Oleaginous bacterial cells and methods for producing lipids

Patent metadata

Field	Value
Publication number	US-9708593-B2
Application number	US-201314646986-A
Country	US
Kind code	B2
Filing date	Oct 16, 2013
Priority date	Nov 26, 2012
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

Abstract

This invention relates to cells and methods for producing lipids using cellulosic carbon source. More specifically the invention relates to oleaginous bacterial cells, wherein genes encoding at least one cellulolytic activity has been introduced. This invention also relates to methods for lipid production by cultivating an oleaginous bacterial strain or strains capable of expressing one or more cellulolytic activity.

First claim

The invention claimed is:

1. An oleaginous bacterial cell that accumulates at least 10% (w/w) of its biomass dry weight as lipids after cultivation in conditions for lipid production, wherein genes encoding at least one endocellulase, at least one exocellulase, at least one cellobiase, and at least one ABC type transporter capable of facilitating cellobiose uptake operably linked to a suitable promoter sequence have been introduced.

2. The oleaginous bacterial cell according to claim 1 , wherein the cell belongs to genus Rhodococcus.

3. The oleaginous bacterial cell according to claim 1 , wherein further the lipid production pathway has been modified.

4. A method for lipid production comprising the steps of
(a) providing a culturing medium where at least part of the carbon source is in cellulosic form;
(b) providing a bacterial cell capable of expressing and secreting one or more cellulolytic activity/cellulolytic activities;
(c) contacting said medium and said cell;
(d) cultivating said medium and said cell in conditions allowing expression of said cellulolytic activities;
(e) providing (i) the cellulolytic bacterial cell of (b) being oleaginous, or (ii) another oleaginous bacterial cell genetically modified to use cellobiose as a carbon source;
(f) contacting the culturing medium of step (c) or the spent culturing medium obtained from step (c) with said cell;
(g) cultivating said cell in conditions allowing lipid production; and
(h) recovering the lipids,
wherein at least one of the bacterial cell secreting cellulolytic activity in step (b) and the oleaginous bacterial cell of step (e) is the oleaginous bacterial cell of claim 1 .

5. The method according to claim 4 , wherein the bacterial cell secreting cellulolytic activity in step (b) and the oleaginous bacterial cell of step (e) are cultivated sequentially.

6. The method according to claim 4 , wherein the bacterial cell secreting cellulolytic activity in step (b) and the oleaginous bacterial cell of step (e) are cultivated simultaneously in the same culturing medium.

7. The method according to claim 4 , wherein the bacterial cell secreting cellulolytic activity in step (b) secretes endoglucanase and exoglucanase activities and the oleaginous bacterial cell according to step (e) expresses cellobiase activity.

8. The method according to claim 4 , wherein the oleaginous bacterial cell according to step (e) further expresses transporter(s) for cellobiose intake.

9. The method according to claim 4 , wherein the bacterial cell secreting cellulolytic activity in step (b) and the oleaginous bacterial cell of step (e) both are the oleaginous bacterial cell of claim 1 .

10. The method according to claim 4 , wherein at least 10%, preferably 30% of the carbon source is in cellulosic form.

11. The method according to claim 4 , wherein the carbon source is pretreated cellulose.

12. A method for lipid production comprising the steps of
(a) contacting the oleaginous bacterial cell of claim 1 with a culture medium wherein at least part of the carbon source is cellobiose, preferably cellobiose from hydrolysis of cellulosic substrate;
(b) cultivating the cell in conditions allowing for the lipid production; and
(c) recovering the lipids.

13. A method for lipid production comprising cultivating the oleaginous bacterial cell according to claim 1 in a culture medium under conditions allowing for the lipid production and recovering the lipids.

14. The oleaginous bacterial cell according to claim 1 , wherein the cell is a modified Rhodococcus opacus.

15. An oleaginous bacterial cell that accumulates at least 10% (w/w) of its biomass dry weight as lipids after cultivation in conditions for lipid production, wherein genes encoding at least one endocellulase, at least one exocellulase, at least one cellobiase, and at least one ABC type transporter capable of facilitating cellobiose uptake operably linked to a suitable promoter sequence have been introduced, and
wherein:
(i) the endocellulase is a polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 (CenA), SEQ ID NO: 3 (CenB), SEQ ID NO: 5 (CenC) or SEQ ID NO: 11 (Ce16A);
(ii) the exocellulase is a polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 9 (Cex) or SEQ ID NO: 7 (CbhA);
(iii) the cellobiase is a polypeptide comprising an amino acid sequence having at least 80% identity to polypeptide SEQ ID NO: 17 (BglC); and
(iv) the ABC type transporter is a polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 13 (Bg1A) or SEQ ID NO: 15 (Bg1B).

16. The oleaginous bacterial cell according to claim 15 ,
wherein:
(i) the endocellulase is a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 (CenA), SEQ ID NO: 3 (CenB), SEQ ID NO: 5 (CenC) or SEQ ID NO: 11 (Ce16A);
(ii) the exocellulase is a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 9 (Cex) or SEQ ID NO: 7 (CbhA);
(iii) the cellulobiose is a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 17 (BglC); and
(iv) the ABC type transporter is a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13 (Bg1A) or SEQ ID NO: 15 (Bg1B).

17. The oleaginous bacterial cell according to claim 15 ,
wherein:
(i) the endocellulase is a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 1 (CenA), SEQ ID NO: 3 (CenB), SEQ ID NO: 5 (CenC) or SEQ ID NO: 11 (Ce16A);
(ii) the exocellulase is a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 9 (Cex) or SEQ ID NO: 7 (CbhA);
(iii) the cellulobiose is a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 17 (BglC); and
(iv) the ABC type transporter is a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 13 (Bg1A) or SEQ ID NO: 15 (Bg1B).

18. The oleaginous bacterial cell according to claim 15 ,
wherein:
(i) the endocellulase is a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NO: 1 (CenA), SEQ ID NO: 3 (CenB), SEQ ID NO: 5 (CenC) and SEQ ID NO: 11 (Ce16A);
(ii) the exocellulase is a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NO: 9 (Cex) and SEQ ID NO: 7 (CbhA); and
(iii) the cellulobiose is a polypeptide comprising an amino acid sequence of SEQ ID NO: 17 (BglC); and
(iv) the ABC type transporter take is a polypeptide comprising an amino acid sequence of SEQ ID NO: 13 (Bg1A) or SEQ ID NO: 15 (Bg1B).

19. A method for lipid production comprising the steps of
(a) contacting the oleaginous bacterial cell of claim 15 with a culture medium wherein at least part of the carbon source is cellobiose, preferably cellobiose from hydrolysis of cellulosic substrate;
(b) cultivating the cell in conditions allowing for the lipid production; and
(c) recovering the lipids.

20. A method for lipid production comprising the steps of
(a) contacting the oleaginous bacterial cell of claim 16 with a culture medium wherein at least part of the carbon source is cellobiose, preferably cellobiose from hydrolysis of cellulosic substrate;
(b) cultivating the cell in conditions allowing for the lipid production; and
(c) recovering the lipids.

21. A method for lipid production comprising the steps of
(a) contacting the oleaginou

Assignees

• Neste Oil Oyj
• Neste Oyj

Inventors

• Hetzler Stephan
• Uthoff Stefan
• Broker Daniel
• Steinbuchel Alexander
• Koskinen Perttu

Classifications

• C12P7/6463

  obtained from glyceride producing microorganisms, e.g. single cell oil · CPC title

• Y02E50/13

  Cross-Sectional Technologies · mapped topic

• C12Y302/01006

  Endo-1,3(4)-beta-glucanase (3.2.1.6) · CPC title

• C12N9/2434

  acting on beta-1,4-glucosidic bonds · CPC title

• C12Y302/01021

  Beta-glucosidase (3.2.1.21) · CPC title