/or

(ii) one or more nucleotides of a non-protein-coding sequence located within 500 base pairs of the 5′-end of said open reading frame.

5. The recombinant microbial cell of claim 3 , wherein said mutation is an insertion that occurs within

(i) an open reading frame encoding said Sou2 sorbitol utilization protein, or

(ii) a non-protein-coding sequence located within 500 base pairs of the 5′-end of said open reading frame.

6. The recombinant microbial cell of claim 1 , wherein said PUFA pathway produces at least one PUFA selected from the group consisting of linoleic acid, alpha-linolenic acid, gamma-linolenic acid, stearidonic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosatetraenoic acid, omega-3 docosapentaenoic acid, omega-6 docosapentaenoic acid and docosahexaenoic acid.

7. The recombinant microbial cell of claim 6 , wherein said PUFA pathway produces eicosapentaenoic acid.

8. The recombinant microbial cell of claim 1 , wherein said sugar alcohols comprise arabitol or mannitol.

9. The recombinant microbial cell of claim 1 , wherein said microbial cell is an oleaginous yeast cell.

10. The recombinant microbial cell of claim 9 , wherein said oleaginous yeast is a Yarrowia lipolytica cell.

11. The recombinant microbial cell of claim 10 , wherein the Sou2 sorbitol utilization protein comprises the amino acid sequence set forth in SEQ ID NO:10.

12. The recombinant microbial cell of claim 10 , wherein said endogenous polynucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO:9.

13. A method of producing a microbial oil comprising a polyunsaturated fatty acid (PUFA), said method comprising:

a) culturing the recombinant microbial cell of claim 1 , wherein a microbial oil comprising a PUFA is produced; and

b) optionally recovering the microbial oil of step (a).

14. The method of claim 13 , wherein the microbial oil comprises eicosapentaenoic acid.

Assignments (3)
CORRECTIVE ASSIGNMENT TO CORRECT THE DESIGNATION OF ASSIGNEE AS A DELAWARE CORPORATION TO A DELAWARE LIMITED LIABILITY COMPANY PREVIOUSLY RECORDED ON REEL 049880 FRAME 0001. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Recorded Aug 28, 2019
From: E. I. DU PONT DE NEMOURS AND COMPANY
To: DUPONT US HOLDING, LLC
Reel/Frame 051502/0610 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 26, 2019
From: E. I. DU PONT DE NEMOURS AND COMPANY
To: DUPONT US HOLDING, LLC
Reel/Frame 049880/0001 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 24, 2014
From: ZHU, QUINN QUN; HONG, SEUNG-PYO; XIE, DONGMING; XUE, ZHIXIONG; YOON, HYERYOUNG; DAUNER, MICHAEL
To: E. I. DU PONT DE NEMOURS AND COMPANY
Reel/Frame 032286/0246 →
Continuity (2)
Provisional Application 61740506 · Dec 21, 2012
Related Publication 20140220645A1 · Aug 7, 2014