Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
NOVEL BETA-OXIDATION PATHWAY AND HEMIASCOMYCETES YEAST MUTANTS
Document Type and Number:
WIPO Patent Application WO/2012/130769
Kind Code:
A1
Abstract:
The present invention relates generally to the fields of microbiology and biotechnology. More precisely, the present invention relates to novel paralog genes of hemiascomycetes yeast designated ECH1, ECH2, and ECH3, which are addressed to the mitochondria, isolated nucleic acid molecules encoding the novel enoyl-CoA hydratase members and corresponding amino acid sequences thereof. The present invention also relates to isolated biologically pure hemiascomycetes yeast strains comprising unique combinations of metabolic pathways for the catabolism or degradation of fatty acid and other substrates. Finally, the present invention provides for novel mutants that can yield improved production of such commodity chemicals, as well as a method of increasing the production of commodity chemicals using the isolated biologically pure hemiascomycetes yeast strains.

Inventors:
NOEL THIERRY (FR)
ACCOCEBERRY ISABELLE (FR)
BESSOULE JEAN JACQUES (FR)
MANON STEPHEN THIERRY (FR)
GABRIEL FREDERIC (FR)
Application Number:
PCT/EP2012/055244
Publication Date:
October 04, 2012
Filing Date:
March 23, 2012
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
UNIV BORDEAUX SEGALEN (FR)
CENTRE NAT RECH SCIENT (FR)
CT HOSPITALIER UNIVERSITAIRE DE BORDEAUX (FR)
NOEL THIERRY (FR)
ACCOCEBERRY ISABELLE (FR)
BESSOULE JEAN JACQUES (FR)
MANON STEPHEN THIERRY (FR)
GABRIEL FREDERIC (FR)
International Classes:
C07K14/40; C12N9/02; C12N9/88; C12P7/64
Other References:
GERALDINE BUTLER ET AL: "Evolution of pathogenicity and sexual reproduction in eight Candida genomes", NATURE, vol. 459, no. 7247, 4 June 2009 (2009-06-04), pages 657 - 662, XP055035717, ISSN: 0028-0836, DOI: 10.1038/nature08064
DATABASE UniProt [online] 8 March 2011 (2011-03-08), XP002681922, Database accession no. C4Y798
DATABASE UniProt [online] 8 March 2011 (2011-03-08), XP002681923, Database accession no. C4XYI9
DATABASE UniProt [online] 8 March 2011 (2011-03-08), XP002681924, Database accession no. C4XZB0
L. Y. YOUNG ET AL: "Disruption of Ergosterol Biosynthesis Confers Resistance to Amphotericin B in Candida lusitaniae", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 47, no. 9, 1 September 2003 (2003-09-01), pages 2717 - 2724, XP055004395, ISSN: 0066-4804, DOI: 10.1128/AAC.47.9.2717-2724.2003
F. PEYRON ET AL: "Sterol and Fatty Acid Composition of Candida lusitaniae Clinical Isolates", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 46, no. 2, 1 February 2002 (2002-02-01), pages 531 - 533, XP055004396, ISSN: 0066-4804, DOI: 10.1128/AAC.46.2.531-533.2002
VILJOEN B C ET AL: "THE SIGNIFICANCE OF CELLULAR LONG-CHAIN FATTY ACID COMPOSITIONS AND OTHER CRITERIA IN THE STUDY OF THE RELATIONSHIP BETWEEN SPOROGENOUS ASCOMYCETE SPECIES AND ASPOROGENOUS CANDIDA SPECIES", SYSTEMATIC AND APPLIED MICROBIOLOGY, vol. 12, no. 1, 1989, pages 80 - 90, XP008140646, ISSN: 0723-2020
M. A. RAMIREZ ET AL: "Mutations in Alternative Carbon Utilization Pathways in Candida albicans Attenuate Virulence and Confer Pleiotropic Phenotypes", EUKARYOTIC CELL, vol. 6, no. 2, 1 February 2007 (2007-02-01), pages 280 - 290, XP055004398, ISSN: 1535-9778, DOI: 10.1128/EC.00372-06
HILTUNEN J K ET AL: "PEROXISOMAL MULTIFUNCTIONAL BETA-OXIDATION PROTEIN OF SACCHAROMYCES-CEREVISIAE MOLECULAR ANALYSIS OF THE FOX2 GENE AND GENE PRODUCT", JOURNAL OF BIOLOGICAL CHEMISTRY, THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, INC., BALTIMORE, MD, US, vol. 267, no. 10, 1 January 1992 (1992-01-01), pages 6646 - 6653, XP002143028, ISSN: 0021-9258
WANDERS RJA ET AL., FEBS J., vol. 278, 2011, pages 182 - 194
HILTUNEN JK; QINYM, MOL. CELL. BIOL. LIPIDS, vol. 1484, 2000, pages 117 - 128
BARTLETT K. ET AL., EUR. J. BIOCHEM., vol. 271, 2004, pages 462 - 469
KENNEDY EP ET AL., J. BIOL. CHEM., vol. 185, 1950, pages 275 - 285
BOISNARD S. ET AL., FUNG.GENET.BIOL., vol. 46, 2009, pages 55 - 66
FERRON G ET AL., FEMS MICROBIOL. LETT, vol. 250, 2005, pages 63 - 69
WANDERS R.J.A. ET AL., J INHERIT METAB DIS, vol. 33, 2010, pages 479 - 494
JACKSON ET AL., BBRC, vol. 214, 1995, pages 247 - 253
SHEN ET AL., FUNCT.LNTEGR.GENOMICS, vol. 73, 2009, pages 1239 - 1242
ENGEL ET AL., EMBO J., vol. 15, 1996, pages 5135 - 5145
MAGGIO-HALL ET AL., MOL.MICROBIOL., vol. 54, 2004, pages 1173 - 1185
"Computational Molecular Biology", 1988, OXFORD UNIVERSITY PRESS
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Computer Analysis of Sequence Data, Part 1", 1994, HUMANA PRESS
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
"Sequence Analysis Primer", 1991, STOCKTON PRESS
CARILLO H ET AL., APPLIED MATH, vol. 48, 1988, pages 1073
HILTUNEN ET AL., JBC, vol. 267, no. 10, 5 April 1992 (1992-04-05), pages 6646 - 6653
HILTUNEN ET AL., J. BIOL. CHEM., vol. 267, 1992, pages 6646 - 53
EL-KIRAT-CHATEL K ET AL., YEAST, vol. 28, 2011, pages 321 - 330
THOMAS D. BROCK: "Biotechnology: A Textbook of Ihdustrial Microbiology", 1989, SINAUER ASSOCIATES
DESHPANDE, MUKUND V., APPL. BIOCHEM. BIOTECHNOL., vol. 36, 1992, pages 227
SCHERER S. ET AL., J.CLIN.MICROBIOL., vol. 25, pages 675 - 679
ALTSCHUL S. ET AL., NUCL. ACIDS RES., vol. 25, 1997, pages 3389 - 3402
CANDIDA GENOME DATABASE, Retrieved from the Internet
FRANQOIS ET AL., YEAST, vol. 39, 2004, pages 3906 - 3914
BRANDT A. ET AL., EUKARYOTIC CELL, vol. 3, 2004, pages 900 - 909
ALTSCHUL S. ET AL., NUCL.ACIDS RES., vol. 35, 1997, pages 14 - 17
DISTEL B. ET AL., METH.MOL.BIOL, vol. 313, 2006, pages 21 - 26
MANON ET AL., EUR.J.BIOCHEM, vol. 172, 1988, pages 205 - 211
HILTUNEN JK ET AL., FEMS MICROBIOL. REV, vol. 27, 2003, pages 35 - 64
PIEKARSKA K ET AL., EUKARYOTIC CELL, vol. 5, pages 1847 - 1856
RAMIREZ MA ET AL., EUKARYOTIC CELL, vol. 6, 2007, pages 280 - 290
LORENZ MC ET AL., NATURE, vol. 412, 2001, pages 83 - 86
RAMIREZ MA. ET AL., EUKARYOTIC CELL, vol. 6, 2007, pages 280 - 290
SHANI N. ET AL., PNAS, vol. 92, 1995, pages 6012 - 6016
Attorney, Agent or Firm:
LECCA, Patricia (21 rue de Fecamp, Paris, FR)
Download PDF:
Claims:
CLAIMS

1. An isolated nucleic acid molecule selected from the group consisting of:

(i) a nucleic acid molecule comprising a nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, or 95 % identity with any one of the nucleotide sequence as set forth in SEQ ID NO:

8, 10, or 12, which are designated ECH1, ECH2, and ECH3 respectively, and encoding a mitochondrial enoyl-CoA hydratase activity, or a complementary nucleotide sequence thereof;

(ii) a nucleic acid molecule comprising a fragment of at least 15 consecutive nucleotides of a nucleotide sequence as set forth in any one of SEQ ID NOs: 7, 8, 10, or 12, or a complementary nucleotide sequence thereof;

(iii) a nucleic acid molecule that hybridizes under high stringency conditions with a nucleotide sequence as set forth in any one of SEQ ID NO: 7, 8, 10, or 12, or a complementary nucleotide sequence thereof, or a fragment thereof;

(iv) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 9, 11, or 13; and

(v) a nucleic acid molecule which encodes a fragment of a polypeptide comprising at least 15 contiguous amino acids of the sequence as set forth in any one of SEQ ID NO: 9, 11, or 13.

2. A nucleotide probe or primer specific of ECH1, ECH2, or ECH3 gene, wherein the nucleotide probe or primer is selected from the group consisting of: (i) a probe or primer comprising a nucleotide sequence as set forth in any one of SEQ ID NO: 9, 10, or 12, or a complementary nucleotide sequence thereof; and (ii) a probe or primer comprising at least 15 consecutive nucleotides of a nucleotide sequence as set forth any one of SEQ ID NOs: 7, 8, 10, or 12, or a complementary nucleotide sequence thereof 3. The probe or primer of claim 2, comprising a marker compound.

4. A method of amplifying a region of the nucleotide sequence according to claim 1, wherein the method comprises:

d) selecting two nucleotide primers from the group consisting of a nucleotide primer comprising at least 15 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NO: 7, 8, 10, or

12;

e) contacting the nucleic acid with two nucleotide primers, wherein a first nucleotide primer hybridizes at a position 5' of the region of the nucleic acid, and a second nucleotide primer hybridizes at a position 3' of the region of the nucleic acid, in the presence of reagents necessary for an amplification reaction; and

f) detecting the amplified nucleic acid region.

5. A vector comprising a nucleotide sequence according to any one of claims 1-4. 6. A recombinant host cell comprising the vector according to claim 5. An isolated polypeptide selected from the group consisting of:

(i) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, or 95 % identity with any one of the nucleotide sequence as set forth in any one of SEQ ID NO: 7, 8, 10, or 12;

(ii) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 9, 11, or 13;

(iii) a polypeptide fragment or functional variant of a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 9, 11, or 13, or catalytically active fragments thereof and

(iv) a polypeptide homologous to a polypeptide comprising amino acid sequence as set forth in any one of SEQ ID NO: 9, 11, or 13, or a functional equivalents thereof or catalytically active fragments thereof.

8. An hemiascomycetes yeast strain comprising at least one of the nucleic acid molecule as defined in claim 1, and comprising as metabolic pathways for the catabolism of fatty acid at least one peroxisomal β- oxidation FOX2 independent pathway, at least one peroxisomal β-oxidation FOX2 dependent pathway, and at least one mitochondrial β-oxidation FOX2 dependent pathway,

9. The hemiascomycetes yeast strain of claim 8, comprising as metabolic pathways for the catabolism of fatty acid (i) at least one FOX2 independent peroxisomal β-oxidation pathway (ii) at least one FOX2 dependent peroxisomal β-oxidation pathway, and (iii) one FOX2 dependent mitochondrial β-oxidation pathway, wherein said strain still retains the capacity to grow on fatty acid carbon source when rendered non-functional for the FOX2 gene. 10. The hemiascomycetes yeast strain according to claim 8 or 9, which is selected among the strains of the genus Candida, Debaryomyces, Clavispora, Metschnikowia, Kluyveromyces, Lodderomyces, Pichia, Hansenula, Lipomyces, Yarrowia, and Geotrichum; wherein strain of the genus Candida is preferably selected among C. tropicalis, C. parapsilosis, C. orthopilosis, C. metapsilosis, C. norvegensis, C. antartica, C. dubliniensis, C. haemulonii, C. kefyr, C. krusei, C. lusitaniae, C. maltosa, and C. oleophila; and most preferably belongs to the species Candida lusitaniae or Clavispora lusitaniae; wherein strain of the genus Debaryomyces is preferably selected among D. hansenii and D. castelli. Yeasts of the genus Clavispora include C. lusitaniae and C. opuntiae; wherein strain of the genus Metschnikowia is preferably M. pulcherrima, wherein strain of the genus Kluyveromyces is preferably selected among K. lactis, K. marxianus, K. yarrowii, and K. wickerhamii; wherein strain of the genus Lodderomyces is preferably L. elongisporus; wherein strain of the genus Pichia is preferably selected among P. guilliermondii, P. anomala, P. fabianii, P. kluyveri, P. norvegensis, P. ohmeri, P. pastoris and P. angusta; wherein strain of the genus Hansenula is preferably selected among H. polymorpha; wherein strain of the genus Lipomyces is preferably selected among L. starkeyi, L. orientalis, L. yarrowii, and L. japonica; wherein strain of the genus Yarrowia is preferably Y. lipolytica; wherein strain of the genus Geotrichum is preferably selected among G. candidum, G. clavatum, and G. fici.

11. The hemiascomycetes yeast strain according to any one of claims 8 to 10, wherein one or more pathways are disrupted or enhanced by at least one genetic modification, preferably by at least one or more genetic mutations, total or partial gene deletions or excisions, gene disruptions or integrations, and/or gene duplications.

12. The hemiascomycetes yeast strain according to any one of claims 8 to 11, comprising one or more genetic modifications that disrupt or enhance the mitochondrial FOX2 dependent pathway and/or the peroxisomal FOX2 dependent pathway.

13. The hemiascomycetes yeast strain according to claim 12, comprising a total or partial gene deletion, excision, disruption insertion, or integration within the FOX2 gene.

14. The hemiascomycetes yeast strain according to claim 12, wherein FOX2 gene is rendered non- functional by insertion of another gene of an unrelated metabolic pathway and/or by an antibiotic resistance gene (ABR).

15. The hemiascomycetes yeast strain according to claim 12, comprising one or more duplication of the FOX2 gene.

16. The hemiascomycetes yeast strain according to any one of claims 8 to 11, comprising one or more genetic modifications that disrupt or enhance of at least one paralog ECH1, ECH2, or ECH3.

17. The hemiascomycetes yeast strain according to claim 16, comprising a total or partial gene deletion, excision, disruption, insertion, or integration of at least one paralog ECH1, ECH2, or ECH3.

18. The hemiascomycetes yeast strain according to claim 16, wherein at least one paralog ECH1, ECH2, or ECH3 is rendered non-functional by insertion of another gene of an unrelated metabolic pathway and/or by an antibiotic resistance gene (ABR).

19. The hemiascomycetes yeast strain according to claim 16, comprising one or more duplication of the said at least one paralog ECH1, ECH2, or ECH3 gene.

20. The hemiascomycetes yeast strain according to any one of claims 8 to 11, comprising one or more genetic modifications that disrupt or enhance of the PXA1 gene.

21. The hemiascomycetes yeast strain according to claim 20, comprising a total or partial gene deletion, excision, disruption, insertion, or integration of the PXA1 gene.

22. The hemiascomycetes yeast strain according to claim 20, wherein the PXAl gene is rendered nonfunctional by insertion of another gene of an unrelated metabolic pathway and/or by an antibiotic resistance gene (ABR). 23. The hemiascomycetes yeast strain according to claim 20, comprising one or more duplication of the PXAl gene.

24. The hemiascomycetes yeast strain according to any one of claims 8 to 11, comprising one or more genetic modifications that disrupt or enhance at least one paralog FOX3-1, FOX3-2, or FOX3-3.

25. The hemiascomycetes yeast strain according to claim 20, comprising a total or partial gene deletion, excision, disruption, insertion, or integration of at least one paralog FOX3-1, FOX3-2, or FOX3-3.

26. The hemiascomycetes yeast strain according to claim 20, wherein at least one paralog FOX3-1, FOX3-2, or FOX3-3 is rendered non-functional by insertion of another gene of an unrelated metabolic pathway and/or by an antibiotic resistance gene (ABR).

27. The hemiascomycetes yeast strain according to claim 20, comprising one or more duplication of at least one paralog FOX3-1, FOX3-2, or FOX3-3.

28. The hemiascomycetes yeast strain according to any one of claims 8 to 11, comprising at least one more genetic modifications that disrupt or enhance at least one gene selected from the group consisting of FOX2, PXA1, OLE2, ECH1, ECH2, ECH3, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3-2, and FOX3-3. 29. The hemiascomycetes yeast strain according to claim 28, comprising a total or partial gene deletion, excision, disruption, insertion, or integration of a least one gene selected from the group consisting of FOX2, PXA1, OLE2, ECH1, ECH2, ECH3, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3-2, and FOX3-3.

30. The hemiascomycetes yeast strain according to claim 28, comprising or wherein a least one gene selected from the group consisting of FOX2, PXA1, OLE2, ECH1, ECH2, ECH3, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3-2, and FOX3-3 is rendered non-functional by insertion of another gene of an unrelated metabolic pathway and/or by an antibiotic resistance gene (ABR).

31. The hemiascomycetes yeast strain according to claim 28, comprising one or more duplication of at least one gene selected from the group consisting of FOX2, PXAl, OLE2, ECH1, ECH2, ECH3, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3-2, and FOX3-3.

32. The hemiascomycetes yeast strain according to any of one of claims 8 to 11, wherein said strain comprises genetic mutation and specific mutants are selected from the group consisting of:

fox2A; fox3-lA;

fox3-2A;

fox3-3A;

fox2A, pxalA;

echlA;

echlA fox2A;

echlA, pxalA;

echlA, fox2A pxalA;

echlA; and a deletion fox3 A A, or fox3-2A, or fox3-3A;

echlA fox2A; and a deletion fox3 -1 Δ, or fox3-2A, or fox3-3A;

echlA, pxalA; and a deletion fox3 A A, or fox3-2A, or fox3-3A;

echlA, fox2A pxalA; and a deletion fox3 A A, or fox3-2A, or fox3-3A;

ech2A;

ech2A; fox2A;

ech2A, pxalA;

ech2A, fox2A pxalA;

ech2A; and a deletion fox3 A A, or fox3-2A, or fox3-3A;

ech2A; fox2A; and a deletion fox3 A A, or fox3-2A, or fox3-3A;

ech2A, pxalA; and a deletion fox3 A A, or fox3-2A, or fox3-3A;

ech2A, fox2A pxalA; and a deletion fox3 A A, or fox3-2A, ox fox3-3A;

ech3A;

ech3A fox2A;

ech3A, pxalA;

ech3A, fox2A pxalA ;

ech3A; and a deletion fox3 A A, or fox3-2A, or fox3-3A;

ech3A fox2A; and a deletion fox3 A A, or fox3-2A, or fox3-3A;

ech3A, pxalA; and a deletion fox3 A A, or fox3-2A, ox fox3-3A; and

ech3A, fox2A pxalA; and a deletion fox3 A A, or fox3-2A, ox fox3-3A. 33. Use of the hemiascomycetes yeast strain according to any one of claims 12-14, 16-18, 20-22, 24-26, and 32 for the production of long chain fatty acids, or for the production of triacylglycerols and ester derivatives, or for the production of phospholipids, phospholipids esters, phospholipids mono and/or di-esters, and biodiesel, or for the production of decalactone, or for the production of lipid derived flavorings and/or fragrances, or for the production of terpenes, sesquiterpenes, as well as aldehydes and esters derivatives thereof.

34. Use according to claim 33, wherein said fatty acids are saturated, mono and/or polyunsaturated fatty acids, or wherein said fatty acids are in C16-C24, such as preferably linoleic acid or γ-linoleic acid. 35. Use of the isolated strain according to claim 15 for the production of lactones.

36. Use according to claim 33, wherein said lipid derived flavourings and/or fragrances are fruit and/or milk flavourings, preferably saturated and unsaturated δ- and γ-lactones, or wherein said lipid derived flavourings and/or fragrances are peach-like flavourings, preferably γ-decalactone, δ-decalactone, and/or γ- octalactone, or wherein said lipid derived flavourings and/or fragrances are coconut milk-like flavourings, preferably 4-dodecanolide and/or 4-octanolide.

37. A method of producing commodity chemicals comprising the following steps:

a. providing the isolated strain according to any one of claims 12-14, 16-18, 20-22, 24-26, and 32;

b. growing said strain in a suitable medium;

c. recovering said commodity chemicals.

38. The method of claim 37, wherein aid suitable medium comprises carbon source and is enriched one or more substrates chosen among palmitic acid, oleic acid, ricinoleic acid, or vegetable oil, and wherein said vegetable oil is preferably selected among sunflower oil, castor oil, coconut oil, Massoi bark oil, and/or hydrolysates thereof.

Description:
NOVEL BETA-OXIDATION PATHWAY AND

HEMIASCOMYCETES YEAST MUTANTS

Technical field of the invention

[001] The present invention relates generally to the fields of microbiology and biotechnology. More precisely, the present invention relates to novel genes in hemiascomycetes yeast designated ECH1, ECH2, and ECH3, isolated nucleic acid molecules encoding the novel enoyl-CoA hydratase members as well as the corresponding amino acid sequences thereof. The present invention also relates to isolated biologically pure hemiascomycetes yeast strains comprising a unique combination of metabolic pathways for the catabolism or degradation of fatty acid and other substrates. Finally, the present invention provides for novel mutants that can yield improved production of such commodity chemicals, and methods of increasing the production of commodity chemicals using the isolated biologically pure hemiascomycetes yeast strains.

Background of the invention

[002] Fatty acids are an important source of energy for all living organisms. For example, the complete degradation of a single palmitic acid molecule in mammals produces 130 ATP molecules. Cellular fatty acid degradation occurs via the β-oxidation pathway in all organisms, β-oxidation is in fact the main catabolism pathway in all organisms, e., mammals, plants, fungal organisms, and bacteria. Other oxidation pathways, such as a- and ω-oxidations exist, but they are minor compared β-oxidation (Figure 29; Wanders RJA et al., 201 1, FEBS J. 278, 182-194).

[003] In both β-oxidation systems of mammals and filamentous fungi, fatty acid β-oxidation begins with the addition of coenzyme A to a fatty acid, and occurs by successive cycles of reactions during each of which the fatty acid is shortened by a two-carbon fragment removed as acetyl coenzyme A, generating trans-2, 3 hydroxyl, and 3-keto intermediates, until only two or three carbons remain, thereby generating acetyl-CoA or propionyl-CoA respectively (Hiltunen JK and QinYM, 2000, Mol. Cell. Biol. Lipids, 1484, 1 17-128).

[004] As showed in Figure 30, β-oxidation pathway comprises the following enzymatic steps:

(1) dehydrogenation of the acyl-coA which is catalyzed by acyl-CoA dehydrogenases or acyl-CoA oxidases producing a trans-2-enoyl-CoA;

(2) hydratation of trans-2-enoyl-CoA by an enoyl-CoA-hydratase producing a 3-hydroxyacyl-CoA; (3) dehydrogenation of the 3-hydroxyacyl-CoA in 3-ketoacyl-CoA by a hydroxyacyl-CoA dehydrogenase; and

(4) thiolysis of 3- ketoacyl-CoA via a 3- ketoacyl thiolase which cuts the carbon chain between the carbones a and β, producing an acetyl-CoA molecule and an acyl-CoA which carbon chain is shortened by two atoms of carbon. The acyl-CoA so obtained will then be subject to another cycle of β-oxidation.

[005] In mammals, short and medium chain fatty acids are exclusively catabolised in mitochondria. Long chain fatty acids are preferably catabolised in mitochondria. However, very long chain fatty acids, such as hexacosanoic acid (C26:0), and unsaturated fatty acids are exclusively catabolised in peroxisomes and their degradation is pursued in mitochondria (Bartlett K. et al., 2004, Eur. J. Biochem., 271 :462-469; Kennedy EP et al. J. Biol. Chem., 185: 275-285, 1950). [006] While a mitochondrial and peroxisomal β-oxidation has been observed in filamentous fungus by Boisnard S. et al., (Fung. Genet. Biol. 46:55-66, 2009) and in one basidiomycete yeast by Ferron G et al., (FEMS Microbiol. Lett, 250:63-69, 2005), it had been so far well-established that β-oxidation of fatty acid in hemiascomycetes yeasts was exclusively located in peroxisomes, thereby leading to the well established dogma that β-oxidation of mammals and plants is located in mitochondria and peroxisomes, whereas and β- oxidation of hemiascomycetes yeasts is located in peroxisomes only. Also, both β-oxidation systems were thus thought to be equipped with different sets of enzymes, since proteins involved in the mitochondrial β-oxidation and in the peroxisomal β-oxidation are different.

[007] Contrary to the well-established and accepted dogma that β-oxidation in yeasts was restricted in the peroxisomes, whereas the β-oxidation in mammals and some filamentous fungi occurs both in mitochondria and peroxisomes, Applicants have demonstrated that some hemiascomycetous yeasts in fact housed β- oxidation in mitochondria, as for mammals and some filamentous fungi. Applicants further demonstrated that the peroxisomal β-oxidation pathway was in fact more complex for some hemiascomycetes and surprisingly revealed the existence of FOX2 independent β-oxidation, in addition to the well-known FOX2 dependent peroxisomal dependent pathway. Specifically, Applicants demonstrated that several pathways of catabolism of fatty acids coexist in the hemiascomycetous yeast, of which one is a fox2p-dependent β-oxidation pathway taking place in both peroxisomes and mitochondria.

[008] In addition, while the presence of Echl enzyme had never been described or demonstrated until now in yeasts, the Applicants have identified and isolated new mitochondrial genes in hemiascomycetes yeasts, having enoyl-CoA hydratase activities. These genes have been shown to be orthologuous of the mitochondrial human ECH genes. These novel genes have been designated ECH1 (Enoyl-CoA Hydratase 1), ECH2 (Enoyl- CoA Hydratase 2) and ECH3 (Enoyl-CoA Hydratase 3). The identification and characterization of these novel genes surprisingly suggests that certain hemiascomycetes yeast strains present mitochondrial enzymes having acetyl-coA dehydrogenase activities.

[009] These new developments greatly contribute to the use of these hemiascomycetes yeast cells for metabolic engineering in preparation for novel biotechnological applications. Indeed, hemiascomycetes yeasts are industrially important and have been used to produce many of the so-called commodity chemicals, such as for example, fatty acids, acyl glycerides, bioflavours, aromas, fragrances, bio-oil, etc... These commodity chemicals are widely used in food industry, animal nutrition, cosmetics, and in the pharmaceutical field.

[0010] In the end of the 1980s, the consumer has developed a "chemophobia" attitude towards chemical or synthetic (even nature-identical) compounds, especially when related to his food and home-care products. The interest of consumers for natural products thus rose. The word "natural" is defined legally by American and European regulations and a substance can be considered as natural when it comes from a plant, animal or microbial origin with a physical, microbial or enzymatic process. Therefore, biotechnological routes may be, if they exclude any chemical steps, a way to get natural products. As a result, the industry has devoted considerable time and effort to develop methods for the production of natural components which have been showed to be useful in various industries.

[0011] By way of examples, the engineered microorganisms are particularly useful for the production of polyunsaturated fatty acids (PUFAs), such as linoleic acid or linolenic acid, and more generally co-3 fatty acids (omega-3 fatty acids) and co-6 fatty acids (omega-6-fatty acids) such as. These are indeed essential fatty acids for mammals, and thus are supplemented to food of humans and animals. These may be derived from fish oils, like herring, mackerel, sardine or salmon, but requires complex extraction and purification procedures, which, especially if carried out on an industrial scale, are expensive, laborious and tend to pollute the environment. Thus, there is a need for improved production of PUFAs in a pure and cost effective form, which also allow large scale and automated process formats.

[0012] The engineered microorganisms are also very useful for production of bio-flavours, fragrances and aromas. It is generally recognized in the industry that a flavour compound having been prepared by microbial processes can be designated as a natural product and therefore have an important place in the commercialization of products containing them. The market for these chemicals has been quite dynamic since a long time, and currently stands at around 7 billion USD. In the recent past, many pathways were investigated and about 400 natural aroma compounds were proposed to the market.

[0013] Numerous fungal and yeast strains, such as Candida tropicalis or Yarrowia lipolytica, have been used to synthesize various commodity substances. Several mutant strains of Candida have been developed by strain selection to produce several substances. The yields of this biotransformation are however often disappointingly low, and are rarely above 100 mg/L, making these processes economically unattractive.

[0014] Therefore, in spite of all the prior work, a bottleneck still exists in the lack of complete knowledge about the biochemical pathways, the enzymes and the metabolic regulation involved for obtaining high yields of commodity chemicals. A better understanding of the fungal biochemistry and enzymes involved, metabolic regulation and genetic modification are primordial to improve yields, in addition to the application of novel fermentation technology.

Summary of the invention

[0015] The present invention is based on the identification and characterization of novel paralog genes in hemiascomycetes yeasts, designated hereinafter ECH1, ECH2, and ECH3, which are addressed to the mitochondria, and have been found to be orthologous to ECHA gene of Aspergillus nidulans and to ECH1 genes in humans, and have enoyl-CoA hydratase activities. In one embodiment, the present invention provides novel nucleic acid molecules encoding the hydratase in hemiascomycetes yeasts, as well as nucleotide probes and primers specific to the novel genes, and method of amplifying and of detecting such genes. Still in another embodiment, the present invention relates to nucleic acid molecules that encode proteins having enoyl-CoA hydratase activities, as well as peptide sequences, antibodies directed against such proteins and methods of detecting the novel proteins.

[0016] According to a second aspect, the present invention relates to an isolated, biologically pure hemiascomycetes yeast strain comprising (i) at least one Fox2p independent peroxisomal β-oxidation pathway (ii) at least one Fox2p dependent peroxisomal β-oxidation pathway, and (iii) one Fox2p dependent mitochondrial β-oxidation pathway, wherein such strain still does grow on fatty acid carbon source when rendered non-functional for the FOX2 gene.

[0017] According to a third aspect, the present invention relates to the use of such isolated, biologically pure hemiascomycetes yeast strains, genetic mutants thereof as well as methods of producing various commodity chemicals, including like fatty acids, PUFA, saturated and unsaturated lipids, phospholipids, phospholipids esters, triacylglycerols, biofuels and/or biodiesel, fragrances and flavouring substances, such as lactones, esters, aldehydes, terpenes, and derivatives thereof, etc... Brief description of the figures

[0018] Figure 1: shows the strategy used for the construction of an OLE2 deletion cassette by overlapping PCR, and for the selection of an ole2A mutant strain after homologous recombination and replacement of the wild OLE2 allele of the recipient strain by the deletion cassette carrying URA3.

[0019] Figure 2: shows the strategy used for the construction of an ECHl deletion cassette by overlapping PCR, and for the selection of an echlA mutant strain after homologous recombination and replacement of the wild ECHl allele of the recipient strain by the deletion cassette carrying URA3.

[0020] Figure 3: shows the nucleotide sequence of the FOX2 gene (SEQ ID NO: 1) in Clavispora lusitaniae.

[0021] Figure 4: shows the amino acid of Fox2p protein (SEQ ID NO: 2) in Clavispora lusitaniae.

[0022] Figure 5: shows the nucleotide sequence of the PXA1 gene (SEQ ID NO: 3) in Clavispora lusitaniae.

[0023] Figure 6: shows the amino acid of Pxalp (SEQ ID NO: 4) in Clavispora lusitaniae.

[0024] Figure 7: shows the nucleotide sequence of the OLE2 gene (SEQ ID NO: 5) in Clavispora lusitaniae.

[0025] Figure 8: shows the amino acid of 01e2p (SEQ ID NO: 6) in Clavispora lusitaniae.

[0026] Figure 9: shows the nucleotide sequence of the ECHl gene (SEQ ID NO: 7) in Clavispora lusitaniae.

[0027] Figure 10: shows the nucleotide sequence of ECHl gene in Clavispora lusitaniae strain CBS

6936 (SEQ ID NO: 8).

[0028] Figure 11: shows the amino acid of the Echlp (SEQ ID NO: 9) in Clavispora lusitaniae.

[0029] Figure 12: shows the nucleotide sequence of ECH2 gene in Clavispora lusitaniae strain CBS

6936 (SEQ ID NO: 10).

[0030] Figure 13: shows the amino acid sequence of Ech2p in Clavispora lusitaniae strain CBS 6936

(SEQ ID NO: 11).

[0031] Figure 14: shows the nucleotide sequence of ECH3 gene in Clavispora lusitaniae strain CBS

6936 (SEQ ID NO: 12).

[0032] Figure 15: shows the amino acid sequence of Ech3p in Clavispora lusitaniae strain CBS 6936 (SEQ ID NO: 13).

[0033] Figure 16: shows the nucleotide sequence of FOX1A gene in Clavispora lusitaniae strain CBS

6936 (SEQ ID NO: 14).

[0034] Figure 17: shows the amino acid sequence of FoxlAp in Clavispora lusitaniae strain CBS

6936 (SEQ ID NO: 15).

[0035] Figure 18: shows the nucleotide sequence of FOX1B gene in Clavispora lusitaniae strain CBS

6936 (SEQ ID NO: 16).

[0036] Figure 19: shows the amino acid sequence of FoxlBp in Clavispora lusitaniae strain CBS 6936

(SEQ ID NO: 17).

[0037] Figure 20: shows the nucleotide sequence of FOX1C gene in Clavispora lusitaniae strain CBS 6936 (SEQ ID NO: 18). [0038] Figure 21: shows the amino acid sequence of FoxlCp in Clavispora lusitaniae strain CBS 6936 (SEQ ID NO: 19).

[0039] Figure 22: shows the nucleotide sequence of FOX3-1 gene in Clavispora lusitaniae strain CBS

6936 (SEQ ID NO: 20)

[0040] Figure 23: shows the amino acid sequence of Fox3-lp in Clavispora Lusitaniae strain CBS 6936 (SEQ ID NO: 21)

[0041] Figure 24: shows the nucleotide sequence of FOX3-2 gene in Clavispora lusitaniae strain CBS 6936 (SEQ ID NO: 22)

[0042] Figure 25: shows the amino acid sequence of Fox3-2p in Clavispora Lusitaniae strain CBS 6936 (SEQ ID NO: 23)

[0043] Figure 26: shows the nucleotide sequence of FOX3-3 gene in Clavispora lusitaniae strain CBS 6936 (SEQ ID NO: 24)

[0044] Figure 27: shows the amino acid sequence of Fox3-3p in Clavispora lusitaniae strain CBS 6936 (SEQ ID NO: 25)

[0045] Figure 28: represents the various fatty acids oxidation pathway.

[0046] Figure 29: represents fatty acids β-oxidation (Wanders RJA et al., 2011, FEBS J. 278, 182- 194).

[0047] Figure 30: shows the mitochondrial β-oxidation in mammals. Wanders R.J. A. et al. (J Inherit

Metab Dis 2010 33:479^-94). CPT1 : carnitine palmitoyl-transferase-I; CPT2: carnitine palmitoyl-transferase II, CACT: carnitine acylcarnitine translocase; VLCAD: very-long-chain acyl-CoA dehydrogenase; ACA9D: acyl-CoA dehydrogenase 9; SCEH: short-chain enoyl-CoA hydratase; LCEH: long-chain enoyl CoA hydratase; LCAD: long-chain hydroxyacyl-CoA dehydrogenase; SCAD: short-chain hydroxyacyl-CoA dehydrogenase; S/MCKAT: short/medium-chain ketoacyl-CoA thiolase; LCKAT: long-chain ketoacyl-CoA thiolase; MTP: mitochondrial trifunctional protein.

[0048] Figure 31: shows fatty acid catabolism in ascomycetes yeasts. The localization of the specific enzymes of the glyoxylate cycle (Icllp and Mlslp) is susceptible to variations according to the species. CAT: carnitine acetyl-transferase.

[0049] Figure 32: shows southern blot analysis of the wild type and Fox2p strains. (A) Panel showing the genomic configurations of the FOX2 (left panel) and URA3 (right panel) loci of the mutants (fox2A::GUN, fox2A ura3A, fox2A), the revertant strain (FOX2Re), and the wild-type strain (6936). Sequences homologous to FOX2 are colored in black, npt sequences are hatched, sequences homologous to URA3 are colored in grey, pGEM-T sequence is colored in white. Genomic DNA was digested with EcoRV (arrows) and analyzed by Southern blotting using: (B) the FOX2 probe (homologous to the entire sequence of FOX2) and (C) URA3 probe (homologous to the entire sequence of URA3).

[0050] Figure 33: shows growth characteristics of mutant, revertant and wild type strains on different carbon sources.

[0051] Figure 34: shows consumption of 14 Ca-palmitoyl-CoA by protein crude extracts of the C. lusitaniae icllA, fox2A, pxalA and wild type strains. (A) 14 Ca-palmitoyl-CoA consumption rates after 15 min at 37°C by crude extracts of the mutant and wild-type strains after analysis of the chromatograms using ImageQuant software. (B) Corresponding chromatograms. TO: Amount of immediate addition of 14Ca- palmitoyl-CoA initially present in the reaction mix. WT: wild-type strain. P: Ca-palmitoyl-CoA. E: Ca- hexadecenoyl-CoA. H: 14 Ca-3-hydroxyhexadecenoyl-CoA. S: start.

[0052] Figure 35: shows purification of peroxisomes and mitochondria from induction-medium grown cells.

[0053] Figure 36: shows consumption of 14 Ca-palmitoyl -CoA by peroxisomal and mitochondrial fractions of C. lusitaniae fox2A , pxalA and wild type strains. (A) 14 Ca-palmitoyl-CoA consumption rates after 15 min at 37°C by peroxisomal and mitochondrial fractions of the mutant and wild -type strains after analysis of the chromatograms using ImageQuant software. The activity observed in the mitochondrial fraction of fox2A and wild-type strains were compared using a Student's t-test (*, p = 0.016). (B) Corresponding chromatograms. TO: Amount of immediate addition of 14 Ca-palmitoyl-CoA initially present in the reaction mix. WT: wild-type strain. P: 14 Ca-palmitoyl-CoA. E: 14 Ca-hexadecenoyl-CoA. H: 14 Ca-3- hydroxyhexadecenoyl-CoA. S: start.

[0054] Figure 37: shows immunodetection of fox2A and icllp by western blot in C. lusitaniae, fox2 A and wild strains. (A) Twenty μg of proteins of the crude extracts and 10 μg of proteins of the peroxisomal and mitochondrial fractions were separated by SDS-PAGE. (B) Signal integration expressed in relative intensity using Quantity One software. The Icllp signal is figured in grey, the Fox2p signal in black. WT: wild type, CE: crude extract, P: peroxisomal fraction, M: mitochondrial fraction.

[0055] Figure 38: shows mitochondrial respiration of C. lusitaniae, fox2 A, pxalA and wild strains. 0 2 consumption rates observed after the addition of inhibitors were expressed in function of those observed after addition of substrate, whose value is fixed at 1 if there was an oxygen consumption and at 0 if not. (A) O 2 consumption rates (expressed in nmol O 2 per minute and per mg of dry weight cells) observed using NADH as substrate were: 2.4 +/- 0.2 for WT spheroplasts, 2.2 +/- 0.52 for fox2A spheroplasts, and 3.08 +/- 0.68 for pxalA spheroplasts. (B) 0 2 consumption rates (expressed in nmol 0 2 per minute and per mg of dry weight cells) observed using palmitoyl-CoA as substrate were: 6.42 +/- 0.4 for WT spheroplasts, 0.084 +/- 0.02 for fox2A spheroplasts, and 4.68 +/- 0.2 for pxalA spheroplasts. (C) 02 consumption rates (expressed in nmol 0 2 per minute and per mg of dry weight cells) observed using stearoyl-CoA as substrate were: 0.6 +/- 0.04 for WT spheroplasts, 0.008 +/- 0.008 for fox2A spheroplasts and 0.8 +/- 0.16 for pxalA spheroplasts.

[0056] Figure 39: shows oxygrams of mitochondrial respiration observed for C. lusitaniae, fox2A and wild strains. (A) Oxygen uptake by wild-type spheroplasts after permeabilization with nystatin. Oxygen consumption rates measured between two vertical lines after addition of the following substrates: 6.42 nmol 02.mg-l.min-l after addition of palmitoyl-CoA (0.2 mM final), 4.28 nmol 02.mg-l.min-l after addition of myxothiazol (0.1 μg/mL final), 8.68 nmol 0 2 .mg-l.min-l after addition of KCN (5mM final). (B) Oxygen uptake by fox2A spheroplasts after permeabilization with nystatin. Oxygen consumption rates measured between two vertical lines after addition of the following substrates: 0.084 nmol 02.mg-l.min-l in the presence of 0.2 mM palmitoyl-CoA, 0.095 nmol 02.mg-l.min-l in the presence of 0.4 mM palmitoyl-CoA.

[0057] Figure 40: shows decalactone production observed for C. lusitaniae, fox3-lA, echlA, pxalA, fox2A and wild strains using different inoculum and growth conditions (conditions A and B). Description of the invention

[0058] According to a first aspect, the present invention relates to the identification and characterization of novel paralog genes in hemiascomycetes yeasts, designated hereinafter ECH1, ECH2, and ECH3, which are addressed into the mitochondria hemiascomycetes yeasts. These novel genes have been hereinafter designated ECH1 (Enoyl-CoA Hydratase 1), ECH2 (Enoyl-CoA Hydratase 2) and ECH3 (Enoyl-CoA Hydratase 3) as they were characterized as being orthologous to ECHA gene of Aspergillus nidulans and to ECH1 gene in human, and as having enoyl-CoA hydratase activities.

[0059] Mitochondrial β-oxidation in mammals starts by the activation of fatty acids into acyl-CoA esters by acyl-CoA synthase, which are then transferred into the mitochondria (Figure 30; Wanders R.J. A. et al., J Inherit Metab Dis 2010 33:479^-94). Two systems of β-oxidation are described in mammals: the first one involves soluble enzymes in the mitochondrial matrix (type I), and the second one involves enzymes that are associated to the internal membrane of mitrochondria (type II). The type I β-oxidation allows for the degradation of fatty acids having short and medium carbon chains and involves four different enzymes. The type II β-oxidation allows for the degradation of fatty acids having long carbon chains. The VLCAD (Very Long Chain Acyl CoA Dehydrogenase) allows for the dehydrogenation of acyl-CoA, whereas the last three steps are catalyzed by a three-functional protein of 460 kDa, comprising two sub-units (α 4 β 4 ). Subunit a comprises activities of 2-enoyl CoA hydratase and 3-hydroxyl-CoA dehydrogenase, and allows binding to the internal membrane of the mitochondria. Subunit β carries the 3-ketoacyl-CoA thiolase activity.

[0060] A particular specificity of mitochondrial β-oxidation in mammals is the hydratation step of trans-2- enoyl-CoA into 3-hydroxyacyl-CoA, which is catalyzed by the enoyl-CoA hydratase (ECH). There are two types of Ech enzyme which differ by the stereospecificity of hydratation of the double bond in the trans-2- enoyl-CoA molecule.

[0061] Type 1 ECH or ECH1 allows for oxidation of trans-2-enoyl-CoA into (L)-3-hydroxyacyl-CoA. The following Echlp isomers have been identified, which differ by their substrate specificity: (i) the crotonase (EC 4.2.1.17) which is a homohexameric protein more active on short carbon chains; (ii) an Echlp which is active on long chains, particularly for esters CoA comprising 8 carbon atoms; and (iii) the human Echlp which is active on esters CoA having medium and long chain (Jackson et al. 1995, BBRC, 214:247-253).

[0062] Type 2 ECH or ECH2 allows for oxidation of trans-2-enoyl-CoA into (D)-3-hydroxyacyl-CoA. Their existence was suggested in S. cerevisiae based on the persistence of an Ech activity in a defective mutant of the peroxisomal MFP. More recently, a bioinformatics study performed by Shen et al. {Funct.Integr.Genomics, 73: 1239-1242, 2009) had suggested the existence of ECH2 in C. Albicans, C. Tropicalis, and Y. Lypolytica, but not in S. cerevisiae or C. lusitaniae. Ech2p is a homodimeric protein and has a C-terminal hot-dog structure (Engel et al., EMBO J. 15:5135-5145, 1996) for substrate having a long carbon chain. One ECH2 has also been described in Aspergillus nidulans by Maggio-Hall et al. (Mol.MicrobioL, 54: 1173-1185, 2004) and a defective mutant for this enzyme was described as being unable to grow on short chain fatty acid as sole carbon source.

[0063] Contrary to the well established knowledge that fatty acid β-oxidation in hemiascomycetes yeasts is restricted to peroxisomes, the Applicants have identified and characterized novel genes suggesting that hemiascomycetes yeast strains present mitochondrial enzymes similar to that of mammals, having mitochondrial acetyl-coA dehydrogenase activity and enoyl-CoA hydratase activities. [0064] The present invention thus relates an isolated nucleic acid molecule selected from the group consisting of:

(i) a nucleic acid molecule comprising a nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, or 95 % identity with any one of the nucleotide sequence as set forth in SEQ ID NO: 8, 10, or 12 which are designated ECH1, ECH2, and ECH3 respectively, and encoding a mitochondrial enoyl-CoA hydratase activity, or a complementary nucleotide sequence thereof;

(ii) a nucleic acid molecule comprising a fragment of at least 15 consecutive nucleotides of a nucleotide sequence as set forth in any one of SEQ ID NOs: 7, 8, 10, or 12, or a complementary nucleotide sequence thereof;

(iii) a nucleic acid molecule that hybridizes under high stringency conditions with a nucleotide sequence as set forth in any one of SEQ ID NO: 7, 8, 10, or 12, or a complementary nucleotide sequence thereof, or a fragment thereof;

(iv) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 9, 11, or 13; and

(v) a nucleic acid molecule which encodes a fragment of a polypeptide comprising at least 15 contiguous amino acids of the sequence as set forth in any one of SEQ ID NO: 9, 11, or 13.

[0065] The term "identity" as used herein is well understood in the art is a relationship between two or more polypeptide sequences or two or more nucleic acid molecule sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or nucleic acid molecule sequences, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M. and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York (1991) and Carillo H et al. (Applied Math, 48: 1073; 1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. Computer programs which can be used to determine identity between two sequences include, but are not limited to, BLAST programs for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and for protein sequence queries (BLASTP and TBLASTN).

[0066] The nucleic acid sequences according to the present invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers. The present invention includes variants including those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Included among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the final proteins or portions thereof. [0067] The present invention also relates to nucleotide probes or primers specific of ECH1, ECH2, or ECH3 gene, wherein the nucleotide probe or primer is selected from the group consisting of: (i) a probe or primer comprising a nucleotide sequence as set forth in any one of SEQ ID NO: 7, 8, 10, or 12, or a complementary nucleotide sequence thereof; and (ii) a probe or primer comprising at least 15 consecutive nucleotides of a nucleotide sequence as set forth any one of SEQ ID NOs: 7, 8, 10, or 12, or a complementary nucleotide sequence thereof. Such probe or primer may comprise a marker compound.

[0068] The above probes or primers may further comprise a marker compound like for example a fluorophore, an organic molecule that fluoresces after absorbing light of lower wavelength/higher energy. The probes can also be indirectly labelled with, e^g., biotin or digoxygenin, or labelled with radioactive isotopes such as 32 P and 3 H.

[0069] The present invention further relates to method of amplifying a region of the nucleotide sequence according to claim 1, wherein the method comprises:

a) selecting two nucleotide primers from the group consisting of a nucleotide primer comprising at least 15 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NO: 7, 8, 10, or 12;

b) contacting the nucleic acid with two nucleotide primers, wherein a first nucleotide primer hybridizes at a position 5' of the region of the nucleic acid, and a second nucleotide primer hybridizes at a position 3' of the region of the nucleic acid, in the presence of reagents necessary for an amplification reaction; and

c) detecting the amplified nucleic acid region.

[0070] The Applicants have further identified the proteins encoded by the novel ECH1, ECH2, and ECH3 genes. The present invention thus further relates to an isolated polypeptide selected from the group consisting of: (i) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, or 95 % identity with any one of the nucleotide sequence as set forth in any one of SEQ ID NO: 7, 8, 10, or 12;

(ii) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 9, 11, or

13;

(iii) a polypeptide fragment or functional variant of a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 9, 11, or 13, or catalytically active fragments thereof; and

(iv) a polypeptide homologous to a polypeptide comprising amino acid sequence as set forth in any one of SEQ ID NO: 9, 11, or 13 or a functional equivalents thereof or catalytically active fragments thereof.

[0071] According to a second aspect, the present invention relates to isolated, biologically pure hemiascomycetes yeast strains comprising mitochondrial and peroxisomal metabolic pathways for the catabolism of fatty acid, carrying and expressing the novel ECH1, ECH2, and ECH3 genes. The isolated, biologically pure hemiascomycetes yeast strains according to the present invention comprise three distinct metabolic pathways for the catabolism of fatty acid: (i) at least one pathway is a peroxisomal β-oxidation FOX2 independent pathway, (ii) at least one peroxisomal β-oxidation FOX2 dependent pathway, and (iii) a mitochondrial β-oxidation FOX2 dependent pathway, and still do not grow on fatty acid carbon source when rendered non-functional for the FOX2 gene. [0072] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0073] The term "yeast" refers to an organism of the kingdom Fungi, and preferably, is directed towards any organism of the phylum Ascomycota and most preferably is directed towards any organism of the class Hemiascomycetes. The ascomycetes generally occur in two significantly distinctive types: those which form a filamentous mycelium, the "euascomycetes" and those which do not, the "hemiascomycetes". Hemiascomycetes as referred to herein correspond to a group including yeast species sharing a small genome size and a low frequency of introns.

[0074] More precisely, hemiascomycetes yeast strains according to the invention include strains of the genus Candida, Debaryomyces, Clavispora, Metschnikowia, Kluyveromyces, Lodderomyces, Pichia, Hansenula,

Lipomyces, Yarrowia, and Geotrichum. By way of examples, yeasts of the genus Candida include C. tropicalis, C. parapsilosis, C. o thopilosis, C. metap silo sis, C. norvegensis, C. antartica, C. dubliniensis, C. haemulonii, C. kefyr, C. krusei, C. lusitaniae, C. maltose, and C. oleophila. Yeasts of the genus Debaryomyces include D. hansenii and D. castelli. Yeasts of the genus Clavispora include C. lusitaniae and C. opuntiae. Yeasts of the genus Metschnikowia include for example M. pulcherrima. Yeasts of the genus Kluyveromyces include K. lactis, K. marxianus, K. yarrowii, and K. wickerhamii. Yeasts of the genus Lodderomyces include for example L. elongisporus. Yeasts of the genus Pichia include P. guilliermondii, P. anomala, P. fabianii, P. kluyveri, P. norvegensis, P. ohmeri, P. pastoris and P. angusta. Yeasts of the genus Hansenula include for example H. polymorpha. Yeasts of the genus Lipomyces include for example L. starkeyi, L. orientalis, L. yarrowii, and L. japonica. Yeasts of the genus Yarrowia include for example Y. lipolytica. Yeasts of the genus

Geotrichum include for example G. candidum, G. clavatum, and G. fici.

[0075] Long- and short-chain fatty acids β-oxidations of hemiascomycetes yeasts were known up to now as being exclusively localized in peroxisomes. Peroxisomes are single-membrane-bound organelles that not only contain the enzymes for the β-oxidation of fatty acids but also can be involved in a variety of other metabolic pathways, such as the inactivation of toxic substrates (H 2 0 2 -based respiration), the synthesis of ether phospholipids (in mammals), and the breakdown of purines and amino acids. Peroxisome biogenesis is a conserved process among eukaryotes, involving the concerted action of at least 32 proteins (peroxins) that are encoded by PEX genes.

[0076] As showed in Figure 31, long chain fatty acids are transported across the peroxisomal membrane via a heterodimeric ABC transporter Pxalp-Pxa2p, while the medium-chain fatty acids are thought to be imported into the peroxisomes as free fatty acids. Fatty acids are activated into acyl-coenzyme A esters (acyl-CoA) by acyl-CoA synthetases. An acyl-CoA-oxidase, which has been designated Poxlp or Foxlp, converts FA-CoA into fran.s-2-enoyl-CoA and transfers electrons directly to oxygen generating H 2 0 2 , which is detoxified by the Ctalp catalase. It has been demonstrated that PoxlA yeasts are unable to grow on fatty acids as sole carbon atoms. Then, a type 2 multifunctional enzyme (Mfe2p) designated Fox2p or Pox2p, having both enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, converts fran.s-2-enoyl-CoA into 3-ketoacyl-CoA. The 2-enoyl-CoA hydratase converts the trans-2-enoyl CoA esters into (3R)-hydroxyacyl-CoA esters, whereas the 3-hydroxyacyl-CoA dehydrogenase produces the 3-ketoacyl-CoA ester. The Fox2p enzyme was first isolated from Candida tropicalis and comprises a duplicated domain organization in its N-terminal region, which contains two dehydrogenase active domains A and B. Domain A was demonstrated to have highest activity with long and medium chain substrates, whereas domain B has the highest activity with short-chain substrates. The C-terminal region of the Fox2p enzyme contains the 2-enoyl-CoA hydratase activity. Hiltunen et al. (JBC, Vol. 267, No. 10, April 5, 1992, pp 6646-6653) showed that fatty acid catabolism in yeast was mainly based on the activity of Fox2p and that disruption of FOX2 resulted in the inability of yeast cells to grow on fatty acids as their sole carbon source. At the next reaction of the β-oxidation cycle, a 3-ketoacyl-CoA thiolase, designated Potlp or Fox3p, converts 3-ketoacyl-CoA into acetyl-CoA and acyl-CoA shortened by two carbon units,. The Potlp/Fox3p is a dimeric protein with a subunit size of 45kDa. A single subunit comprises three domains: two core domains, and a loop domain of 120 residues. The active site of yeast thiolase is shaped by residues from the two core domains and surrounded by the loop domain. The products of this last step are thus acetyl-CoA and a C2-shortened acyl-CoA. The shortened acyl-CoA then becomes the substrate of Poxlp/Foxlp during a further β-oxidation cycle. The acetyl-CoA integrates the glyoxylate cycle, thereby allowing the transformation of acetyl-CoA into oxaloacetate, which can be used to synthesize carbohydrates via gluconeogenesis, or can be exported outside the peroxisome by the carnitine acetyl transferase shuttle system, or under the form of citrate. These reactions are catalyzed by two main enzymes: isocitrate lyase (Icllp) and malate synthase (Mlslp) which permits the use of two carbon atoms such as acetate, in the neoglucogenese.

[0077] Contrary to the well-established dogma that β-oxidation in yeasts is restricted in the peroxisomes, whereas the β-oxidation in mammals and in some filamentous fungi occurs both in mitochondria and peroxisomes, the Applicants have demonstrated that hemiascomycetous yeasts actually also housed β- oxidation in mitochondria, similarly to mammals. The Applicants further demonstrated that the peroxisomal β- oxidation pathway was in fact more complex for some hemiascomycetes, and surprisingly revealed the existence of FOX2 independent β-oxidation, in addition to the well-known FOX2 dependent peroxisomal pathway.

[0078] The novel isolated biologically pure yeast strains are surprisingly capable of growing and producing desired molecules via a peroxisomal β-oxidation FOX2 independent pathway, while having their β-oxidation FOX2 dependent pathways blocked. The isolated, biologically pure hemiascomycetes yeast strains have been characterized as having, in addition to the peroxisomal β-oxidation FOX2 dependent pathway, at least one peroxisomal β-oxidation FOX2 independent pathway, as well as a mitochondrial β-oxidation FOX2 dependent pathway.

[0079] The Applicants have fully characterized nucleotide sequences of the enzymes -encoding genes involved in the peroxisomal and mitochondrial fatty acid β-oxidation pathways in hemiascomycetes yeasts as well as the deduced amino acid sequences of enzymatic proteins. These enzyme-encoding genes include three paralogs of FOX1, ej>., FOX1A, FOX1B and FOX1C (Fatty-acyl coenzyme A oxidase); the unique gene FOX2 (multifunctional enzyme). The multifunctional enzyme (hereinafter FOX2 gene) encodes the second enzyme of the β-oxidation pathway. Fox2p is the enzyme with dual activity: 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase (Seem Hiltunen, et al., J. Biol. Chem. 267:6646-53 (1992)). The gene encoding Fox2p of C. lusitaniae has been isolated and sequenced and encodes a 903 amino acid protein. Furthermore, three paralogs FOX3, e_^_., FOX3-1, FOX3-1, and FOX3-3 (3-ketoacyl-CoA thiolase) which encode the third enzyme of the β-oxidation pathway. Finally, the Applicants have identified three paralogs ECH, ej>., ECH1, ECH2, and ECH3 which are addressed to the mitochondria. [0080] The isolated biologically pure hemiascomycetes yeast strains according to the present invention may further comprise or a genetic modification of the PXAl gene. Preferably, such strains comprise a partial or total deletion of the PXAl gene. Yeast contains two ALDP (Adrenoleukodystrophy Protein) homologues: PXA1 (Peroxisomal ABC-transporter 1), also known as LPI1, PAT2, SSH2 or PALI, and PXA2, also known as PATl (Protein Associated with Topoisomerase 1). The Pxalp-Pxa2p transporter is reported to bear sufficient similarity to the human adrenoleukodystrophy protein ALDP. During β-oxidation, the two peroxisomal half ABC transporters Pxalp and Pxa2p form a heterodimeric complex in the peroxisomal membrane. The gene of C. lusitaniae encoding Pxalp has been isolated and sequenced and encodes 809 amino acid protein.

[0081] More specifically, the Applicants have characterized in Candida lusitaniae the nucleotide sequences of FOX2 gene (SEQ ID NO: 1), PXA1 gene (SEQ ID NO: 3), OLE2 gene (SEQ ID NO: 5), ECH1 gene (SEQ ID NO: 7 or 8), ECH2 gene (SEQ ID NO: 10), ECH3 gene (SEQ ID NO: 12), FOX1A gene (SEQ ID NO: 14), of FOX1B gene (SEQ ID NO: 16), of FOX1C gene (SEQ ID NO: 18), FOX3-1 gene (SEQ ID NO:20), FOX3-2 gene (SEQ ID NO:22), and FOX3-3 gene (SEQ ID NO: 24). The Applicants have also characterized the deduced amino acid sequences of Fox2p (SEQ ID NO: 2), Pxalp (SEQ ID NO: 4), 01e2p (SEQ ID NO: 6), Echlp (SEQ ID NO: 9), Ech2p (SEQ ID NO: 11), Ech3p (SEQ ID NO: 13), FoxlAp (SEQ ID NO: 15), FoxlBp (SEQ ID NO: 17), FoxlCp (SEQ ID NO: 19), Fox3-lp (SEQ ID NO: 21), Fox3-2p (SEQ ID NO: 23), Fox3-3p (SEQ ID NO: 25).

[0082] The present invention thus also provides a nucleic acid molecule comprising a nucleotide sequence selecting from the group consisting of

(i) a nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, or 95 % identity with any one of the nucleotide sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 18, 20, 22, or 24;

(ii) a nucleic acid molecule comprising a fragment of at least 15 consecutive nucleotides of a nucleotide sequence as set forth in any one of SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 18, 20, 22, or 24; or a complementary nucleotide sequence thereof;

(iii) a nucleic acid molecule that hybridizes under high stringency conditions with a nucleotide sequence as set forth in any one of SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 18, 20, 22, or 24, or a complementary nucleotide sequence thereof, or a fragment thereof;

(iv) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 17, 19, 21, 23, or 25; and

(v) a nucleic acid molecule which encodes a fragment of a polypeptide comprising at least 15 contiguous amino acids of the sequence as set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 17, 19, 21, 23, or 25.

[0083] The Applicants have further identified, in C. Lusitaniae, the proteins encoded by the FOX2, PXA1,

OLE2, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3-2, and FOX3-3 genes. The present invention thus further relates to an isolated polypeptide selected from the group consisting of:

(i) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, or 95 % identity with any one of the nucleotide sequence as set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 17, 19, 21, 23, or 25;

(ii) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 2, 4, 6, 9,

11, 13, 15, 17, 19, 21, 23, or 25; (iii) a polypeptide fragment or functional variant of a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 17, 19, 21, 23, or 25, or catalytically active fragments thereof, and

(iv) a polypeptide homologous to a polypeptide comprising amino acid sequence as set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 17, 19, 21, 23, or 25, or a functional equivalents thereof or catalytically active fragments thereof.

[0084] The present invention further relates to hemiascomycetes yeast strains comprising at least one of the nucleotide sequence selected from the sequences as set forth in SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 18, 20, 22, or 24, and comprising as metabolic pathways for the catabolism of fatty acid at least one peroxisomal β-oxidation FOX2 independent pathway, at least one peroxisomal β-oxidation FOX2 dependent pathway, and at least one mitochondrial β-oxidation FOX2 dependent pathway.

[0085] According to a third aspect, the present invention relates to the isolated biologically pure hemiascomycetes yeast strains wherein one or more pathways as described above, Le_., either the peroxisomal β-oxidation FOX2 dependent pathway and/or the mitochondrial β-oxidation FOX2 dependent pathway, disrupted or enhanced by at least one genetic modification.

[0086] Preferably, the isolated, biologically pure hemiascomycetes yeast strains belong to the genus Candida, and more preferably to the species Candida lusitaniae, and comprise one or more genetic modifications which may disrupt or enhance the mitochondrial FOX2 dependent pathway and/or the peroxisomal FOX2 dependent pathway.

[0087] By "genetic modifications" is meant genetic mutations, total or partial gene deletions or excisions, gene disruptions or integrations, and/or gene duplications. Such genetic modifications are accomplished by genetic engineering methods which are well known in the art.

[0088] Such genetic modifications may be performed for example by mutagenesis or other genetic engineering methods well known methods in the art, followed by appropriate selection or screening to identify the desired mutants. Method of deletion may be preferably performed according to the strategy described in El-Kirat-Chatel K et al. (2011, Yeast 28, 321-330. PMID 21456057).

[0089] In a specific embodiment, the Applicants have identified a mutant of C. lusitaniae lacking the mutifunctional enzyme fox2p of the β-oxidation pathway which was still able to grow on fatty acids as sole carbon source, suggesting that C. lusitaniae had an alternative pathway for fatty acid catabolism. Using 14Ca- palmitoyl-CoA, applicants have demonstrated that fatty acid catabolism took place in both peroxisomal and mitochondrial subcellular fractions, and that a fox2A null mutant was unable to catabolize fatty acids in the mitochondrial fraction, indicating that the mitochondrial pathway was Fox2p-dependent, as also confirmed by western blotting experiments. Finally, oxymetry measurements showed that mitochondria were able to consume oxygen when fed with palmitoyl-CoA. Accordingly, this invention constitutes the first demonstration of the existence of a mitochondrial β-oxidation pathway in hemiascomycetous yeasts.

[0090] Though, a mitochondrial β-oxidation pathway was suspected for a long time in the ascomycetous yeast C. tropicalis, when both acyl-CoA oxidase and acyl-CoA dehydrogenase, which were thought to be linked to peroxisomal and mitochondrial β- oxidation pathway, respectively, were characterized at the biochemical level (44). However, it was argued that acyl-CoA dehydrogenases were not specific to mitochondrial β-oxidation, as they can be also involved in the degradation of valine, leucine, and isoleucine, as recently confirmed in A. nidulans (45). It was then definitely considered that β-oxidation was exclusively located to the peroxisomes in C. tropicalis, because the four main enzymes of Boxidation, i.e. acyl-CoA oxidase, enoyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase were localized in the same subcellular compartment than the typical peroxisomal enzymes catalase and isocitrate lyase.

[0091] Using a polyclonal antibody, western blot analysis of protein extracts obtained from organelle fractions of C. lusitaniae allowed the applicants to detect the Fox2 protein in both the peroxisomal and mitochondrial fractions. Another evidence that supported a mitochondrial localization of Fox2p was provided by the significant defect (p<0.05) of 14Ca- palmitoyl-CoA catabolism in the fox2A mitochondrial fraction. This demonstrated that the mitochondrial B-oxidation pathway of C. lusitaniae was Fox2p-dependent. The presence of Fox2p in the mitochondria of C. lusitaniae constitutes an unique phenotype in eukaryotes, for which the mitochondrial B-oxidation is known to involve two other sets of enzymes, depending on the organism and on the length of the fatty acyl chain.

[0092] Finally, Applicants used oximetry to show that a mitochondrial respiration could be induced by long chain acyl-CoA in spheroplasts of both the wild-type and the pxalA mutant strains. The oxygen uptake induced by palmitoyl-CoA was unaffected by the presence of phenylsuccinate, a competitive inhibitor of the succinate shuttle between peroxisomes and mitochondria, and of valinomycin, a mitochondrial inner- membrane uncoupling agent and also a H+ dependent transport inhibitor. This strongly suggested that 0 2 consumption was not dependent from the peroxisomal B-oxidation of palmitoyl-CoA. The lack of mitochondrial respiration after induction by acyl-CoA in the fox2A mutant, in contrast to the pxalA and wild- type spheroplasts, provided an additional proof that a fatty acyl B-oxidation pathway was taking place in the mitochondria of C. lusitaniae. Surprisingly, attempts to inhibit the acyl-CoAinduced mitochondrial respiration with KCN failed. Inversely, cyanide caused a marked increase of 0 2 consumption when palmitoyl-CoA was used as respiratory substrate, whereas it completely inhibited the respiration when NADH was used as respiratory substrate. Alternative cyanide-insensitive respiratory pathways were already described in Candida yeasts, and were shown to be induced after cell growth in presence of KCN or after over- reduction of the coenzyme Q pool in C. albicans and C. parapsilosis, respectively. When these pathways are recruited for the transfer of electrons from the Q pool, the respiratory rates are quite similar before and after addition of KCN, whatever the substrate. The existence of such an alternative cyanide-insensitive respiratory pathway is possible in C. lusitaniae. Indeed, a BLAST analysis allowed the identification of an ORF of C. lusitaniae homolog (58% identity, 73% similarity) to the alternative oxidase 1 of C. albicans (orfl9.4774, Candida Genome Database). However, the sole existence of an alternative oxidase activity is not sufficient to explain the strong increase of 0 2 consumption by KCN when palmitoyl-CoA was used as respiratory substrate. We assume that the decrease of 0 2 consumption resulting from the inhibition of the cytochrome c oxidase by KCN could be largely overbalanced by the concomitant inhibition of the peroxisomal catalase, which is required to detoxify the hydrogen peroxide generated from molecular oxygen in the first reaction of B-oxidation taking place in peroxisomes.

[0093] Another unique feature of the catabolism of fatty acids in C. lusitaniae is the existence of a palmitoyl- CoA catabolism in purified peroxisomes of the fox2A mutant. This alternative peroxisomal pathway is Fox2p- independent, and allows the C. lusitaniae fox2 A mutant to grow and assimilate the saturated fatty acids C12:0, C14:0, C16:0, C18:0, whereas afox2A mutant of C. albicans cannot. Fox2p is a multifunctional protein, which possesses three catalytic domains, one for enoyl-CoA hydratase and two for 3-hydroxyacyl-CoA dehydrogenase activities. One can imagine that these activities could also be harbored by independent proteins, as in the mitochondrial β-oxidation pathway of filamentous fungi. Alternatively, two other mechanisms are known to drive fatty acid oxidation. The first one, co- oxidation, occurs in peroxisomes of mammalian cells, and was already described in the yeast C. tropicalis. Through this pathway, the monocarboxylic acids are first transformed into dicarboxylic acids and after activation by a dicarboxylyl-CoA synthetase, the dicarboxylyl- CoA esters then need a functional β-oxidation to be shortened. Accordingly, ω-oxidation cannot explain the breakdown of the COOH- extremity of 14Ca-palmitoyl-CoA observed in the fox2A mutant. The second one, a- oxidation, is involved in the shortening of fatty acids that cannot directly undergo β-oxidation, because of a methyl group in position 3 or a hydroxyl group in position 2, by the removal of a single carbon from the carboxyl end through four enzymatic steps. Both peroxisomes and mitochondria were reported to host a- oxidation, but this pathway was only described in mammals, where its deficiency may lead to the Refsum's disease.

[0094] Applicants have surprisingly found that the pxalA mutant, deleted for a gene encoding a protein responsible for peroxisomal long chain fatty acid uptake, could still grow on long-chain fatty acids as sole carbon source. However, fatty acid import into peroxisome seemed affected since triacylglycerol, involved in carbon storage from fatty acid , were significantly more accumulated in pxalA (p < 0.05) than in wild-type after growth on C16:0 medium. A C. lusitaniae pxalA fox2A double mutant had the fox2A phenotype, as it was able to grow on C14:0, C16:0 and C18:0 as the wild-type strain (data not shown). Since the mitochondrial β-oxidation was inhibited in the pxalA fox2A mutant, fatty acids were probably catabolized into the peroxisomes, and therefore imported via a transport system alternative to Pxa. Furthermore, the localization of fatty acid catabolism in two distinct organelles in C. lusitaniae, each housing specific metabolic cycles, i.e. glyoxylate cycle in peroxisomes and TCA cycle in mitochondria, suggests a crucial role of the shuttling systems required for exchanges of carbon units between mitochondria, peroxisomes and cytoplasma. Besides acyl-CoA, citrate, succinate and malate transporters, the acetyl-CoA shuttles have attracted most interest thus far. In C. albicans and A. nidulans, it seems that the transport of acetyl units between the cellular compartments is only mediated by carnitine acetyltransferases, while in S. cerevisiae, it also involves the citrate shuttle via the peroxisomal citrate synthase which catalyses the condensation of acetyl-CoA with oxaloacetate to form citrate.

[0095] Contrary to common thinking, the Applicants have surprisingly demonstrated that some hemiascomycetous yeasts also present the mitochondrial pathway of fatty acid degradation.

[0096] According to a preferred embodiment of the present invention, the isolated biologically pure hemiascomycetes yeast strains comprise at least one more genetic modification within at least one gene selected from the group consisting of F OX2, PXA1, OLE2, ECH1, ECH2, ECH3, FOXIA, FOXIB, FOXIC, FOX3-1, FOX3-2, and FOX3-3 having the nucleotide sequences as set forth in SEQ ID NOs: 1, 3, 5, 8, 10, 12, 14, 16, 18, 20, 22, and 24 respectively. As described above, at least one of FOX2, PXA1, OLE2, ECH1, ECH2, ECH3, FOXIA, FOXIB, FOXIC, F OX3-1, FOX3-2, and FOX3-3 gene may be rendered non functional and the strains comprise a total or partial gene deletion, excision, disruption insertion or integration within at least one of these genes. Preferably, at least one gene selected from FOX2, PXAl, OLE2, ECH1, ECH2, ECH3, FOXIA, FOXIB, FOXIC, F OX3-1, FOX3-2, and FOX3-3 genes is partially or totally deleted. Most preferably, at least one gene selected from FOX2, PXA1, OLE2, ECH1, ECH2, ECH3, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3- 2, and FOX3-3 genes is rendered non functional by insertion of another gene of an unrelated metabolic pathway by an antibiotic resistance gene (AB R ).

[0097] Alternatively, the present invention provides a hemiascomycetes yeast strain wherein at least one gene selected from FOX2, PXA1, OLE2, ECH1, ECH2, ECH3, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3-2, and FOX3-3 genes is enhanced and the strain comprises one or more duplication of the nucleotide sequences thereof as set forth in the nucleotide sequences as set forth in SEQ ID NOs: 1, 3, 5, 8, 10, 12, 14, 16, 18, 20, 22, or 24. In another preferred embodiment the present invention provides a hemiascomycetes yeast strain wherein at least one gene selected from FOX2, PXA1, OLE2, ECH1, ECH2, ECH3, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3-2, and FOX3-3 genes is duplicated by insertion along with another gene of an unrelated metabolic pathway and/or by an antibiotic resistance gene (AB R ).

[0098] The isolated biologically pure hemiascomycetes yeast strains and genetically modified strains as described above are capable of accumulating lipids and are thus particularly advantageous for several biotechnology applications, particularly for the production of commodity chemicals. The present invention thus also relates to the use of the biologically pure strains and mutants thereof as well as to methods of producing of commodity chemicals.

[0099] "Commodity chemical" as used herein includes any lipids that can be produced either directly or as a by-product and can be used in various industries. These include for example free fatty acids, long chain fatty acids, saturated, mono or polyunsaturated fatty acids (PUFAs), triacylglycerols (TAGs), phospholipids, phospholipids esters, phospholipids mono and/or diesters, biofuels/biodiesels, lipid derived fragrances and/or flavourings (bioflavours).

[00100] The term "fatty acids" refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C u to C 2 4, and preferably having chain lengths from about between C i6 to C 2 4- These may be saturated fatty acids, unsaturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids.

[00101] PUFAs refer to fatty acids comprising at least 18 carbon atoms and at least two double bonds, more preferably the term relates to omega-3 and omega-6 fatty acids comprising at least 18 carbon atoms, even more preferably the term relates to linoleic acid, conjugated linoleic acid (CLA), gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), arachidonic acid (ARA), alpha-linolenic acid (ALA), stearidonic acid (STA), eicosatetraenoic acid (ETA), eicosapentaenoic acid, docosapentaenoic acid (DP A) or docosahexaenoic acid (DHA). The terms "omega-3" and "omega-6" relates to double bonds in a fatty acid, which are located on the 3 rd or 6 th carbon-carbon bond, respectively, counting from the co, (methyl carbon) end of the fatty acid molecule.

[00102] Preferably, the fatty acids synthesized by the mutated strains according to the present invention are polyunsaturated fatty acids such as linoleic acid, conjugated linoleic acid, gamma-linolenic acid, dihomo- gamma-linolenic acid, arachidonic acid, alpha-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid.

[00103] TAGs refer to the lipids composed of three fatty acyl resides esterified to a glycerol molecule. TAGs can contain long chain PUFAs and saturated fatty acids, as well as shorter chain saturated and unsaturated fatty acids. [00104] The isolated biologically pure hemiascomycetes yeast strains and genetically modified strains are preferably used to produce long chain fatty acids, preferably unsaturated, mono and/or polyunsaturated fatty acids (PUFAs), most preferably Ci 6 -C24 fatty acids, and most specifically are linoleic acid and/or γ-linoleic acid.

[00105] Lipid derived fragrances and/or flavourings are natural fragrances, natural aromas, or bioflavours and refer to compounds resulting from the extraction of natural materials and/or to be transformed by natural means. The include for example terpenes, sesquiterpenes, aldehydes, esters, lactones and derivatives thereof.

[00106] The terms "terpenes and derivatives" refer to a large and varied class of organic compounds derived biosynthetically from units of isoprene, which has the molecular formula C 5 H 8 . The basic molecular formulae of terpenes are multiples of (C 5 H 8 ) n These may be selected from Hemiterpenes C 5 H 8 , Monoterpenes Ci 0 Hi 6 , Sesquiterpenes Q5H24, Diterpenes C 2 oH 32 , Sesterterpenes C25H40, Triterpenes C 30 H 48 , Tetraterpenes C^H^, Polyterpenes (C 5 H 8 )n. Derivatives include and are not restricted to iridoids, tetrahydrocannabinol, guaianolides, pseudoguaianolides, eudesmanolides, eremophilanolides, and xanthanolides etc... Flavour- terpenes compounds include for example citronellol, geraniol, linalol nerol, and a-terpineol.

[00107] Lactones are a family of flavors and fragrances. Lactones are cyclic esters of primarily γ- and 5- hydroxy acids and are produced via a key step is the hydroxylation of fatty acids. Most of the other lactones are also encountered in fermented products but are far more difficult to produce. Macrocyclic lactones are used for their musk notes in cosmetics and perfumes and their production also uses a yeast metabolic pathway. Depending on the compounds, they exhibit fruity and oily properties with peach, apricot, milky or coconut notes. Fruit and/or milk flavourings or fragrances generally comprise saturated and unsaturated δ- and γ- lactones.

[00108] By way of example, peach-like fragrances comprise γ-decalactone (4-decanolide), 4-hydroxy-cis- dodecanoic acid-y-lactone, δ-decalactone, and/or γ-octalactone, whereas coconut milk-like fragrances comprise 4-dodecanolide and/or 4-octanolide.

[00109] δ-decalactone and δ-dodelactone are highly appreciated flavouring compounds occurring in minute quantities in milk products and are responsible for bringing out the typical flavour in these products. Due to their low natural occurrence and their highly appreciated organoleptic characteristics, there exists a strong need for processes capable of providing industrial quantities of these lactones.

[00110] γ-Decalactone is present naturally in many fruits and fermented products. It is particularly important in the formulation of peach, apricot, and strawberry flavours. Microbial processes to produce this compound have been described, although the metabolic pathways involved were not completely defined so far.

[00111] Other lactones of interest may include for examples γ-valerolactone, γ-hexalactone, γ-heptalactone, γ- nonalactone, γ-undecalactone, γ-dodecalactone, γ-tridecalactone, γ-tetradecalactone, δ-hexalactone, δ- heptalactone, δ-octalactone, δ-nonalactone, δ-undecalactone, δ-dodecalactone, δ-tridecalactone, decanolides and δ-tetradecalactone.

[00112] The present invention is directed to strategies for increasing the production of lactones. In particular, the method of the present invention may be used for the production of various lactones, such as δ-decalactone, and/or γ-octalactone, which may be specifically produced by culturing the genetically modified strain according to the current invention in a medium enriched with vegetable oil and/or ricinoleic acid substrates, preferably castor oil, a castor oil hydrolysate, and the like. [00113] δ-decalactone may alternatively be produced by comprising culturing the strains according to the present invention in a medium enriched with massoi bark oil and/or hydroxypalmitic acid substrates like 1 1- hydroxypalmitic acid and/or ethyl 1 1-hydroxypalmitate.

[00114] Flavour-esters include for example volatile branched acetates, such as 3-methylbutyl-acetate which has a banana aroma, isobutylacetate, and 2-methylbutylacetate.

[00115] The recitation "biofuels/biodiesel" as used herein includes solid, liquid, or gas fuels derived, at least in part, from a biological source, such as a yeast cell. Trans-esterification of lipids yields long-chain fatty acid esters useful as biodiesel. For production of such biofuels, suitable known and commercially available media may be employed. The media may comprise suitable monosaccharide or oligosaccharide, biomass selected from marine biomass (kelp, giant kelp, sargasso, seaweed, algae, marine microflora, microalgae, and sea grass), vegetable/fruit/plant biomass, cellulose, hemicellulose, pectin, lignocellulose etc...

[00116] Furthermore, the strains as described above may comprise a genetic modification of one or more of desaturase genes. Desaturase genes encode polypeptides which can desaturate, Le_., introduce a double bond, in one or more fatty acid or precursors. It is usual to indicate activity of the desaturase by counting from the carboxyl end of the substrate. For example Δ5 desaturases desaturate a fatty acid between the fifth and sixth carbon atom numbered from the carboxyl-terminal end of the molecule and can catalyze the conversion of dihomo gamma linolenic acid (DGLA) to arachidonic acid (ARA), and/or eicosatetraenoic acid (ETA) to eicosapentaenoic acid (EPA).

[00117] Of particular interest herein are Δ6 desaturases that catalyze the conversion of linoleic acid (LA) to gamma linolenic acid (GLA) and/or a-linolenic acid (ALA) to stearidonic Acid (STA); Δ12 desaturases that catalyze the conversion of oleic acid to linoleic acid; and Δ15 desaturases that catalyze the conversion of linoleic acid (LA) to (a-linolenic acid) ALA and/or gamma linolenic acid (GLA) to stearidonic Acid (STA).

[00118] Other useful fatty acid desaturases include, for example: Δ8 desaturases that can desaturate eicosadienoic acid (EDA) to dihomo-y-linolenic acid (DGLA) and/or eicosatrienoic acid (EtrA) to eicosatetraenoic acid (ETA); Δ4 desaturases that catalyze the conversion of DPA to DHA; Δ-17 desaturases that catalyze the conversion of arachidonic acid (ARA) to eicosapentaenoic acid (EPA) and/or dihomo gamma linolenic acid (DGLA) to eicosatetraenoic acid (ETA).

[00119] The strains as described above thus comprise a genetic modification of selected from a group comprising Δ6 desaturase, Δ9 desaturase, Δ12 desaturase, and/or Δ15 desaturase. Preferably, the isolated biologically pure hemiascomycetes yeast strains according to the present invention may further comprise a genetic modification of the OLE2 gene which encodes a Δ9 desaturase. Most preferably, the OLE2 gene is a partially or totally deleted. The nucleotide and amino acid sequences are are as set forth in Figures 7 and 8, respectively.

[00120] Strains that contain non functional desaturase genes have improved production of commodity chemicals of interest. Particularly, strains according to the present invention comprising a mutation of the OLE2 gene are particularly useful in improved yield and production of lactones, such as decalactones when the yeasts are grown on suitable substrates like ricinoleic acid.

[00121] The isolated biologically pure hemiascomycetes yeast strains according to the present invention may thus comprise a genetic modification of the ECH1 gene, preferably a partial or total deletion of this gene. These genetically modified strains result in an increase in the production of lactones like decalactone, when the strains are grown in a medium containing suitable substrates like ricinoleate.

[00122] According to the present invention, some of the preferred isolated biologically pure hemiascomycetes yeast mutant strains according are as follows:

fox2A;

foxlAA;

foxlBA;

foxlCA;

fox3-lA;

f ox3-2 A;

fox3-3A;

pxalA;

fox2A, pxalA;

fox2A;foxlAA;

fox2A;foxlBA;

fox2A;foxlCA;

pxalA; foxlAA;

pxalA; foxlBA;

pxalA; foxl CA;

fox2A, pxalA; foxlAA;

fox2A, pxal A; foxlBA;

fox2A, pxal A; foxl C A;

fox2A;fox3-lA;

fox2A;fox3-2A;

fox2A;fox3-3A;

pxal A; fox3-lA;

pxal A; fox3-2A;

pxal A; fox3-3A;

fox2A, pxa 1 A; fox3 -1A;

fox2A, pxa 1 A; fox3 -2 A;

fox2A, pxal A; f ox3 -3 A;

ole2A;

ole2A, fox2A;

ole2A, pxal A;

ole2A, fox2A, pxalA;

ole2A;foxlAA;

ole2 A, foxlBA;

ole2A; foxl CA; ole2A;fox3-lA;

ole2A;fox3-2A;

ole2A;fox3-3A;

ole2A, fox2A; foxl A A;

ole2A, fox2A; foxl B A;

ole2A, fox2A; foxl CA;

ole2A, fox2A; fox3-lA;

ole2A, fox2A; fox3-2A;

ole2A, fox2A; fox3-3A;

ole2A, pxal A; foxl AA;

ole2A, pxal A; foxlBA;

ole2A, pxal A; foxl C A;

ole2A, fox2A, pxal A; foxlAA; ole2A, fox2A, pxal A; foxlBA; ole2A, fox2A, pxal A; foxl CA; ole2A, fox2A, pxalA;fox3-lA. ole2A, fox2A, pxal A; fox3-2A. ole2A, fox2A, pxal A; fox3-3A. echlA;

echl A; foxlAA;

echl A; foxlBA;

echl A; foxl C A;

echl A; fox3-lA;

echl A; f ox3 -2 A;

echl A; f ox3 -3 A;

echl A, fox2A;

echl A, fox2A; foxlAA;

echl A, fox2A; foxlBA;

echl A, fox2A; foxl CA;

echl A, fox2A; fox3-lA;

echl A, fox2A; fox3-2A;

echl A, fox2A; fox3-3A;

echl A, pxal A;

echl A, pxal A; foxlAA;

echl A, pxal A; foxlBA;

echl A, pxal A; foxl C A; echl A, pxal A; f ox3-l A; echl A, pxal A; f ox3 -2 A; echlA, pxalA;fox3-3A;

echlA, fox2A, pxalA;

echlA, fox2A, pxal A; foxl AA;

echlA, fox2A, pxal A; foxlBA;

echlA, fox2A, pxal A; foxl CA;

echlA, fox2A, pxalA;fox3-lA;

echlA, fox2A, pxal A; fox3-2A;

echlA, fox2A, pxal A; fox3-3A;

echlA; ole2A,

echlA; ole2A, foxl A A;

echlA; ole2A, foxlBA;

echlA; ole2A, foxl C A;

echlA; ole2A, fox3-lA;

echlA; ole2A, fox3-2A;

echlA; ole2A, fox3-3A;

echlA, ole2A, fox2A;

echlA, ole2A, fox2A; foxl A A;

echlA, ole2A, fox2A; foxlBA;

echlA, ole2A, fox2A; foxl CA;

echlA, ole2A, fox2A; fox3-lA;

echlA, ole2A, fox2A; fox3-2A;

echlA, ole2A, fox2A; fox3-3A;

echlA, ole2A, pxal A;

echlA, ole2 A, pxal A; foxl A A;

echlA, ole2A, pxal A; foxlBA;

echlA, ole2A, pxalA;foxlCA;

echlA, ole2A, pxalA;fox3-lA;

echlA, ole2A, pxalA; fox3-2A;

echlA, ole2A, pxalA; fox3-3A;

echlA, ole2A, pxalA; fox2A;

echlA, ole2A, pxal A; fox2 A; foxl A A; echlA, ole2A, pxal A; fox2 A; foxlBA; echlA, ole2A, pxal A; fox2A; foxl CA; echlA, ole2A, pxal A; fox2A; fox3-l A; echlA, ole2A, pxalA;fox2A;fox3-2A; echlA, ole2A, pxal A; fox2A; fox3-3 A; ech2 A;

ech2A; foxl A A; ech2A; foxlBA;

ech2A; foxlCA;

ech2A; fox3-lA;

ech2A; fox3-2A;

ech2A; fox3-3A;

ech2A, fox2A;

ech2A, fox2A; foxlAA;

ech2A, fox2A; foxlBA;

ech2A, fox2A; foxl CA;

ech2A, fox2A; fox3-lA;

ech2A, fox2A; fox3-2A;

ech2A, fox2A; fox3-3A;

ech2A, pxalA;

ech2A, pxalA; foxlAA;

ech2A, pxal A; foxlBA;

ech2A, pxal A; foxl C A;

ech2A, pxal A; f ox3-l A; ech2A, pxal A; f ox3 -2 A; ech2A, pxal A; f ox3 -3 A; ech2A, fox2A, pxal A;

ech2A, fox2A, pxal A; foxlAA; ech2A, fox2A, pxal A; foxlBA; ech2A, fox2A, pxal A; foxl CA; ech2A, fox2A, pxalA;fox3-lA: ech2A, fox2A, pxal A; fox3-2A: ech2A, fox2A, pxalA;fox3-3A: ech2A; ole2A,

ech2A; ole2A, foxlAA;

ech2A; ole2A, foxlBA;

ech2A; ole2A, foxl C A;

ech2A; ole2A, fox3-lA;

ech2A; ole2A, fox3-2A;

ech2A; ole2A, fox3-3A;

ech2A, ole2A, fox2A;

ech2A, ole2A, fox2 A; foxlAA; ech2A, ole2A, fox2 A; foxlBA; ech2A, ole2A, fox2A; foxl CA; ech2A, ole2A, fox2A; fox3-lA; ech2A, ole2A, fox2A;fox3-2A;

ech2A, ole2A, fox2A; fox3-3A;

ech2A, ole2A, pxalA;

ech2A, ole2 A, pxal A; foxlAA;

ecti2A, ole2A, pxalA; foxlBA;

ech2A, ole2 A, pxal A; foxlCA;

ech2A, ole2A, pxalA;fox3-lA;

ech2A, ole2A, pxalA; fox3-2A;

ech2A, ole2A, pxalA; fox3-3A;

ecti2A, ole2A, pxalA; fox2A;

ech2A, ole2A, pxal A; fox2 A; foxlAA; ech2A, ole2A, pxal A; fox2 A; fox IB A; ech2A, ole2A, pxal A; fox2 A; foxlCA; ech2A, ole2A, pxal A; fox2A; fox3-l A: ech2A, ole2A, pxalA; fox2A; fox3-2A: ech2A, ole2A, pxal A; fox2A; fox3-3 A: ech.3 A;

ech3A; foxlAA;

ech3A; foxlBA;

ech3 A; foxlCA;

ech3A; fox3-lA;

ech.3 A; fox3-2A;

ech.3 A; fox3-3A;

ech3A, fox2A;

ech3A, f ox2 A; foxlAA;

ech3A, fox2A; foxlBA;

ech3A, fox2A; foxl CA;

ech3A, fox2A; fox3-lA;

ech3A, fox2A; fox3-2A;

ech3A, fox2A; fox3-3A;

ech3A, pxal A;

ech3A, pxal A; foxlAA;

ech3A, pxal A; foxlBA;

ech3A, pxal A; foxlCA;

ech3A, pxalA; fox3-lA;

ech3A, pxal A; f ox3 -2 A;

ech3A, pxal A; f ox3 -3 A;

ech3A, fox2A, pxal A; ech3A, fox2A, pxalA; foxlAA;

ech3A, fox2A, pxalA; foxlBA;

ech3A, fox2A, pxalA; foxl CA;

ech3A, fox2A, pxalA;fox3-lA;

ecti3A, fox2A, pxalA; fox3-2A;

ech3A, fox2A, pxalA; fox3-3A;

ech3A; ole2A,

ech3A; ole2A, foxlAA;

ech3A; ole2A, foxlBA;

ech3A; ole2A, foxl CA;

ech3A; ole2A, fox3-lA;

ech3A; ole2A, fox3-2A;

ech3A; ole2A, fox3-3A;

ech3A, ole2A, fox2A;

ech3A, ole2A, fox2 A; foxlAA;

ech3A, ole2A, fox2 A; foxlBA;

ech3A, ole2A, fox2A; foxl CA;

ech3A, ole2A, fox2A; fox3-lA;

ech3A, ole2A, fox2A; fox3-2A;

ech3A, ole2A, fox2A; fox3-3A;

ech3A, ole2A, pxalA;

ech3A, ole2A, pxalA; foxlAA;

ech3A, ole2A, pxalA; foxlBA;

ech3A, ole2A, pxalA; foxl C A;

ecti3A, ole2A, pxalA; fox3-lA;

ech3A, ole2A, pxalA; fox3-2A;

ech3A, ole2A, pxalA; fox3-3A;

ech3A, ole2A, pxalA; fox2A;

ech3A, ole2A, pxalA; fox2A; foxlAA;

ech3A, ole2A, pxalA; fox2A; foxlBA;

ech3A, ole2A, pxal A; fox2A; foxl CA;

ech3A, ole2A, pxal A; fox2A; fox3-l A;

ech3A, ole2A, pxalA; fox2A;fox3-2A; and

ech3A, ole2A, pxal A; fox2A; fox3-3 A.

[00123] Furthermore, the Applicant has showed that the mutant yeasts were able to accumulate high amount of lipid compared to their dry weight, when grown under some specific conditions. The present invention thus relates to method of producing lipids and derived commodity materials using mutated yeasts for industrial purposes and method of optimizing such lipid production yield and rate.

[00124] Furthermore, the Applicant has showed that the mutant yeasts were able to accumulate high amount of lipid compared to their dry weight, when grown under some specific conditions. The present invention thus relates to method of producing lipids and derived commodity materials using mutated yeasts for industrial purposes and method of optimizing such lipid production yield and rate.

[00125] The present invention further relates to a method of increasing the production of lipids and/or commodity materials as described above comprising the following steps:

[00126] The method of the present invention hence facilitates the effective production of many commodity chemicals in high purity and yield including but not restricted to free fatty acids, long chain fatty acids, saturated, mono or polyunsaturated fatty acids (PUFAs), triacylglycerols (TAGs), phospholipids, phospholipids esters, phospholipids mono and/or diesters, biofuels/biodiesels, lipid derived fragrances and/or flavourings (bioflavours).

[00127] The method of producing commodity chemicals according to the present invention comprises the following steps:

a. providing the isolated strain according to the present invention,

b. growing said strain in a suitable medium, and

c. recovering said commodity chemicals.

[00128] The processes reported with the strains of the invention can be easily scaled up and hence by following the process of the present invention one can improve the working efficiency in industrial production.

[00129] The method of the present invention hence facilitates the effective production of many commodity chemicals in high purity and yield including but not restricted to free fatty acids, long chain fatty acids, saturated, mono or polyunsaturated fatty acids (PUFAs), triacylglycerols (TAGs), phospholipids, phospholipids esters, phospholipids mono and/or diesters, biofuels/biodiesels, lipid derived fragrances and/or flavourings (bioflavours). The processes reported with the strains of the invention can be easily scaled up and hence by following the process of the present invention one can improve the working efficiency in industrial production.

[00130] In preferred embodiments the present invention provides the use of the hemiascomycetes yeast strain according comprising genetic modifications in FOX2, PXA1, FOX3 (e.g., any of the paralogs FOX-1, FOX3-2, and FOX3-3), and ECH (any of the paralogs ECHl, ECH2, and ECH3) or in either two or all three genes, for the production of long chain fatty acids, or for the production of triacylglycerols and ester derivatives, or for the production of phospholipids, phospholipids esters, phospholipids mono and/or di-esters, and biodiesel, or for the production of decalactone, or for the production of lipid derived flavorings and/or fragrances, or for the production of terpens, sesquiterpene, as well as adehydes and esters derivatives thereof. In some embodiments the said fatty acids are saturated, mono and/or polyunsaturated fatty acids, or wherein said fatty acids are in Ci 6 -C 2 4, such as preferably linoleic acid or γ-linoleic acid. In yet another embodiment, the said lipid derived flavourings and/or fragrances are fruit and/or milk flavourings, preferably saturated and unsaturated δ- and γ- lactones, or wherein said lipid derived flavourings and/or fragrances are peach-like flavourings, preferably γ- decalactone, δ-decalactone, and/or γ-octalactone, or wherein said lipid derived flavorings and/or fragrances are coconut milk-like flavourings, preferably 4-dodecanolide and/or 4-octanolide. [00131] In some embodiments, the present invention also provides strains and modified strains comprising at least one more genetic modification within at least one gene selected from the group consisting of FOX2, PXA1, OLE2, ECHl, ECH2, ECH3, FOX1A, FOX1B, FOX1C, FOX3-1, FOX3-2, and FOX3-3 having the nucleotide sequences as set forth in SEQ ID NOs: 1, 3, 5, 8, 10, 12, 14, 16, 18, 20, 22, and 24 respectively.

[00132] Preferably, the present invention provides strains and modified strains comprising a genetic modification of the FOX2 gene, or of at least one gene chosen among the paralogs FOX3-1, FOX3-2, and FOX3-3, or at least of one gene chosen among of the paralogs ECHl, ECH2, and ECH3 for the production of lactones.

[00133] Most preferably, the present invention provides strains and modified strains comprising a double genetic modification within one of the gene chosen among the paralogs FOX3-1, FOX3-2, and FOX3-3, and within another gene chosen among the paralogs ECHl, ECH2, and ECH3 (FOX3/ECH double-mutated strains), for the production of lactones, and more particularly of γ-decalactones as described above.

[00134] The media and the fermentation conditions used for production of commodity chemicals is routine and well known in art. Such media may contain at least one carbon source which may be selected from a group comprising of castor oil, a castor oil hydrolysate, ricinoleic acid, palmitic acid, hydroxyl palmitic acid, stearic acid, hydroxyl stearic acid, oleic acid and derivatives, Massoi bark oil, lesquerolic acid etc... Preferably, the medium comprises carbon sources and is enriched in palmitic acid.

[00135] The media may also contain additional components like yeast extract, malt extract, polypeptone, and saccharides such as glucose. The media may contain a suitable nitrogen source which may be selected from a group comprising yeast extract, urea, corn steep liquor, ammonium sulfate, diammonium hydrogen phosphate, and the like. Chosen media may additionally contain cofactors selected from a group comprising inorganic salts such as manganese sulfate, calcium chloride, ferric chloride, ferrous sulfate, ferric sulfate, zinc sulfate, copper sulfate, magnesium sulfate, cobalt chloride, sodium molybdate, boron, and potassium iodide; coenzymes such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), and coenzyme A (CoA); nucleotides such as adenosine triphosphate (ATP); and vitamins such as L-carnitine, and vitamins of the group B.

[00136] A variety of fermentation process designs may be applied for commercial production of commodity chemicals using the strains as described above. For example, commercial production of commodity chemicals may be produced by a batch, fed-batch or continuous fermentation process.

[00137] A batch fermentation process is a closed system wherein the media composition is set at the beginning of the process and not subject to further additions beyond those required for maintenance of pH and oxygen level during the process. Thus, at the beginning of the culturing process the media is inoculated with the strains and growth or metabolic activity is permitted to occur without adding additional sources (Le^ carbon and nitrogen sources) to the medium. In batch processes the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. In a typical batch process, cells proceed through a static lag phase to a high-growth log phase and finally to a stationary phase, wherein the growth rate is diminished or halted. Left untreated, cells in the stationary phase will eventually die. A variation of the standard batch process is the fed-batch process, wherein the carbon source is continually added to the fermenter over the course of the fermentation process. [00138] A fed-batch process is also suitable in the present invention. Fed-batch processes are useful when catabolite repression is apt to inhibit the metabolism of the cells or where it is desirable to have limited amounts of source in the media at any one time. Measurement of the carbon source concentration in fed-batch systems is difficult and therefore may be estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases (ej>., CO 2 ). Batch and fed-batch culturing methods are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., (1989) Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992), herein incorporated by reference.

[00139] Commercial production of commodity chemicals using the strains according to the present invention may also be accomplished by a continuous fermentation process wherein a defined media is continuously added to a bioreactor while an equal amount of culture volume is removed simultaneously for product recovery. Continuous cultures generally maintain the cells in the log phase of growth at a constant cell density. Continuous or semi-continuous culture methods permit the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one approach may limit the carbon source and allow all other parameters to moderate metabolism. In other systems, a number of factors affecting growth may be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth and thus the cell growth rate must be balanced against cell loss due to media being drawn off the culture. Methods of modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[00140] In preferred embodiments the said suitable medium comprises carbon source and is enriched one or more substrates chosen among palmitic acid, oleic acid, ricinoleic acid, or vegetable oil, and wherein said vegetable oil is preferably selected among sunflower oil, castor oil, coconut oil, Massoi bark oil, and/or hydrolysates thereof.

[00141] In general, means for the purification of commodity chemicals may include extraction with organic solvents, sonication, supercritical fluid extraction (ej>., using carbon dioxide), saponification, fractional crystallization, HPLC, fractional distillation, silica gel chromatography, high-speed centrifugation or distillation and physical means such as presses, or combinations of these techniques. After extraction, the organic solvents can be removed by evaporation under a stream of nitrogen.

[00142] The following example describes in details the methods and techniques illustrative of the present invention. EXAMPLES

Example 1

Example 1.1: Strains and media

The C. lusitaniae strains as constructed were listed in Table 1. All the mutant strains were derived from the wild-type strain 6936 MATa (Centraalbureau voor Schimmelcultures, Utrecht, The Nederlands). Yeasts were routinely cultivated in YPD (1 % (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose). Carbon assimilation tests were performed using YNB (0.17% (w/v) yeast nitrogen base without amino acids and without ammonium sulfate (Difco laboratories), 0.5% (w/v) ammonium sulfate) supplemented with 2% (w/v) of either glucose (so called YNB glucose) or other carbon sources. Counterselection of uracil auxotrophs after pop-out of the URA3 marker gene in transformants was done on YNB glucose supplemented with 1 mg/ml of 5-fluoroorotic acid (5-FOA, Sigma Chemicals Co.) and 50 g/ml uracil (1). For subcellular fractionation, the WOYglu medium made of YNB supplemented with 0.1 % (w/v) yeast extract and 0.3% (w/v) glucose and the Induction medium containing 0.5% (w/v) bactopeptone, 0.3% (w/v) yeast extract, 0.12% (v/v) oleic acid, 0.2% (v/v) Tween 40 and 0.5% (w/v) KH 2 PO 4 (adjusted to pH 6.0 with NaOH) was used. Yeasts were cultivated at 30°C, 35°C or 37°C, under constant agitation (230 rpm) for liquid cultures.

Table 1 : Genotypes of the Candida lusitaniae strains as constructed according to the present invention

Mutant strains of C. C. lusitaniae

Strains Genotype

6936 MATa, URA3, ICL1, FOXla, FOXlb, FOXlc, FOX2, FOX3-1, FOX3-2, vvt from CBS FOX3-3, PXA1

CL38 MATa, URA3, ICL1, FOXla, FOXlb, FOXlc, FOX2, FOX3-1, FOX3-2,

FOX3-3, PXA1

MATa, wra3[A360], ICL1, FOXla, FOXlb, FOXlc, FOX2, FOX3-1, FOX3-2, ?[A360]

FOX3-3, PXA1

MATa, wra3[A990], ICL1, FOXla, FOXlb, FOXlc, FOX2, FOX3-1, FOX3-2, ura3[A990]

FOX3-3, PXA1

MATa, wra3[A360], FOXla, FOXlb, FOXlc, FOX2, icllA-.-.GVN

FOX3-1, FOX3-2, FOX3-3, PXA1

MATa, icll Ar.npt, wra3[A360], FOXla, FOXlb, FOXlc, FOX2, FOX3-1, icllA, ura3A

FOX3-2, FOX3-3, PXA1

MATa, icllAv.npt, um3[A36G]:: URA3 , FOXla, FOXlb, FOXlc, FOX2, icllA

FOX3-1, FOX3-2, FOX3-3, PXA1

MATa, icllA::ICLl-URA3, wra3[A360], FOXla, FOXlb, FOXlc, FOX2,

ICLIRe

FOX3-1, FOX3-2, FOX3-3, PXA1

MATa,fox2A::npt-URA3-npt, wra3[A360], ICL1, FOXla, FOXlb, FOXlc, fox2A:: GUN

FOX3-1, FOX3-2, FOX3-3, PXA1

MATa, j vx2 A::npt, wra3[A360], ICL1, FOXla, FOXlb, FOXlc, FOX3-1, fox2A, ura3A

FOX3-2, FOX3-3, PXA1

MATa,fox2A::npt, um3[A36G]:: URA3 , ICL1, FOXla, FOXlb, FOXlc, FOX3- fox 2 A

1, FOX3-2, FOX3-3, PXA1

MATa, f ox2 A::FOX2-URA3, wra3[A360], ICL1, FOXla, FOXlb, FOXlc,

FOX2Re

FOX3-1, FOX3-2, FOX3-3, PXA1

MATa, fox2A:: URA3 -npt, wra3[A990], ICL1, FOXla, FOXlb, FOXlc, FOX3- fox2A_EL

1, FOX3-2, FOX3-3, PXA1

MATa, f ox2 A, wra3[A990], ICL1, FOXla, FOXlb, FOXlc, FOX3-1, FOX3-2, fox2A_c ura3A

FOX3-3, PXA1

MATa, f ox2 , um3[A990] URA3, ICL1, FOXla, FOXlb, FOXlc, FOX3-1, f ox2 A_cl

FOX3-2, FOX3-3, PXA1

pxalA MATa, pxalA::pGEMT-URA3-pxal [cove], ura3 A[A990], ICL1, FOXla,

Example 1.2: DNA extraction

C. lusitaniae genomic DNA was extracted as described by Scherer S. et al. (J.Clin.Microbiol. 25:675-679) from spheroplasts prepared using zymolyase (Euromedex). DNA concentrations were determined with a spectrophotometer at λ 2 , using a conversion ratio of 1 OD 2 eo for 50 mg/ml DNA.

Example 1.3: PCR amplifications

Hot-Star (Qiagen) or Pfu (Promega) Taq DNA polymerase was used for the PCR amplification of DNA fragments. PCR were performed as recommended by the supplier. All the primers were synthesized by Eurofins MWG Operon and are listed in Table 2.

Table 2: Oligonucleotides

PRIMERS Sequence (5' to 3') * USES

GACATGCATGCGGCCGCACAGAGGAGTA

NotURA3

AGACAGG

Insertion of URA3 in pGEM-T

GCATCACACTAGTACCCGTTGATGGCAGA

SpeURA3

GTTG

TCGAGATGATCACCCGAGGTCGCATGCTC

BclGUN

C Insertion of URA3 for mutation of

CACGTGAGGCCTCCAAGCTATTTAGGTGA ICLl

StuGUN

CAC

TCGAGACAATTGCCCGAGGTCGCATGCTC

MfeGUN

C Insertion of URA3 for mutation of

CGCGCGAGATCTCCAAGCTATTTAGGTGA FOX2

Bgl2GUN

C

3'URA3F CTTCTCTATCCGTCTCTGTTCC PCR of transformants 5'U A3R ACATCATCCTTCACCAACTCTGC

URA3del ACCGGTGACTCCATGAGCGTTG

URA3ex CTCAGCGTCAGGTGTTTACG

5Ura3 cccgttgatggcagaptggtgaa

PCR of wt URA3- URA3 probe

3Ura3 atgacattctccattcaaatgc

FICLI CGGTACATTACCTGTGCTTGC Insertion of ICL1 in pGEM-T -

RICLl GAGCAGTTCAGGTAAATCCG ICL1 probe

FICLlex ACTTCCAGATGGCTTCTCCA

PCR of transformants icll

RICLlex TTCAGTCCACATGGATGGTC

Insertion of FOX2 in pGEM-T -

FFOX2 CGAGGTGTCACCATATAAGCC

FOX2 probe

Insertion of FOX2 in pGEM-T - PCR of the downstream fragment

RFOX2 GCTCACGAACGAAAGCCTAC

for preparing/o^2A _EL - FOX2 probe

5amFox2 GCGTCGTAACAACATCGCATAATT

PCR of the upstream fragment for atggaaaacatcatccttcaccaactctgccatcaacgg AG A

3amFox2Ura preparing fox2A _EL

GAAGCGAGTTAGTTCGAG

5Fox2niche GAGTTTCGTTCCTCTTTCAAGGTT Nested PCR for preparing/o^2 Δ

3Fox2niche ACATTTCACACTCCTGAGACA JEL

5AmFox2clean CTG AGGC ACC AAGTG ATTG AGGT A

TTTCAGAAACAGAAACATTTCACACTCCT PCR of the upstream fragment for

3amFox2clean GAGACATAAAAGAGAAGCGAGTTAGTTC preparing fox2 A _cl

GAG

TTTATGTCTCAGGAGTGTGAAATGTTTCTG PCR of the downstream fragment

5AvFox2clean

TTTCTGAAAGTAGGCTTTCtTTCGTGAGC for preparing/ot2A _cl

PCR of the downstream fragment

3AvFox2clean CGGTTCCGTTACGAATACCAAGGC for preparing/ot2A _cl, - FOX2-2 probe

5AvFox2Southern GTAGGCTTTCGTTCGTGAGC FOX2-2 probe

5Fox2cleanniche GCAAGAAGTCCTCTCACCGCACAC

Nested PCR for obtaining fox2A _cl

3Fox2cleanniche CACATTGATGGTGCCATGCACGCA

5exFox2clean CTAGACACTCATTGTTGCTCAAGT

3exFox2clean CTGCACGACCTGAGCTTTGTGCCG

PCR of transformants fox2

FFOX2ex ATCTCGGTCCGTCATGAGTG

RFOX2ex ATATGGCCTCCGAACAGAG

5'PxalNcoI CATGCCATGGCACCAAGACGAAGGCGAG PXA1 in pG-URA3

3TxalSacII TCACCGCGGGCTCAACATATCGTCGTACG PXA1 probe

5'Pxalex GACAGGTGCTATTGTTGGC

3'Pxalex CGTATCCCCTTTGATCTCCAACAC PCR of transformants pxal

3 FOX3-2b GTTGGCCAAGCCCACAACAGA

ATGGTGACAGACGCCCAAGAAGAGCAAT

5AmFoxla

C PCR of the upstream fragment to ttcaccaactctgccatcaacgggCACAl ACGCAAGGG prepare foxla A

3amFoxlaUra

CAACAGACTG

gcatttgaatggagaatgtcatAlGGCGCAAAlCCAC

5AvFoxlaUra PCR of the downstream fragment to

CGACGTTAG

prepare foxla A

3AvFoxla CTTGTTGATGAGAGTGATTTCCGCCG

5Foxla_niche AGCTTCTTCCATGGTGACAGACGC

Nested PCR for foxlaA

3Foxla niche GCCAGACCACGCGATGTTATTCCA

5Foxla ex GAATTCCGAAGTATCCTAAAGTTGCG

PCR of transformants foxla

3Foxla_ex CTTCTCATAATGCGACATGATGCG

5amFoxlb CGAAGTGTTGATCACACGTATTGG

PCR of the upstream fragment for atggaaaacatcatccttcaccaactctgccatcaacgggG AA

3amFoxlbUra preparing foxlbA

GGAGGGGAGTGATATCG

atttgtgtttgccaaagcatttgaatggaeaatgtcatGCGTCG PCR of the downstream fragment

5AvFoxlbUra

TCGCAtTCAGGTATA for preparing /«ζ/

3AvFoxlb GAGGCAAGAGGATAGCTGGGGTGG PCR of the upstream fragment for

' The sequences in bold characters correspond to a restriction endonuclease site whose name is included in the name of the primer (NotI, Spel, Bell, Stul, Mfel, Bglll, SacII).

2 Sequences in lower case letter are homologous to the LJRA3 gene. In silica BLAST analysis of the C. lusitaniae database Q SlU^^^E^^^ ^ Mi^ ^ ^ ye n< ui i e/c a n ( I i cl gro ιψ/ ) thereafter named BROAD Institute Candida genome database, allowed identification of the following ORFs:

(i) a 1650-bp ORF CLUG_01411.1 encoding a predicted protein of 549 amino acids (61 kDa) that bore a significant identity (79%) and 89% similarity with isocitrate lyase Icllp (orfl9.6844) of C.

Albicans; and

(ii) a 2709-bp ORF CLUG_01348.1 encoding a predicted protein of 902 amino-acids (98 kDa) that bore significant identity (71 %) and 85% similarity with the multifunctional- β -oxidation protein Fox2p (orfl9.1288)) of C. albicans.

The 1650-bp intronless C. lusitaniae ICL1 gene was located on supercontig 2 from positions 409109 to 410755 (CLUG_01411.1) and the 2709-bp intronless C. lusitaniae FOX2 gene was located on supercontig 2 from positions 281008 to 283713 (CLUG_01348.1). The complete ICL1 and FOX2 genes with their 5' and 3' non coding regions (300 and 500 bp for ICL1 and 200 and 400 bp for FOX2) were isolated from the wild-type strain 6936 by PCR amplification and cloned into pGEM-T (Promega), to yield the plasmids pG-ICLl and pG-FOX2.

The genotypes of all the strains were verified by PCR and Southern blot analysis. Figure 32 provides southern blot analysis of the wild type and Fox2p strains.

The C. lusitaniae Icllp putative protein showed conservation of the K208, K209, C210, H212 amino acids, which were previously identified as the catalytic residues in the isocitrate lyase of Escherichia coli. As in other yeasts, the C. lusitaniae Fox2p contains two dehydrogenase domains (DHaseA at aa 24-180, and DHaseB at aa 323-485), which are shortchain alcohol dehydrogenase/reductase superfamily members, and one enoyl-CoA hydratase domain (aa 780-895) Both Icllp and Fox2p of C. lusitaniae contain a type I peroxisomal targeting signal (-GKL) at the COOH extremity of the protein. Synteny was conserved in the close vicinity of CLUG_01411.1 and CLUG_01348.1 in C. lusitaniae and in C. albicans, which reinforced the idea that these ORF are the true orthologs of the ICL1 gene and FOX2 gene of C. albicans. Furthermore, other paralog genes having significant homology with ICL1 and FOX2 could not be detected in the genome of C. lusitaniae. Example 3: Construction of IcllA and fox2A mutants

Icll and fox2A null-mutants, and the respective revertant strains having a reconstituted functional allele were constructed by using an improved integrative transformation system based upon the " JTRAi-blaster" strategy, adapted for C. lusitaniae by Francois et al. (Yeast, 2004, 39:3906-3914). The URA3 gene was used as an auxotrophic marker because its removal from the deleted loci can be easily counterselected on a medium containing 5-fluoro-orotic acid, and it can be further reintroduced at its endogenous locus to avoid positional effects of URA3 expression (Brandt A. et al., 2004, Eukaryotic Cell, 3:900-909). The central part of the coding region of each cloned gene was deleted by digestion with adequate restriction enzymes from New England Biolabs (Bell and Stul for ICL1, resulting in 701-bp deletion ; Bglll and Mfel for FOX2, resulting in 1543-bp deletion) and was replaced by the GUN fragment ; this fragment consisted of the C. lusitaniae URA3 gene flanked on both sides by a noncoding 327 -bp repeat (npt) obtained by amplification from the prokaryotic NPTI gene encoding neomycin phosphotransferase. The resulting disruption cassettes (IcllA-GUN, Fox2A-GUN) were liberated from the plasmid backbone using Ndel and Sphl and were separately used to transform the strain ura3 [A360] to prototrophy (Francois et al. (Yeast, 2004, 39:3906-3914), in order to obtain the mutant strains icllA::GUN and/o 2A::GUN. All the genotypes were confirmed by PCR and Southern Blot analysis as shown for FOX2 and URA3 loci (Figure 32). Nucleotide sequencing was also used to confirm the genotype of the fox2A mutant.

Example 4: Complementation of icllA and fox2A null mutant alleles

Selection of mutant strains that had excised the URA3 marker was achieved by plating yeast cells from the mutant strains icllA::GUN and /o 2A::GUN onto YNB glucose plates containing 5FOA and uracil. Ura ~ clones resistant to 5FOA were selected and their genetic organization, Le_., the loss of the URA3 gene and of one of the flanking npt repetitive fragments, was confirmed by PCR and Southern Blot hybridization. The resulting mutant strains icllA, ura3A and fox2A, ura3A were used further in two ways. First, they were transformed to uracil prototrophy by the complementation plasmid pG-URA3 to restore a functional URA3 at its resident locus, allowing the selection of two new mutant strains having the genotypes icllA, ura3 [A36 o : URA3 (abbreviated icllA) and fox2A, ura3 iA36Q] :: URA3 (abbreviated fox2A). This methodology was proven to be phenotypically neutral and avoids a possible position effect on the expression level of URA3. Second, the strains icllA, ura3A and fox2A, ura3A were transformed to uracil prototrophy by the complementation plasmids pG-ICLl-URA3 and pG-FOX2-URA3, obtained after insertion of an URA3 allele at the Notl and Spel restriction site of pG-ICLl and pG-FOX2. Integration of the plasmids into the genome of the recipient strains was targeted at the icllA and the fox2A loci, respectively, to give the "revertant" strains ICLlRe and FOX2Re. All genotypes were verified by PCR and Southern Blot hybridization; the genotype of the fox2A strain was also confirmed by nucleotide sequencing of the deleted locus. Example 5: Identification, cloning of the PXA1 gene of C. lusitaniae and construction of the pxalA mutant strain

The gene PXA1 encoding part of an ABC transporter responsible for peroxisomal long chain FA uptake was identified in the genome of C. lusitaniae (http://www.broadinstitute.org/) with a BLAST analysis (Altschul S. et al. (1997) Nucl. Acids Res. 25:3389-3402) using as query the orthologous proteins of C. albicans {Candida Genome Database, £ΐΙ1^^ίί^Μ^ £!^Π^ί Μ£}-

BLAST analysis of the C. lusitaniae database (BROAD Institute Candida genome database) allowed identification of a 2430-bp ORF CLUG_01238.1 encoding a predicted protein of 809 amino acids that bore a significant identity (66%) and 79% similarity with the protein (Pxalp (orfl9.7500) of C. albicans. The C. lusitaniae PXA1 gene was located on supercontig 2 from positions 55051 to 57480 (CLUG_01238.1).

Pxalp belongs to the superfamily of ATP-binding cassette (ABC) proteins, and to the adrenoleukodystrophy protein (ALDp) subfamily. The C. lusitaniae Pxalp putative protein had highly conserved residues corresponding to the Walker A (aa 566-589) and Walker B (aa 687-734) domains, possessed an ABC signature (aa 668-716) and a motif that resembled (aa 603-635) a sequence described as "new motif for the ALDp subfamily . Syntheny was partially conserved in the close vicinity of Pxalp in C. lusitaniae and in C. albicans.

Disruption of the PXA1 gene of C. lusitaniae

The pxalA mutant was constructed by disruption of the coding sequence of the wild allele. A 564bp fragment corresponding to the core of the coding region of PXA1 was amplified by PCR and cloned to the Ncol and Sacll restriction sites of the plasmid pG-URA3 (corresponding to a pGEM-T plasmid harbouring a cloned URA3 gene). The resulting plasmid pG-URA3-pxal [core] was then linearized by Aflll, a single restriction site located within the PXA1 cloned sequence, before transforming the strain ura3 iA36Q] to prototrophy. The pxal A:\ URA3 mutant strain was obtained after homologous integration of the plasmid at the PXA1 locus.

Example 6: Identification of the OLE1 and OLE2 gene of C. lusitaniae and construction of the ole2A mutant strain.

BLAST analysis of the C. lusitaniae database (BROAD Institute Candida genome database) allowed identification of two genes encoding proteins that bore a significant identity with the Olelp and 01e2p proteins of C. albicans. The C. lusitaniae OLE1 gene (CLUG_00176.1) was located on supercontig 1 from positions 345711 to 344260. The gene is intronless, has a size of 1452 bp, and encodes a predicted protein of 483 amino acids having 78.6% of identity with Olelp of C. albicans. The C. lusitaniae OLE2 gene (CLUG_05828.1) was located on supercontig 8 from positions 512253 to 513689. The gene is intronless, has a size of 1437 bp, and encodes a predicted protein of 478 aa having 45.2% of identity with 01e2p of C. albicans.

Several attempts to disrupt the OLE1 gene of C. lusitaniae failed, which strongly suggested that OLE1 was an essential gene for C. lusitaniae, as for C. albicans.

Deletion of OLE2 was performed by homologous integration of a DNA cassette constructed by the cloning- free PCR-based method as previously described (21), using URA3 as the marker gene of transformation. An OLE2 deletion DNA cassette made of a 1400 bp fragment carrying the URA3 gene, flanked by two 700 bp fragments corresponding to the upstream and downstream region of OLE2, was constructed by overlapping PCR (Fig. 1) using the specific oligonucleotides listed in Table 2. The ole2 A:\ URA3 mutant strain was obtained after transformation of the wra3A[990] strain to uracil prototophy by the homologous replacement of the wild OLE2 locus with the OLE2 deletion cassette. Example 7: Cloning the ECH1 gene of C. lusitaniae and construction of the echlA mutant strain

BLAST analysis of the C. lusitaniae database (BROAD Institute Candida genome database) allowed identification of a 1452-bp gene encoding a predicted protein of 483 amino acids that was homologous to the echAp mitochondrial enoyl-CoA hydratase of Aspergillus nidulans (30% identity, 46% similarity over partial alignment) and to the Ehd3p mitochondrial 3-hydroxyisobutyryl-CoA hydrolase (member of a family of enoyl- CoA hydratase/isomerases) of Saccharomyces cerevisiae (38.5% identity, 67.5% similarity). The C. lusitaniae ECH1 gene was located on supercontig 4 from positions 1636544 to 1637992 (CLUG_04032.1). The echlA::URA3 mutant was obtained by homologous integration of a DNA cassette constructed by the cloning- free PCR-based method as previously described (21), according to the scheme of Fig. 2, and using the oligonucleotides listed in Table 2. Additionally, the URA3 marker of the echlA::URA3 mutant was excised by transforming the mutant strain with a cassette made of the upstream DNA fragment of ECH1 fused to the downstream fragment of ECH1. Integration of the cassette by a double crossing-over at the echlA locus led to the excision and loss of the URA3 marker. Transformants that had excised URA3 (genotype echlA, ura3A) were selected onto YNB supplemented with 5FOA and uracil. The URA3 wild allele was then reintroduced by homologous recombination at its own locus to give the final echlA mutant.

Example 8: Cloning the FOX3-1 gene of C. lusitaniae, construction of the fox3-lA mutant strain and of the double mutant \fox3-l, echl]A

BLAST analysis of the C. lusitaniae database (BROAD Institute Candida genome database) allowed identification of a 1317-bp gene (CLUG_05112) encoding a predicted protein of 438 amino acids that was homologous to the Fox3p protein of C. albicans (34% identity, 52% similarity). The fox3-l A:: URA3 mutant was obtained by homologous integration of a DNA cassette constructed by the cloning-free PCR-based method as previously described (21), according to the strategy whose the principle is illustrated in Figure 2. The double mutant \fox3-l, echl]A is obtained by invalidating the FOX3 gene in a echlA, ura3A mutant (constructed in Example 7).

Example 9: Constructing mutants invalidated for several genes

Mutants bearing deletions in several genes (2, 3, or more) were obtained according to the strategy given in Examples 7 and 8. It is based on the JT/Mi-blaster strategy which allows the recurrent use of the URA3 gene for disrupting one or several genes of interest in a single strain.

Example 10 Southern hybridizations

A least 2 g of C. lusitaniae DNA was digested with Hindlll or EcoRV and separated by electrophoresis in a 0.8% agarose gel. The DNA was transferred onto a nylon membrane (Hybond N + ; Roche Molecular Biochemicals) and hybridized with digoxigenin-labeled DNA probes synthesized with a PCR DIG probe synthesis kit (Roche Molecular Biochemicals), as recommended by the supplier. ICL1, FOX2, PXA1, ECH1, OLE2 and URA3 DNA probes were generated by PCR amplification with the relevant primers (see Table 2). The bla DNA probe, which was specific for the plasmidic ampicillin resistance gene, was also generated with specific primers (Francois et ai, Yeast, 2004, 39:3906-3914).

Example 11: DNA sequencing and sequence analysis

The nucleotide sequence of FOX2, ICL1, PXAl, ECH1 and OLE2 were determined, along with intergenic surrounding regions, and were provided with their predicted translation products. Sequence reaction was realized using ABI Prism Dye Terminator Cycle Sequencing Ready Reaction ® vl . l Kit (Applied Biosystems) from plasmidic DNA (300 a 600 ng) or PCR amplification (100 ng), as recommended by the supplier. Sequencing was done by the genotyping-sequencing facility of Bordeaux Segalen University. The similarity searches in databases were performed with the Basic Local Alignment Search Tool (BLAST) programs (Altschul S. et al., Nucl. Acids Res., 1997,35: 14-17) available on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) and on the BROAD Institute website (http://www.broadinstitote.org/annotation/genome/candida_gro up/Blast.html).

Consensus multiple alignments for nucleotide and amino acid sequences were done with the ClustalW program, available on the European Bioinformatics Institute website (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

Example 12: In vitro growth assays

Growth assays were performed on solid YNB media containing 2% of either glucose, potassium acetate, ethanol, citrate, glycerol, oleic acid (C18: l), stearic acid (C18:0), palmitic acid (C16:0), myristic acid (C14:0), or capric acid (C10:0) as the sole carbon source. Inoculated media were incubated at room temperature, 30°C, or 37°C for 3 to 7 days, depending on the carbon source, as indicated in the figure legends. For the spot dilution assays, the strains were grown to mid-log phase in YPD, collected by centrifugation, washed with water, and resuspended in water to an OD 60 o of 0.5. Cells were transferred to 96-well plates, and serially diluted fivefold. Drops of 7 μΐ of each dilution were deposited onto solid media. For growth assays in liquid media, strains were grown in YNB-glucose at 30°C or 37°C overnight. The next day, cells were harvested by centrifugation, washed twice with water, and resuspended at an OD 60 o of 0.08 in YNB media containing the appropriate carbon source. Growth of the cultures was assessed over a period of 5 to 7 days by measuring the OD at 600 nm.

Example 13: Purification of subcellular organelle fractions with sucrose density gradient

Preparation of subcellular fraction was adapted from Distel & Kragt protocol (Distel B. et al., Meth.Mol.Biol, 2006, 313:21-26). Briefly, yeast strains were grown for 2 days in WOYglu at 35°C with agitation. When OD 60 o reached 1.0, cells were harvested by centrifugation (4000 g, 5 min) and resuspended in 10 ml of induction medium. Then, 5 ml were used to inoculate one liter of induction medium. After 16 h of incubation at 30°C, yeasts were harvested, washed twice (4000 g, 5 min) with distilled water. Wet weight of the cell pellet was determined and cells were resuspended in 10 ml of buffer SH (β-mercapto-ethanol 0.5M, Tris-HCl 2.5 M, pH 9.3) per gram and incubated 10 minutes at 30°C under agitation. Then, cells were washed twice (4000 g, 5 min) in Tris-KCl buffer (KCl 0.5M, Tris 10 mM, pH 7). Yeasts were converted in spheroplasts after 1 hour of incubation at 30°C under agitation in 10 ml of Digestion Buffer (Sorbitol 1.35 M, EDTA 1 mM, Citrate phosphate 10 mM, pH 5.8) supplemented with 10 mg zymolyase per gram of fresh cells. All subsequent steps, including the separation of peroxisomes, were performed at 4°C. Spheroplasts were washed (4000 g, 10 min) twice in Spheroplast Buffer (Sorbitol 1.2 M in KEM (MES 5 mM, EDTA 1 mM, KCl 1 mM, pH 5.5)). Then, spheroplasts were broken by incubation 1 hour on ice in 10 ml homogenization buffer (Sorbitol 0.6 M in KEM, PMSF 1 mM) per gram of initial fresh cells. The homogenate was centrifuged at 1500 g for 10 min. The supernatant was saved, and the pellet was homogenized again in 20 ml of homogenization buffer. After 15 min on ice, the suspension was centrifuged; the supernatants were pooled and were further centrifuged at 20,000 g for 30 min. The organelle pellet was resuspended in 2 ml of sucrose 20% (w/w, in KEM).

Sucrose density gradient were adapted from Kamiryo et al. (9). The organelle fraction was applied onto a discontinuous gradient consisting of 4,5-, 4,5-, 9-, and 4,5- ml sucrose solutions of 25, 35, 42, and 53% (w/w in KEM), respectively. The tubes were centrifuged at 100,000 g for 1 h with an AH -629 swinging -bucket rotor (Sorvall). The small visible band at the 53 to 42% sucrose interphase contained the peroxysomal fraction, the larger one at the 42 to 35% interphase contained the mitochondrial fraction. Protein concentrations were estimated by the Bradford method, using bovine serum albumin as standard.

Example 14: Assay of enzymes

Catalase (EC 1.11.1.6) activity was assayed using a Cary 100 scan (Varian) spectrophotometer at λ=240 nm and 20°C, as described by Aebi (11). The reaction mixture for catalase assay contained 10 g protein sample, 50 mM potassium phosphate (pH 7) and 11 mM H 2 0 2 was used to start the reaction. Cytochrome c oxidase (EC 1.9.3.1) activity was measured at λ 550 nm and 37 °C. Cytochrome c (Sigma) was previously reduced by equimolar addition of ascorbic acid. The reaction mixture contained 100 mM potassium phosphate (pH 6.9), 1 mM EDTA, and 28 μΜ cytochrome c reduced with ascorbic acid. The addition of protein sample was used to start the reaction. One enzyme unit is defined as the amount which catalyzes the conversion of 1 μιηοΐ of substrate per min.

Example 15: Palmitoyl-CoA catabolism assay

Crude extracts (150 g) or organelle fraction lysates (10 μg) were incubated at 37°C in 100 μΕ of reaction mixture containing 20 mM NAD, 10 mM Co ASH, 1 mM FAD, 100 mM KCN, 2% Triton, 100 mM Tris-HCl pH 7.4. Reaction was started by the addition of ΙμΕ of 14 C a -palmitoyl-CoA 10 μΜ (20 μθϊ/ητΐ, Sigma- Aldrich). Reaction was stopped by the addition of 100 μΕ KOH 5M. After saponification (one hour at 65 °C), and acidification (addition of 100 μΕ H 2 S0 4 36 N), fatty acids were extracted with 2 mL of CHC1 3 . Extracts were evaporated (under N 2 ), dissolved in 50 μΕ CHCI 3 / CH 3 OH 2: 1 (v/v) and the compounds were separated by thin layer chromatography (TLC) on 10x10 cm silica gel plates using 75:24: 1 hexane/diethyl ether/acetic acid as solvent (v/v/v). Revelation was done using Phosphorimager SI system (Amersham Biosciences). Analysis of 14 C a -palmitoyl-CoA consumption was then done using ImageQuant analysis software (Amersham Biosciences).

Example 16: Lipid fatty acid composition

After growth on YPD or YNB + 2% palmitic acid, yeast were harvested and washed 3 times in water; amount of cells equivalent to 15 mg dry weight were then transformed into spheroplasts using zymolyase, as described above. To extract lipids from whole cells, two ml of chloroform/methanol (2: 1, v/v) were added to the cell suspensions. After shaking and centrifugation, the organic phase was isolated and the remaining lipids were further extracted twice by the addition of 2 ml of chloroform to the aqueous phase and by shaking. The organic phases were then pooled and evaporated to dryness. Next, the lipids were redissolved in 70 μΕ of chloroform/ methanol (2: 1 v/v). Neutral lipids were purified from the extracts by one-dimensional TLC on silica gel plates (10x10 cm; Merck) using hexane / diethylether / acetic acid (90: 15: 2) as solvent. The lipids were then visualized by spraying the plates with a solution of 0.001 % (w/v) primuline in 80% acetone, followed by exposure of the plates to UV light. The silica gel zones corresponding to the various lipids were then scraped from the plates and added to 1 mL of methanol/2.5% H 2 S0 4 containing 5 μg of heptadecanoic acid. After 1 h at 80°C, 1.5 mL of H 2 0 was added and fatty acid methyl esters (FAMES) were extracted with 0.75 mL of hexane. Separation of FAMES was performed by gas chromatography (GC) (Hewlett Packard 5890 series II; Hewlett-Packard, Palo Alto, CA, USA). Example 17: Lipid analysis by mass-spectrometry (MS)

Qualitative analyses were performed using an Agilent 6850 gas chromatograph equipped with an HP-5MS column (30 m x 0.25 mm x 0.25 μιη) and an Agilent 5975 mass spectrometric detector (70 eV, mass-to-charge ratio 50-750). The initial temperature of 50°C was held for 1 min, increased at 50°C min "1 to 200°C, held for 1 min at 200°C, increased again at 10°C min "1 to 320°C, and held for 8 min at 320°C, with helium (1.5 mL min " : ) as carrier gas.

Example 18: Mitochondrial respiration

This method was adapted from Manon et al. (Eur.J.Biochem, 1988, 172:205-211). Assays were performed using spheroplasts instead of purified mitochondria, because the FA-CoA, at the concentrations used, behaved as a detergent inducing lysis of mitochondria.

Spheroplasts obtained as above were washed three times (2000 g, 10 min) in R buffer (sorbitol 1 M, EGTA 0.5 mM, MgS0 4 2 mM, NaCl 1.7 mM, BSA 0.1 % and KH 2 P0 4 10 mM pH 7.2) and resuspended in the same buffer at a concentration of 125 mg/ml. Spheroplasts were then permeabilized with nystatin (40 g ml) during 10 minutes under gentle agitation. The respiratory activity was measured at 28°C with a Clark-type electrode connected to a computer giving an on-line display of rate values. Different substrates and inhibitors were added to the following final concentrations: NADH (4 mM), palmitoyl-CoA (0.2 mM), stearoyl-CoA (0.2 mM), valinomycin (5 μg/ml), myxothiazol (0.1 μg/ml), phenylsuccinate (5 mM). O 2 consumption rates are expressed in nmol O 2 per milligram of proteins and per milliliter (nmol Oi.mg ' mT 1 )

Example 19: Immunolocalization or immunodetection of Fox2p and Icllp by western-blotting

Ten g of proteins of the peroxisomal and mitochondrial fractions were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and transferred to polyvinylidene difluoride membrane. After a saturation phase in PBS-Tween-Milk (Phosphate 15 mM, pH 7.4, NaCl 154 mM, Tween 20 0.05% (v/v), milk powder 5%), the membrane was incubated with primary rabbit antiserum. The polyclonal antibody against Fox2p, designed against the Candida tropicalis multifunctional beta-oxidation enzyme, was graciously provided by W. Schliebs (Medizinisch Fakultat Ruhr-Universitat Bochum, Germany). The polyclonal antibody against Icllp, designed against the Ashbya gossypii isocitrate lyase, was a gift from S. Nieland (Hochschule Lausitz, Senftenberg, Germany). After 3 washing steps and incubation with a secondary anti-rabbit antibody coupled with peroxidase, immunodetection was realized in presence of 3,3'-diamino benzidine tetrahydrochloride and H 2 0 2 . The Quantity One software (BioRad) was used for the signal quantification.

Example 20: Growth of C. lusitaniae fox2A mutant and icllA mutant on medium-chain and long- chain saturated FA as sole carbon source

S. cerevisiae and C. albicans mutants with defect in peroxisomal β-oxidation (Le_., fox2A) or in the glyoxylate (i.e., icllA) are not able to utilize FA as the sole carbon source as reported by Hiltunen JK et al. (2003, FEMS Microbiol. Rev 2003, 27:35-64), Piekarska K et al. (Eukaryotic Cell, 20065: 1847-1856), and Ramirez MA et al. (2007, Eukaryotic Cell 6:280-290). The ability of the C. lusitaniae icllA and fox2A mutants to grow on minimal medium (YNB) containing glucose, glycerol, acetate, saturated short-chain FA (C10:0), saturated medium-chain FA (C12:0, C14:0), saturated long-chain FA (C16:0, C18:0), or unsaturated long-chain FA (C18: l), as exclusive carbon source was tested (Figure 33).

All strains grew equally on medium containing glucose and none of them was able to grow on medium containing C10:0. C. lusitaniae icllA was unable to assimilate acetate and FA as it was previously described for C. albicans (Lorenz MC et al., 2001, Nature 412:83-86; Ramirez MA. et al., 2007, Eukaryotic Cell 6:280- 290), but was still able to grow on glycerol, whereas the C. albicans mutant did not. Surprisingly, the deletion of FOX2 did not abolish growth on medium-chain and long-chain saturated FA in C. lusitaniae. Growth on C18: l was alterated in a temperature-dependent manner, with a growth defect marked at 37°C. The fox2A mutant also grew on acetate and glycerol. The wild-type phenotypes were restored in the revertant strains ICLIRe (icllAv.ICLl) and FOX2Re The growth phenotypes of the fox2A mutants of C. lusitaniae, C. albicans and S. cerevisiae were so divergent that we constructed and characterized another C. lusitaniae f ox2 A null-mutant harboring a full deletion of the FOX2 open reading frame. The two fox2A mutants had the same growth phenotypes. These results suggested the possible existence of a Fox2p-independent FA catabolism pathway in C. lusitaniae.

Example 21: Growth of C. lusitaniae pxalA long-chain saturated FA as sole carbon source

Shani N. et al. (PNAS, 1995, 92:6012-6016) reported that the disruption of PXA1 encoding a long-chain FA peroxisomal import protein, in S. cerevisiae resulted in impaired growth on oleic acid and reduced ability to oxidize oleate.

In contrast, disruption of PXA1 in C. lusitaniae had no effect onto fatty acid assimilation. The pxalA mutant was able to grow on C18: l, C18:0 and C16:0 as sole carbon source (Figure 33). One possibility was that long- chain fatty acids could still penetrate peroxisomes using another transporter. However, analysis of the lipid content revealed that the pxalA mutant strongly accumulated triacylglycerol (TAG) when compared to the wild-type strain (the TAG content was increased by 300%). This suggested that the transport, and possibly the catabolism of fatty acids, was impaired in the pxalA mutant. Alternatively, the growth phenotype of pxalA raised the possibility that a functional catabolic pathway could take place in a subcellular compartment differing from peroxisomes.

Example 22: Functionality of long chain FA CoA catabolism in C. lusitaniae fox2A and pxalA mutants

The C. lusitaniae f ox2 A and pxalA mutants being able to assimilate long-chain FA, the consumption of 14 Ca- palmitoyl-CoA by crude protein extracts was measured. There was no significant difference between the specific activities observed for the icllA, fox2A, pxalA and wild-type strains grown on glucose medium (Figure 33). The fox2A, pxalA and wild-type strains were then cultivated in C18: l medium to induce FA β- oxidation. The specific activities after induction were increased 2 to 3 fold, but no significant difference could be detected between the mutant and the wild-type strains. Example 23: Functionality of long chain FA CoA catabolism in the peroxisomal fraction of the C. lusitaniae fox2A mutant

Mitochondrial and peroxisomal fractions were obtained from mutant and wild-type strains using a discontinuous sucrose gradient. The purity of each organelle fraction was estimated by assaying in parallel catalase and cytochrome c oxidase which were considered as enzymatic markers specific to the pexoxisomal and mitochondrial fraction, respectively (Figure 35). The peroxisomal fraction of the fox2A, pxalA and wild- type strains exhibited a high specific activity of catalase (ranging from 404 to 658 U/mg of protein) and a high ratio catalase/cytochrome c oxidase activities (from 918 to 1834). The mitochondrial fraction exhibited nearly a 20-fold lower ratio. The contamination of the mitochondrial fraction by peroxisomes did not exceed 20% (for example, for the wild-type strain, the ratio of the catalase activity of the mitochondrial fraction versus the catalase activity of the peroxisomal fraction was 0.21). The consumption of 14 Ca-palmitoyl-CoA was then assayed with the protein extracts obtained from each fraction (Figure 36). It was noteworthy that the consumptions of 14 Ca-palmitoyl-CoA by peroxisomal fractions of the fox2A, pxalA and wild-type strains were similar. This result supported the idea that the peroxisomes of C. lusitaniae harbored a Fox2p-independent FA- CoA catabolism pathway.

It was also observed that long-chain fatty acids can be catabolized in the mitochondrial fraction of the C. lusitaniae wild-type strain, but not in the mitochondrial fraction of the fox2A mutant.

Unexpectedly, a consumption of 14 Ca-palmitoyl-CoA was observed in the mitochondrial fractions of the pxalA and wild-type strains (Figure 36). The specific activities were similar to those measured in the peroxisomal fractions, even when assays were performed with up to 100-fold lower protein concentrations, thus ruling out the possibility that the whole 14 Ca-palmitoyl-CoA consumption observed in the mitochondrial fraction could be derived from a peroxisomal contamination. A FA-CoA catabolism was therefore functional in the mitochondrial extract of C. lusitaniae. In the mitochondrial fractions of the fox2A mutant, 14 Ca- palmitoyl-CoA catabolism was significantly decreased (p < 0.05) (Figure 36). These results strongly suggested that the mitochondrial catabolism of FA-CoA in C. lusitaniae was Fox2pdependent.

Example 24: Demonstration of co-localization of Fox2p in peroxisomes and mitochondria in C. lusitaniae

Western-blotting analysis was performed using polyclonal anti-Fox2p and anti-Icllp antibodies on crude extracts, peroxisomal or mitochondrial fractions of the fox2A mutant and of the wild-type strains. In C. albicans, it was shown that the two key enzymes of the glyoxylate cycle, Icllp and Mlslp, were localized to peroxisomes.

It was then confirmed that Icllp was present in the peroxisomal fractions of both the C. lusitaniae fox2A mutant and wild-type strains. A faint signal, not exceeding 10 to 20% of the amount of Icllp present in the peroxisomal fraction, was detected in the mitochondrial fraction (Figure 37, 19% for the wild-type strain and 10% for fox2A). This corresponded to the level of contamination of the mitochondrial fraction by peroxisomes that had been already estimated using catalase assays. On the other hand, and as expected, Fox2p was undetectable in the fox2A mutant.

Interestingly, Fox2p was detected in both peroxisomal and mitochondrial fractions of the wild type strain (Figure 37). The amount of Fox2p detected in the mitochondrial fraction was nearly 50 % of the amount detected in the peroxisomal fraction. Western-blot thus confirmed that Fox2p was localized in both peroxisomal and mitochondrial fractions.

Example 25: Demonstration of Fatty acyl-CoA used as respiratory substrate inducing mitochondrial 0 2 consumption in the wild-type strain, but not in the fox2A mutant

The mitochondrial respiratory chain oxidizes NADH and FADH 2 , and reduces 0 2 to water, through electron transfer processes. This results in a substantial release of energy, which generates a proton gradient across the mitochondrial inner membrane that is used to synthesize ATP. In FA β-oxidation, Fox2p uses NAD+ as cofactor, releasing NADH (Figure 31). The question of whether FACoA could support mitochondrial respiration in C. lusitaniae was addressed. Monitoring of the 0 2 consumption rates was performed by oximetry using nystatin-permeabilized spheroplasts from the fox2A, pxalA and wild-type strains (Figures 38 and 39). First, respiration rates were assayed using NADH as respiratory substrate, and valinomycin as uncoupling agent. These assays confirmed that mitochondria of the fox2A the pxalA and the wild-type strain were coupled efficiently (Figure 38A). Then, the oxygen uptake was measured using palmitoyl-CoA as respiratory substrate. The oxygen uptake increased with wild-type and pxalA spheroplasts, until the addition of myxothiazol, an inhibitor of the bcl complex of the mitochondrial respiratory chain, which reduced the 0 2 consumption by 33% (Figure 39A). In contrast, no oxygen uptake could be detected with spheroplasts of the fox2A mutant (Figure 39B). Similarly, the use of stearoyl-CoA induced 0 2 consumption in the wild-type and pxal Astrains, but not in the fox2A mutant.

To ascertain that 0 2 consumption was the consequence of the mitochondrial metabolic activity of the permeabilized spheroplasts, we used potassium cyanide as a second respiratory inhibitor. The cyanyl radical can bind and inhibit the heme centers of cytochrome c oxidase and catalase. As expected, the NADH-induced respiration of spheroplasts of the wildtype and mutant strains was completely inhibited by KCN. In contrat, KCN caused a marked increase of 0 2 consumption when palmitoyl-CoA was used as respiratory substrate. This paradoxical effect can be explained by the first step of β-oxidation taking place in peroxisomes, during which the convertion of palmitoyl-CoA into fran.s-2-hexadecenoyl-CoA by the acyl-CoA oxidase needs 0 2 to generate H 2 0 2 , and by the KCN mediated inhibition of catalase, which is normally required for the breaking- down of H 2 0 2 to H 2 0 and 0 2 , Figure 31). Thus, inhibition of the peroxisomal catalase by KCN could certainly result in an increaseof the apparent 0 2 consumption. Then, we verified that phenylsuccinate (a competitive inhibitor of the succinate shuttle between peroxisomes and mitochondria) and valinomycin (a mitochondrial inner-membrane uncoupling agent and also an H+ dependent transport inhibitor) had no significant effect on the oxygen uptake induced by palmitoyl-CoA on the wild-type and pxal A strains. This was a supplemental indication that the 02 consumption that we measured in our experiments was not due to the sole peroxisomal catabolism of FA-CoA. Finally, the lack of mitochondrial respiration of the fox2A mutant in the presence of FA-CoA confirmed that the mitochondrial FA-CoA catabolism pathway in C. lusitaniae was Fox2p dependent.

Example 26: Gamma-decalactone production

After growth on YNB + 2% ricinoleic acid using different inoculums and growth conditions (Figure 40), 1.5 ml samples were centrifuged (10,000 g, 5 min), and the supernatants (both aqueous and oil phases) were mixed. Fifty g of heptadecanoic acid methyl ester (used as internal standard) were added, and the mixture was extracted with diethyl ether, in 4-ml glass vials, by shaking for 90 s. The ether phase was analyzed by gas chromatography (Hewlett Packard 5890 series II (Hewlett-Packard, Palo Alto, CA, USA).

Statistical analysis

All the data obtained from the assay were analyzed using the two-tailed Student's f-test with a statistical significance at p < 0.05. All assays and experiments were carried out at least in triplicate.