PRODUCTION OF DICARBOXYLIC ACIDS BY IMPROVED MUTANT STRAINS OF YARROWIA LIPOLYTICA

• US 20100041115A1
• Filed: 12/13/2005
• Published: 02/18/2010
• Est. Priority Date: 12/15/2004
• Status: Abandoned Application

• Current Assignee: Centre National De La Recherche Scientifique, Institut Francais Du Petrole, Institut National de La Recherche Agronomique
• Original Assignee: Centre National De La Recherche Scientifique, Institut Francais Du Petrole, Institut National de La Recherche Agronomique
• Inventors: Thevenieau, France, Marchal, Rémy, Le Dall, Marie-Thérèse, Nicaud, Jean-Marc
• Application Number:
  US11/721,726
• Publication Number:
  US 20100041115A1
• Time in Patent Office: Days
• US Class Current: 435/145
• CPC Class Codes: C12N 9/0042 NADPH-cytochrome P450 reductase (1.6.2.4)
• CPC Class Codes: C12N 9/0077 with a reduced iron-sulfur protein as one donor (1.14.15)
• CPC Class Codes: C12P 7/44 Polycarboxylic acids

Abstract

The invention concerns a method for producing dicarboxylic acids (DCA) with long hydrocarbon chains, also called diacids, which consists in culturing a mutant strain of Yarrowia lipolytica obtained by mutagenesis directed and more particularly disrupted at least for the POX2, POX3, POX4 and POX5 genes encoding acyl-CoA oxydase, in a medium consisting essentially of an energetic substrate including at least one carbon source and one nitrogen source and in subjecting said strain to a bioconversion substrate selected among n-alkanes of at least 10 carbon atoms, fatty acids of at least 10 carbon atoms, their alkyl esters and natural oils.

Claims

1. A method of producing dicarboxylic acids, comprising:

(a) a growth stage wherein a mutant strain of Yarrowia lipolytica disrupted at least for the POX2, POX3, POX4 and POX5 genes (coding for acyl-CoA oxidase) is cultured in a culture medium essentially consisting of an energetic substrate comprising at least a source of carbon and a source of nitrogen,

(b) a bioconversion stage wherein said strain is subjected to a bioconversion substrate selected from among the n-alkanes having at least 10 carbon atoms, fatty acids having at least 10 carbon atoms, alkyl esters having 1 to 4 carbon atoms of these fatty acids and natural oils, in the presence of an energetic substrate, and

(c) a stage of recovering the dicarboxylic acid formed.

2. A method as claimed in claim 1, characterized in that the mutant used is MTLY37.

3. A method as claimed in claim 1, characterized in that the mutant used is MTLY79 overexpressing the CPR and ALK1 genes.

4. A method as claimed in claim 1, characterized in that the mutant used is MTLY80 overexpressing the CPR and ALK2 genes.

5. A method as claimed in claim 1, characterized in that the mutant used is MTLY81 overexpressing the CPR gene.

6. A method as claimed in claim 1, characterized in that the mutant used is FT120 overexpressing the CPR gene.

7. A method as claimed in claim 1, characterized in that the mutant used is FT130 overexpressing the CPR gene.

8. A method as claimed in claim 1, characterized in that said bioconversion substrate consists of a mixture of methyl esters or of a mixture of ethyl esters.

9. A method as claimed in claim 1, characterized in that said bioconversion substrate consists of an oleic sunflower oil.

10. A method as claimed in claim 1, characterized in that, in the bioconversion stage, the culture medium comprises peptone.

11. A method as claimed in claim 1, characterized in that, in the bioconversion stage, the culture medium comprises a supply of secondary energetic substrate consisting of at least one polyhydroxyl compound.

12. A method as claimed in claim 9, characterized in that said polyhydroxyl compound is glycerol or a sugar.

13. A method as claimed in claim 1, characterized in that, in stage (c), the dicarboxylic acid is recovered by precipitation in form of calcium salt.

14. A method of obtaining a mutant Yarrowia lipolytica auxotrophic strain MTLY66, Leu−

Ura−

, from the prototrophic mutant MTLY37, usable for transformation with, as the selection markers, the LEU2 and URA3 genes, characterized in that the conversion operations of stages 1 to 3 of the table hereafter are carried out;

Conversion operations Stage Mutant to be converted Conversion with Converted mutant 1 MTLY37, Leu+, Ura+, Fragment of PCR ura3- MTLY40, Leu+, Ura−

, Δ

5- Δ

5-PT, Δ

2-PT, Δ

3-PT, 41, 5FOA selection PT, Δ

2-PT, Δ

3-PT, Δ

4-PUT Δ

4-Pura3-41T 2 MTLY40, Leu+, Ura−

, Δ

5- PHTleu2 cassette, MTLY64, Leu−

, Ura−

, PT, Δ

2-PT, Δ

3-PT, hygromycin selection Hyg+, Δ

5-PT, Δ

2-PT, Δ

3- Δ

4-Pura3-41T PT, Δ

4-Pura3-41T, Leu2;

;

Hyg 3 MTLY64, Leu−

, Ura−

, pRRQ2 vector, Leu+ MTLY66, Leu−

, Ura−

, Δ

5- Hyg+, Δ

5-PT, Δ

2-PT, Δ

3- selection, checking Hyg−

, PT, Δ

2-PT, Δ

3-PT, PT, Δ

4-Pura3-41T, loss of plasmid pRRQ2 Δ

4-Pura3-41T, Δ

leu2 Leu2;

;

Hyg on YPD, isolation of Leu−

15. A method of obtaining, from mutant MTLY66, a mutant Yarrowia lipolytica strain MTLY74 Leu+ Ura−

that overexpresses the CPR gene coding for NADPH-cytochrome reductase under the bioconversion conditions, by conversion of the JMP21-CPR vector containing selection marker LEU2 and the expression cassette with the CPR gene under the control of promoter pPOX2 inducible by the fatty acids, fatty acid esters or natural oils.

16. A method of obtaining, from mutant Yarrowia lipolytica strain MTLY74, a mutant Yarrowia lipolytica strain MTLY79 that overexpresses the genes coding for NADPH-cytochrome reductase and for cytochrome P450 monooxygenase under the bioconversion conditions, by conversion of the JMP21-ALK1 vector containing selection marker URA3 and the expression cassette with the ALK1 gene under the control of promoter pPOX2 inducible by the fatty acids, fatty acid esters or natural oils.

17. A method of obtaining, from mutant Yarrowia lipolytica strain MTLY74, a mutant Yarrowia lipolytica strain MTLY80 that overexpresses the genes coding for NADPH-cytochrome reductase and for cytochrome P450 monooxygenase ALK2 under the bioconversion conditions, by conversion of the JMP61-ALK2 vector containing selection marker URA3 and the expression cassette with the ALK2 gene under the control of promoter pPOX2 inducible by the fatty acids, fatty acid esters or natural oils.

18. A method of obtaining a mutant Yarrowia lipolytica strain MTLY81 from mutant strain MTLY74, that overexpresses the CPR gene coding for NADPH-cytochrome reductase under the bioconversion conditions, by expressing it under the control of promoter pPOX2 induced by the bioconversion substrates, of fatty acid, fatty acid ester or natural oil type and that is prototrophic, by making mutant MTLY 74 prototrophic by transformation with the JMP61 plasmid carrying marker URA3.

19. A method of obtaining, from mutant Yarrowia lipolytica strain MTLY66, a mutant Yarrowia lipolytica strain FT120 Leu−

Ura−

, characterized in that the conversion operations of stages 1 to 4 of the table hereafter are carried out;

Conversion operations Stage Mutant to be converted Conversion cassette Converted mutant 1 MTLY66, Leu−

, Ura−

, POX1-PHT MTLY82, Leu−

, Ura−

, Hyg−

, Δ

5-PT, Δ

2-PT, Δ

3- Hyg+, Δ

5-PT, Δ

2-PT, Δ

3- PT, Δ

4-Pura3-41T PT, Δ

4-Pura3-41T, Δ

1- PHT 2 MTLY82, Leu−

, Ura−

, pRRQ2 vector, Leu+ MTLY85 Leu−

, Ura−

, Hyg−

, Hyg+, Δ

5-PT, Δ

2-PT, Δ

3- selection, checking Hyg−

, Δ

5-PT, Δ

2-PT, Δ

3-PT, PT, Δ

4-Pura3-41T, Δ

1- loss of plasmid pRRQ2 Δ

4-Pura3-41T, Δ

1-PT PHT on YPD, isolation of Leu−

3 MTLY85 Leu−

, Ura−

, Hyg−

, POX6-PHT MTLY92 Leu−

, Ura−

, Δ

5-PT, Δ

2-PT, Δ

3-PT, Hyg+, Δ

5-PT, Δ

2-PT, Δ

3- Δ

4-Pura3-41T, Δ

1-PT PT, Δ

4-Pura3-41T, Δ

1- PT, Δ

6-PHT 4 MTLY92 Leu−

, Ura−

, pRRQ2 vector, Leu+ MTLY95 Leu−

, Ura−

, Hyg−

, Hyg+, Δ

5-PT, Δ

2-PT, Δ

3- selection, checking Hyg−

, Δ

5-PT, Δ

2-PT, Δ

3-PT, PT, Δ

4-Pura3-41T, Δ

1- loss of plasmid pRRQ2 Δ

4-Pura3-41T, Δ

1-PT, PT, Δ

6-PHT on YPD, isolation of Leu−

Δ

6-PT 5 MTLY95 Leu−

, Ura−

, Hyg−

, Expression cassette FT101, Leu+, Ura−

, Hyg−

, Δ

5-PT, Δ

2-PT, Δ

3-PT, pPOX2-CPR of JM21- Δ

5-PT, Δ

2-PT, Δ

3-PT, Δ

4-Pura3-41T, Δ

1-PT, LEU2ex-CPR Δ

4-Pura3-41T, Δ

1-PT, Δ

6-PT Δ

6-PT, CPR-LEU2ex 6 FT101, Leu+, Ura−

, Hyg−

, pUB4-CRE vector, Hyg+ FT120, Leu−

, Ura−

, Hyg−

, Δ

5-PT, Δ

2-PT, Δ

3-PT, selection, checking Leu−

, Δ

5-PT, Δ

2-PT, Δ

3-PT, Δ

4-Pura3-41T, Δ

1-PT, loss of plasmid pUB4- Δ

4-Pura3-41T, Δ

1-PT, Δ

6-PT, CPR-LEU2ex CRE on YPD, isolation of Δ

6-PT, CPR Hyg−

and in that construction of the mutant strain FT101 Leu+ Ura−

is performed from Yarrowia lipolytica strain MTLY95, that overexpresses the gene coding (CPR) for NADPH-cytochrome reductase under the bioconversion conditions, by conversion of the JMP21-LEU2ex-CPR vector containing the excisable selection marker LEU2 and the CPR gene under the control of promoter pPOX2 inducible by the fatty acids, fatty acid esters or natural oils, strain FT120 being obtained after excision of marker LEU2ex by conversion with the pUB4-CRE plasmid, Hyg+ selection, loss of plasmid on YPD and finally isolation of a clone Leu−

.

20. A method of obtaining from mutant Yarrowia lipolytica strain FT120, a mutant Yarrowia lipolytica strain FT130, characterized in that the following conversion stage is carried out:

Conversion operations Conversion Stage Mutant to be converted cassette Converted mutant 1 FT120, Leu−

, Ura−

, DGA1-PUT FT130, Leu−

, Ura+, Hyg−

, Hyg−

, Δ

5-PT, Δ

2-PT, Δ

5-PT, Δ

2-PT, Δ

3-PT, Δ

3-PT, Δ

4-Pura3-41T, Δ

4-Pura3-41T, Δ

1-PT, Δ

1-PT, Δ

6-PT, CPR Δ

6-PT, CPR, Δ

dga1-PUT

21. A new mutant Yarrowia lipolytica strain MTLY66.

22. A new mutant Yarrowia lipolytica strain MTLY81.

23. A new mutant Yarrowia lipolytica strain FT120.

24. A new mutant Yarrowia lipolytica strain FT130.