Yeast strains with reduced fatty alcohol oxidase activity and method for the production of omega-hydroxy fatty acids and dicarboxylic acids

Abstract

The invention relates to various new yeast strains of the type Yarrowia lipolytica as well as relevant methods for the biocatalytic preparation of -hydroxy fatty acids or dicarboxylic acids with the aid of these strains, whereby the formation of -hydroxy fatty acids or dicarboxylic acids is advantageously increased.

Claims

1. A genetically engineered Yarrowia lipolytica cell, comprising: a. a disrupted fatty alcohol oxidase encoding FAO1 YALI0B14014g gene; and b. at least one disrupted acyl-CoA oxidase encoding gene selected from the group consisting of: POX1, POX2, POX3, POX4, POX5, and POX6, wherein the engineered Yarrowia lipolytica cell has reduced fatty alcohol oxidase activity relative to a wild type Yarrowia lipolytica cell.

2. The engineered Yarrowia lipolytica cell according to claim 1, wherein the fatty alcohol oxidase encoding FAO1 YALI0B14014g gene is partially deleted.

3. The engineered Yarrowia lipolytica cell according to claim 1, wherein the fatty alcohol oxidase encoding FAO1 YALI0B14014g gene is completely deleted.

4. The engineered Yarrowia lipolytica cell according to claim 1, wherein at least one acyl-CoA oxidase encoding gene selected from the group consisting of: POX1, POX2, POX3, POX4, POX5, and POX6 is partially deleted.

5. The engineered Yarrowia lipolytica cell according to claim 1, wherein at least one acyl-CoA oxidase encoding gene selected from the group consisting of: POX1, POX2, POX3, POX4, POX5, and POX6 is completely deleted.

6. The engineered Yarrowia lipolytica cell according to claim 1, having reduced acyl-CoA oxidase activity relative to a wild type Yarrowia lipolytica cell.

7. The engineered Yarrowia lipolytica cell according to claim 1, comprising: at least one disrupted fatty alcohol dehydrogenase encoding gene selected from the group consisting of: FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7.

8. The engineered Yarrowia lipolytica cell according to claim 1, wherein at least one fatty alcohol dehydrogenase encoding gene selected from the group consisting of: FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7 is partially deleted.

9. The engineered Yarrowia lipolytica cell according to claim 1, wherein at least one fatty alcohol dehydrogenase encoding gene selected from the group consisting of: FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7 is completely deleted.

10. The engineered Yarrowia lipolytica cell according to claim 1, further comprising: at least one disrupted fatty aldehyde dehydrogenase encoding gene selected from the group consisting of FALDH1, FALDH2, FALDH3 and FALDH4.

11. The engineered Yarrowia lipolytica cell according to claim 1, wherein at least one fatty aldehyde dehydrogenase encoding gene selected from the group consisting of FALDH1, FALDH2, FALDH3 and FALDH4 is partially or completely deleted.

12. The engineered Yarrowia lipolytica cell according to claim 1, having reduced fatty alcohol dehydrogenase activity relative to a wild type Yarrowia lipolytica cell.

13. The engineered Yarrowia lipolytica cell according to claim 1, comprising: at least one of the following genes disrupted selected from the group consisting of: phosphatidic acid dephosphohydrolase (PAH1), phospholipid diacylglycerol acyltransferase (LRO1), diacylglycerol acyltransferase (DGA1), and glycerol-3-phosphate acyltransferase (SCT1).

14. The engineered Yarrowia lipolytica cell of claim 13, further comprising: a disrupted acyl-CoA oxidase encoding gene selected from the group consisting of: POX1, POX2, POX3, POX4, POX5, and POX6.

15. The engineered Yarrowia lipolytica cell of claim 13, further comprising: at least one disrupted fatty alcohol dehydrogenase encoding gene selected from the group consisting of: FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7.

16. The engineered Yarrowia lipolytica cell of claim 13, wherein the disruption of the genes reduces the size of the lipid bodies.

17. The engineered Yarrowia lipolytica cell according to claim 1, comprising: a disrupted peroxisome biogenesis factor 10 protein (PEX10).

18. The engineered Yarrowia lipolytica cell of claim 17, further comprising: a disrupted acyl-CoA oxidase encoding gene selected from the group consisting of: POX1, POX2, POX3, POX4, POX5, and POX6.

19. The engineered Yarrowia lipolytica cell of claim 17, further comprising: at least one disrupted fatty alcohol dehydrogenase encoding gene selected from the group consisting of: FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7.

20. A genetically engineered Yarrowia lipolytica cell, comprising: a disrupted fatty alcohol oxidase encoding FAO1 YALI0B14014g gene.

21. The engineered Yarrowia lipolytica cell of claim 20, further comprising a disrupted peroxisome biogenesis factor 10 protein (PEX10).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the figures:

(2) FIG. 1: shows the metabolic paths for the breakdown of hydrophobic substrates by Y. lipolytica.

(3) FIG. 2: shows the accumulation of dicarboxylic acids and -hydroxy fatty acids in Y. lipolytica H222P, H222PA and H222PF using various hydrophobic substrates.

(4) FIG. 3: shows the bioconversion of pentadecanoic acid to -hydroxypentadecanoic acid by Y. lipolytica H222PF.

(5) FIG. 4: shows the formation of lipid bodies in Y. lipolytica H222PF, H222PFD, H222PFAH and H222PFS.

(6) FIG. 5 A-C: shows the accumulation of dodecanoic diacid and -hydroxydodecanoic acid in the cultivation of various strains of the yeast Y. lipolytica using dodecane as substrate. FIG. 5AY. lipolytica H222PAF formed only small quantities of dicarboxylic acids and is thus the preferred initial strain for the construction of production strains for -hydroxy fatty acids. FIG. 5BThe deletion of DGA1 in the strain Y. lipolytica H222P (resulting strain: Y. lipolytica H222PD) resulted in an increased production of dicarboxylic acids whereas the deletions of PAH1 and SCT1 (resulting strains: Y. lipolytica H222PH and H222PAS) did not significantly increase the production of dicarboxylic acids. FIG. 5CThe overexpression of CPR1 in the strain Y. lipolytica H222PAF (resulting strain: Y. lipolytica H222PAFoC) resulted in an increased production of dicarboxylic acids but not of -hydroxy fatty acids.

(7) FIG. 6 A-B: shows the (fatty) alcohol dehydrogenase and oxidase enzyme activity in the cultivation of various strains of the yeast Y. lipolytica using dodecane as substrate. FIG. 6ASurprisingly no clear difference of the (fatty) alcohol dehydrogenase was determined between the strains. FIG. 6BAs was expected, the (fatty) alcohol oxidase activity could only be detected in the strains Y. lipolytica H222P and H222PA and not in the FAO1-deletion strains Y. lipolytica H222PF and H222PAF.

(8) FIG. 7: shows the bioconversion of dodecane to -hydroxydodecanoic acid dodecanoic diacid by Y. lipolytica H222PF.

EXPLANATIONS ON THE GENETIC MODIFICATIONS

(9) P Deletion of the acyl-CoA oxidase genes (POX1-6) F Deletion of the (fatty) alcohol oxidase gene (FAO1) A Deletion of the (fatty) alcohol dehydrogenase genes (FADH, ADH1-7) D Deletion of the diacylglycerol acyltransferase gene (DGA1) H Deletion of the phosphatidic acid dephosphohydrolase gene (PAH1) S Deletion of the glycerol-3-phosphate acyltransferase gene (SCT1) oC Overexpression of the NADPH-cytochrome P450-reductase gene CPR1 oF Overexpression of the (fatty) alcohol oxidase gene FAO1
Explanations on the Sequence Protocol SEQ ID NO. 1: DNA sequence of the plasmid pJET1.2/blunt (Fermentas). SEQ ID NO. 2: DNA sequence of the plasmid pUCBM21 (Boehringer Ingelheim). SEQ ID NO. 3: DNA sequence of the artificial plasmid pUC-Lys2-DK2. SEQ ID NO. 5: DNA sequence of the artificial plasmid pINTB_HMG1.

(10) FIG. 1 shows that n-alkanes are assimilated into the cell and are then converted to the corresponding fatty acid with the same chain length in the course of primary alkane oxidation. The fatty acids are then broken down in the course of the -oxidation to acetyl-CoA (or propionyl-CoA). The diterminal oxidation of fatty acids (-oxidation) can take place in parallel. The names of the chemical substances formed are also given as are the designations of the enzymes involved and the corresponding genes. ALK1: YALI0E25982g, ALK2: YALI0F01320g, ALK3: YALI0A20130g, ALK4: YALI0B13816g, ALK5: YALI0B13838g, ALK6: YALI0B01848g, ALK7: YALI0A15488g, ALK8: YALI0C12122g, ALK9: YALI0B06248g, ALK10: YALI0B20702g, ALK11: YALI0C10054g, ALK12: YALI0A20130g, CPR1: YALI0D04422g, FADH: YALI0F09603g, ADH1: YALI0D25630g, ADH2: YALI0E17787g, ADH3: YALI0A16379g, ADH4: YALI0E15818g, ADH5: YALI0D02167g, ADH6: YALI0A15147g, ADH7: YALI0E07766g, ADH8: YALI0C12595g, FAO1: YALI0B14014g, FALDH1: YALI0A17875g, FALDH2: YALI0E15400g, FALDH3: YALI0B01298g, FALDH4: YALI0F23793g, FAA1: YALI0D17864g, POX1: YALI0E32835g, POX2: YALI0F10857g, POX3: YALI0D24750g, POX4: YALI0E27654g, POX5: YALI0C23859g, POX6: YALI0E06567g, MFE2: YALI0E15378g, POT1: YALI0E18568g.

(11) In FIG. 2 Y. lipolytica H222P, H222PA and H222PF were cultivated in minimal medium with glycerol and various n-alkanes and fatty acids (DD: dodecane; DDS: dodecanoic acid; PD: pentadecane; PDS: pentadecanoic acid; HD: hexadecane; HDS: hexadecanoic acid) in a shaking flask (3% (v/v) glycerol+0.5% (v/v) glycerol after 48 h, 1% (v/v) n-alkane or 1% (v/v) fatty acid, 20 g 1.sup.1 CaCO.sub.3, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride). The quantities of the dicarboxylic acid and -hydroxy fatty acids formed (DDDS: dodecanoic diacid; w-HDDS: -hydroxydodecanoic acid; PDDS: pentadecanoic acid; -HPDS: -hydroxypentadecanoic acid; HDDS: hexadecanoic acid; -HHDS: -hydroxyhexadecanoic acid) were determined after cultivation for 96 h by means of gas chromatography.

(12) In FIG. 3 Y. lipolytica H222PF was cultivated in minimal medium with glucose and pentadecanoic acid (PDS) as carbon sources in a shaking flask (3% (v/v) glucose, 1% (w/v) PDS, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride). The glucose concentration was held between 2 and 3% (w/v) by regular after-feeding (arrows). After 5 days 1% (w/v) pentadecanoic acid is after-fed. The pentadecanoic acid derivates formed, -hydroxypentadecanoic acid (w HPDS) and pentadecanoic acid (PDDS), were quantified by means of gas chromatography. The cells were examined under a microscope towards the end of the cultivation, where the lipid bodies were stained with Nile red.

(13) FIG. 4 shows the cultivation of Y. lipolytica H222PF, H222PFD, H222PFH and H222PFS in minimal medium with glucose and pentadecanoic acid (PDS) as carbon sources (3% (v/v) glucose, 1% (w/v) PDS, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride). The glucose concentration was held between 2 and 3% (w/v) by regular after-feeding. After 3 days 1% (w/v) pentadecanoic acid was after-fed. The cells were examined under a microscope after 5 days, where the lipid bodies were stained with Nile red (top: optical micrograph; bottom: fluorescence micrograph).

(14) In FIG. 5 Y. lipolytica H222P, H222PA, H222PF, H222PAF, H222PoF, H222PoC, H222PD, H222PH, H222PS, H222PAFoC, H222PAFD, H222PAFH and H222PAFS was cultivated in a fermenter. The cultivation medium here was minimal medium with glucose (5% (w/v) glucose, 1 g KH.sub.2PO.sub.4, 0.16 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride). Cultivation was carried out in a fermenter at 28 C. The oxygen saturation was set at 55%. The cells were inoculated with an OD.sub.600 of 1 and incubated for 24 h at pH 5.5 (automated titration of HCl and NaOH). Then 15 g 1.sup.1 DD was added and the pH was set to 8.0. Glucose was added every 24 h to adjust a final concentration of 5% (w/v). The quantities of dodecanoic acid (DDDS) and -hydroxydodecanoic acid formed (w-HDDS) were determined after cultivation for 2 d, 3 d and 4 d by means of gas chromatography.

(15) FIG. 6 shows the enzyme activities of the (fatty) alcohol dehydrogenases and oxidase in cell lysates of the strains Yarrowia lipolytica H222P, H222PA, H222PF, H222PAF and H222PoF. The cells were cultivated as described in Example 8 and harvested and macerated after three days.

(16) In FIG. 7 Y. lipolytica H222PF was cultivated in minimal medium with glucose and dodecane (DD) as carbon sources in a fermenter (5% (w/v) glucose, 2 g 1.sup.1 KH.sub.2PO.sub.4, 0.32 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 6 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 1.4 g 1.sup.1 MgSO.sub.47 H.sub.2O, 1 g 1.sup.1 NaCl, 0.8 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 1 mg 1.sup.1 H.sub.3BO.sub.3, 0.08 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.2 mg 1.sup.1 KI, 0.8 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.4 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.8 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 12 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.6 mg 1.sup.1 thiamine hydrochloride). The glucose was adjusted to 5-10% (w/v) every 24 h by regular after-feeding (arrows). After 2 d the pH was set to 8.0 and 15 g 1.sup.1 of dodecane was added after 2 d and 3 d. Glucose was added every 24 h in order to adjust a final concentration of 5-10% (w/v).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1

Prevention of the Breakdown of Fatty Acids by Blocking of -Oxidation

(17) For construction of the deletion cassettes for the POX genes, the respective promoter and terminator region was amplified by PCR, where the primers pXXX_fw/pXXX_rv and XXXt_fw/XXXt_rv were used (XXX stands for the gene to be deleted). Genomic DNA of Y. lipolytica H222 was used as template here.

(18) By using overhang primers, an I-SceI-restriction interface was inserted at the end of the promoter and at the beginning of the terminator region. Promoter and terminator fragment were fused together by overlap-PCR with the primers pXXX_fw and XXXt_rv. The overlap fragment was then ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1). The resulting plasmid was cut with I-SceI and the loxP-URA3-loxP cassette, which was obtained from the pJMP113 plasmid (Fickers P, Le Dall M T, Gaillardin C, Thonart P & Nicaud J M (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55: 727-737) using the restriction enzyme I-SceI was inserted.

(19) The respective deletion cassette was obtained by PCR or restriction and transformed in Y. lipolytica H222-S4, which can be produced from Y. lipolytica H222 (Mauersberger, S., H. J. Wang, et al. (2001), J Bacteriol 183(17): 5102-5109), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York).

(20) Before a renewed transformation, the marker was recovered with the aid of the Cre-lox recombination systems (Fickers P, Le Dall M T, Gaillardin C, Thonart P & Nicaud J M (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55: 727-737).

(21) The successful deletion of a gene was confirmed by PCR where the primers pXXX_fw and XXXt_rv as well as pXXX_fw and XXXt_rv2. XXXt_rv2 binds in the region of the gene to be deleted but outside the deletion cassette.

(22) The strain which carried the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 was called H222P and forms the basis for the construction of strains which can be used for the biotechnological production of -hydroxy fatty acids and dicarboxylic acids.

(23) TABLE-US-00001 TABLE1 ontherecoveryanddetectionofPOXdeletioncassettes Name SEQID Sequence(5.fwdarw.3) RS pPOX1_fw SEQIDNO7 TCCAGAAGCGCTACAAAGAG pPOX1_rv SEQIDNO8 attaccctattatccctaTGAAGGTTGCAGTCGTAGTC I-SceI POX1t_fw SEQIDNO9 tagggataacagggtaatTGCGATCTCGATGAGTGATG I-SceI POX1t_rv SEQIDNO10 GCCCAGAAGATTGGAATGAC pPOX2_fw SEQIDNO11 atataccgcggGATTCCGCCAAGTGAGACTG Cfr42I pPOX2_rv SEQIDNO12 attaccctgttatccctaCGTCGAGGAAGTAGGICATC I-Scel POX2t_fw SEQIDNO13 tagggataacagggtaatGCGAGCTTGATGAGGAATAG I-SceI POX2t_rv SEQIDNO14 atataccgcggCCTGACGCCAATTTGAAGAG Cfr42I pPOX3_fw SEQIDNO15 atataccgcggCTGGGCTGTTCGGTCGATAG Cfr42I pPOX3_rv SEQIDNO16 tagggataacagggtaatAGGACGCACAACGCCATCAC I-SceI POX3t_fw SEQIDNO17 attaccctgttatccctaCGCTCCCATTGGAAACTA I-SceI POX3t_rv SEQIDNO18 atataccgcggTCTCTTCGCTGTGGTCTAGG Cfr42I pPOX4_fw SEQIDNO19 atataccgcggTCCACCGTTCTCCTTCATAC Cfr42I pPOX4_rv SEQIDNO20 tagggataacagggtaatATGTCTCTAGGGTCGAAGTC I-SceI POX4t_fw SEQIDNO21 attaccctattatccctaTGGCAAGCCTCACTACTACG I-SceI POX4t_rv SEQIDNO22 atataccgcggTGCGGCGGAACTACTGTATC Cfr42I pPOX5_fw SEQIDNO23 atataccgcggGGGATTCTCCGGGTTATTTG Cfr42I pPOX5_rv SEQIDNO24 tagggataacagggtaatACGTCTCGGACCTTGAATTG I-SceI POX5t_fw SEQIDNO25 attaccctgttatccctaCCTTCAACCTGTCCGACTTC I-SceI POX5t_rv SEQIDNO26 atataccgcggGAAGCGGTCCTCGTTGTATG Cfr42I pPOX6_fw SEQIDNO27 GTGTAGCAACTCGGATACAG pPOX6_rv SEQIDNO28 tagggataacagggtaatGGTCCATAAGCAGAGTGTTC I-SceI POX6t_fw SEQIDNO29 attaccctgttatccctaACCCTCGACCTCCTTATTAC I-SceI POX6t_rv SEQIDNO30 CTCTTCTTGACTGGCATAGC

Example 2

Prevention of the Oxidation of -Hydroxy Fatty Acids to Fatty Acid Aldehyde or to Dicarboxylic Acid in the Course of -Oxidation

(24) For construction of the deletion cassettes for the (fatty) alcohol dehydrogenase and oxidase genes, the respective promoter and terminator region was amplified by PCR, where the primers pXXX_fw/pXXX_rv and XXXt_fw/XXXt_rv were used (XXX stands for the gene to be deleted). Genomic DNA of Y. lipolytica H222 was used as template here.

(25) For the construction of the deletion cassettes for the genes ADH1-ADH6 and FAO1 a BamHI restriction interface was inserted by using overhang primers at the end of the promoter and at the beginning of the terminator region. In addition, an HindIII-restriction interface was attached at the beginning of the promoter region and an NdeI-restriction interface was attached at the end of the terminator region (ADH1: NotI, FAO1: EcoRI). The fragments were ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1) or pUCBM21 (Boehringer Ingelheim; SEQ ID NO 2). The plasmids thus obtained were linearized with BamHI and the URA blaster (TcR-URA3-TcR-Kassette), which was obtained from the plasmid pUC-Lys2-DK2 (SEQ ID NO 3) by restriction with BamHI and BglII, was inserted.

(26) For the construction of the ADH7 deletion vector, the complete gene (including promoter and terminator region) was amplified by means of PCR, where the primers pADH7_fw and ADH7t rv were used. The fragment obtained was ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1), the open reading frame of the ADH7 gene was removed by restriction with SanDI and NsiI and the URA blaster, which was obtained from the plasmid pUC-Lys2-DK2 by restriction with SanDI and NsiI, was inserted.

(27) The respective deletion cassette was obtained by PCR or restriction and transformed in Y. lipolytica H222-S4, which can be produced from Y. lipolytica H222 (Mauersberger, S., H. J. Wang, et al. (2001), J Bacteriol 183(17): 5102-5109), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York).

(28) Before a renewed transformation, the marker was recovered by FOA selection (Boeke J D, La-Croute F & Fink G R (1984) Mol Gen Genet 197: 345-346).

(29) The successful deletion of a gene was confirmed by PCR, where the primers pXXX_fw and XXXt_rv as well as pXXX_fw and XXXt_rv2 were used. XXXt_rv2 binds in the region of the gene to be deleted but outside the deletion cassette.

(30) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FADH, AHD1, AHD2, AHD3, AHD4, AHD5, AHD6 and AHD7 was called H222PA.

(31) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 was called H222PF.

(32) TABLE-US-00002 TABLE2 ontherecoveryanddetectionof(F)ADH-andFAOdeletioncassettes Name SEQID Sequence(5.fwdarw.3) RS pFADH_fw SEQIDNO31 atataaagctTGCGGCTCGGCACGTGATCTG HindIII pFADH_rv SEQIDNO32 atataggatccATCGTGCGTACGTCGCTAGTG BamHI FADHt_fw SEQIDNO33 atataggatccCGACCGGCACGATCAATTGG BamHI FADHt_rv SEQIDNO34 atatacatatgGGTGCATCTCAGCCCGACCTC NdeI FADHt_rv2 SEQIDNO35 TCCCGAAACACAGAACTTCC pADH1_fw SEQIDNO36 atataaagctTGGTGGACGTTCCGGCAGACAG HindIII pADH1_rv SEQIDNO37 atataggatccCTCCCAGGCATCTCCACACTC BamHI ADH1t_fw SEQIDNO38 atataggatccCACTTACAGGCTTAGCAAGG BamHI ADH1t_rv SEQIDNO39 atatagcggccgcGGAAATCACGCTTGATTCG NotI ADH1t_rv2 SEQIDNO40 TAGGCGCTGGTACAGAAGAG pADH2_fw SEQIDNO41 atataaagctTGAGTACAGTAGGTGGTACTC HindIII pADH2_rv SEQIDNO42 atataggatccAGTGGTGGTGGTGGTGGTAG BamHI ADH2t_fw SEQIDNO43 atataggatccTTTACGTGCAACAGGAGGAG BamHI ADH2t_rv SEQIDNO44 atatacatatgGCCTGTCTTGAGTTCTTTGG NdeI ADH2t_rv2 SEQIDNO45 AGGGTCGTAGATAACGAGTC pADH3_fw SEQIDNO46 atataaagctTCACGTGGCTGCTGGGCCAACC HindIII pADH3_rv SEQIDNO47 atataggatccCGCACGGTATCGGAGCATCG BamHI ADH3t_fw SEQIDNO48 atataggatccCGCGGCTATTGACGCTGAGG BamHI ADH3t_rv SEQIDNO49 atatacatatgCCCGTCAGCTCCATCGACGAGTG NdeI ADH3t_rv2 SEQIDNO50 AGGTGTACTGTAGCCACCCTGAC pADH4_fw SEQIDNO51 atataaagctTCCGGCCAGCCGCTGGCAACG HindIII pADH4_rv SEQIDNO52 atataggatccACACGACAGCTGCACCTGAC BamHI ADH4t_fw SEQIDNO53 atataggatccCAGCCATGAGCCAGGCATTG BamHI ADH4t_rv SEQIDNO54 atatacatatgGGCGCCAGCCACATTTGCCCTC NdeI ADH4t_rv2 SEQIDNO55 AGCGATACAGCAGTTGACTC pADH5_fw SEQIDNO56 TCAGCCGTCTACTTGTAGAG pADH5_rv SEQIDNO57 atataggatccGTGGCTCGGATACTCCTGAC BamHI ADH5t_fw SEQIDNO58 atataggatccAGCCGGAGGTCAGATCAAGC BamHI ADH5t_rv SEQIDNO59 atatacatatgGCGCAATAGTTCGCCGGCCTG NdeI ADH5t_rv2 SEQIDNO60 CTCGTGTTGTGCCTTTCTTG pADH6_fw SEQIDNO61 atataaagctTGCGCGACAACCCATAGCGATGGC HindIII pADH6_rv SEQIDNO62 atataggatccGATAAGAGGGCGCTCTGACC BamHI ADH6t_fw SEQIDNO63 atataggatccGGCGTGACATCGAGTTTGG BamHI ADH6t_rv SEQIDNO64 atatacatatgCTACGTCTCGCCGCAGAGGG NdeI ADH6t_rv2 SEQIDNO65 AGCGAGAGGTTATACGGAAG pADH7_fw SEQIDNO66 CTCCTACAGCCTCTCAAGAC ADH7t_rv SEQIDNO67 GTCTACAAGACAGCCCAGAG pADH7_fw2 SEQIDNO68 CCGCTTGAGAAGAGCAATAC pFAO1_fw SEQIDNO69 atataaagctTCGCCACCTGTCCACGTCTCG HindIII pFAO1_rv SEQIDNO70 atataggatccGCGAAGCGACGTGTGGTGAG BamHI FAO1t_fw SEQIDNO71 atataggatccGCTGAGCACGCGAGTACACC BamHI FAO1t_rv SEQIDNO72 atatagaattcGATCTGTCGTACAACTAAGG EcoRI FAO1t_rv2 SEQIDNO73 CAGAAGTTACGACGCCAAGG

Example 3

Comparative Cultivation of Various Production Strains in a Shaking Flask

(33) In order to check whether the initial strains produced Yarrowia lipolytica H222P, H222PA and H222PF are suitable for the production of larger quantities of -hydroxy fatty acids and dicarboxylic acids, these were cultivated in minimal medium with glycerol and various n-alkanes (dodecane, pentadecane, hexadecane) and fatty acids (dodecanoic acid, pentadecanoic acid, hexadecanoic acid) (3% (v/v) glycerol+0.5% (v/v) glycerol after 48 h, 1% (v/v) n-alkane or 1% (v/v) fatty acid, 20 g 1.sup.1 CaCO.sub.3, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride). The quantities of -hydroxy fatty acids (-hydroxydodecanoic acid, -hydroxypentadecanoic acid, -hydroxyhexadecanoic acid) and dicarboxylic acids formed (dodecanoic diacid, pentadecanoic diacid, hexadecanoic diacid) were determined by means of gas chromatography 96 h after cultivation (FIG. 2A-F).

(34) Y. lipolytica H222P here formed relatively large quantities of dicarboxylic acids and can thus be used as initial strain for the construction of a corresponding production strain for dicarboxylic acids.

(35) Both Y. lipolytica H222PA and also H222PF formed increased quantities of -hydroxy fatty acids and can thus be used as initial strains for the construction of corresponding production strains for -hydroxy fatty acids. Y. lipolytica H222PF is to be preferred to H222PA here.

(36) Both n-alkanes and also fatty acids can be converted from all strains to -hydroxy fatty acid or dicarboxylic acid having the same chain length.

Example 4

Use of Different Cultivation Conditions and Media

(37) Different strains (e.g. Yarrowia lipolytica H222P and H222PF) were cultivated in different media under various conditions, where it was found that the media and conditions used are variously well suited.

(38) The yeasts were cultivated comparatively in minimal medium with glucose (3% (w/v) glucose, 1% (w/v) pentadecanoic acid, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride) and glycerol (3% (v/v) glycerol, 1% (w/v) pentadecanoic acid, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1 K2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride) as carbon source, where after 48 h 1% (w/v) glucose, or 1% (v/v) glycerol were after-fed. After 96 h, all the strains studied (e.g. Y. lipolytica H222P, H222PA and H222PF) formed larger quantities of -hydroxy fatty acid or pentadecanoic diacid for growth in minimal medium with glucose than for growth in minimal medium with glycerol.

(39) The yeasts were then cultivated in full medium with glucose and pentadecanoic acid (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose and 1% (w/v) pentadecanoic acid in 1% Tween 80), where 1% (w/v) glucose was after-fed when this was used up. After 4 days, 1% (w/v) of pentadecanoic acid in 1% Tween 80 was after-fed. Under these conditions, almost no -hydroxy pentadecanoic acid or pentadecanoic diacid was formed (each<0.1 g 1.sup.1).

(40) The yeasts were cultivated in minimal medium with glucose and pentadecanoic acid (3% (v/v) glucose, 1% (w/v) pentadecanoic acid in 1 Tween 80, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride), where 1% (w/v) glucose was after-fed when this was used up. After 4 days, 1% (w/v) pentadecanoic acid in 1% Tween 80 was after-fed. Under these cultivation conditions, the strain Y. lipolytica H222PF after 10 days formed around 0.7 g 1.sup.1-hydroxy pentadecanoic acid and 2.4 g 1.sup.1 pentadecanoic diacid.

(41) The yeasts were cultivated in minimal medium with glucose and pentadecanoic acid (3% (v/v) glucose, 1% (w/v) pentadecanoic acid in 1 Tween 80, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride), where the glucose was after-fed so that its concentration in the medium was between 1 and 3% (w/v). After 5 days 1% (w/v) pentadecanoic acid in 1% Tween 80 was after-fed. Under these cultivation conditions the strain Y. lipolytica H222PF after 7 days formed around 3.9 g 1.sup.1 -hydroxy pentadecanoic acid and 1.3 g 1.sup.1 pentadecanoic diacid.

(42) To sum up, it should be noted that minimal medium is better suited for the cultivation than full medium. Furthermore, glucose is more suitable than glycerol as carbon source for the energy preparation. The glucose should be after-fed in the course of the cultivation so that the glucose is not used up (i.e. its concentration does not drop to 0 g 1.sup.1).

Example 5

Reduction in the Formation of Lipid Bodies

(43) For construction of the deletion cassettes for the genes DGA1, PAH1 and SCT1 the respective promoter and terminator region was amplified by PCR where the primers pXXX_fw/pXXX_rv and XXXt_fw/XXXt_rv were used (XXX stands for the gene to be deleted). Genomic DNA of Y. lipolytica H222 was used as template here.

(44) For the construction of deletion cassettes for the genes DGA1, PAH1 and SCT1 a BamHI-restriction interface was inserted by using overhang primers at the end of the promoter region and at the beginning of the terminator region. In addition a HindIII-restriction interface was attached at the beginning of the promoter region and an EcoRI-restriction interface was attached at the end of the terminator region. The fragments were ligated into the vector pUCBM21 (Boehringer Ingelheim; SEQ ID NO 2). The plasmids thus obtained were linearized with BamHI and the URA blaster (TcR-URA3-TcR-cassette), which was obtained from the plasmid pUC-Lys2-DK2 (SEQ ID NO 3) by restriction with BamHI and BglII was inserted. The respective deletion cassette was obtained by PCR or restriction and transformed in Y. lipolytica H222-S4, which can be produced from Y. lipolytica H222 (Mauersberger, S., H. J. Wang, et al. (2001), J Bacteriol 183(17): 5102-5109), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York).

(45) Before a renewed transformation, the marker was recovered by FOA selection (Boeke J D, La-Croute F & Fink G R (1984) Mol Gen Genet 197: 345-346).

(46) The successful deletion of a gene was confirmed by PCR, where the primers pXXX_fw and XXXt_rv as well as pXXX_fw and XXXt_rv2 were used. XXXt_rv2 binds in the region of the gene to be deleted but outside the deletion cassette.

(47) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also DGA1 was called H222PD.

(48) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also PAH1 was called H222PH.

(49) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also SCT1 was called H222PS.

(50) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and DGA1 was called H222PFD.

(51) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and PAH1 was called H222PFH.

(52) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and SCT1 was called H222PFS.

(53) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and DGA1 was called H222PAFD.

(54) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and PAH1 was called H222PAFH.

(55) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and SCT1 was called H222PAFS.

(56) TABLE-US-00003 TABLE3 PrimersforrecoveryanddetectionofDGA1-,PAH1-andSCT1- deletioncassettes Name SEQID Sequence(5.fwdarw.3) RS pDGA1_fw SEQIDNO74 atataaagcttCCGTAA- HindIII pDGA1_rv SEQIDNO75 atataggatccAGGTCGAT- BamHI DGA1t_fw SEQIDNO76 atataggatccGGTTAGGCAAA- BamHI DGA1t_rv SEQIDNO77 atatagaattcGCCTGGAGCGAG- EcoRI DGA1t_rv2 SEQIDNO78 TCGCAAGGGCCATAGAGGTG pPAH1_fw SEQIDNO79 atataaagcttCCTT- HindIII pPAH1_rv SEQIDNO80 atataggatccGGTGTTACGCCACCAC- BamHI PAH1t_fw SEQIDNO81 atataggatccGGGACCTGCGATAC- BamHI PAH1t_rv SEQIDNO82 atatagaattcGCTTGCGCAGCCGGTG- EcoRI PAH1t_rv2 SEQIDNO83 GGCGTTGTGGAGCTATCACC pSCT1_fw SEQIDNO84 atataaagcttGCGTGCGGTGCGTGCG- HindIII pSCT1_rv SEQIDNO85 atataggatccCAGCACCAC- BamHI SCT1t_fw SEQIDNO86 atataggatccGTGCGCTTACATGTG- BamHI SCT1t_rv SEQIDNO87 atatagaattcCAGGCAGCTTCTT- EcoRI SCT1t_rv2 SEQIDNO88 GAGATAGGAGGTTCCCATAC

(57) The strains Y. lipolytica H222PFD, H222PFH and H222PFS as well as the initial strain Y. lipolytica H222PF were cultivated in minimal medium with glucose and pentadecanoic acid (3% (v/v) glucose, 1% (w/v) pentadecanoic acid, 17.3 g 1.sup.1 KH.sub.2PO.sub.4, 1.35 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride). Every 24 h the glucose concentration was adjusted to 3% (w/v) and after 3 days 1% (w/v) of pentadecanoic acid was after-fed. The quantities of -hydroxy fatty acid and pentadecanoic acid were determined after cultivation for 8 days by means of gas chromatography. The cells were examined under the microscope, where the lipid bodies were stained with Nile red and could thus be detected by fluorescence microscopy (FIG. 4).

(58) Here it can be identified that the size of the lipid bodies was significantly reduced due to deletions of PAH1 and SCT1 under the said cultivation conditions. As already shown, this could not be determined for the already described deletion of DGA1 (Thevenieau F (2006) Institut National Agronomique Paris-Grignon, PhD thesis; Athenstaedt K (2011) Biochim Biophys Acta 1811: 587-596).

(59) The availability of strains with reduced lipid bodies forms the basis for the development of strains for the efficient production of -hydroxy fatty acids and dicarboxylic acids since the esterification of the supplied substrates with glycerol and its incorporation into the lipid bodies reduces the conversion rate.

Example 6

Increased Expression of FAO1 in Yarrowia lipolytica H222P

(60) For construction of a vector for overexpression of FAO1 a part of the constitutive promoter of the translation elongation factor 1 alpha-gene (TEF1: YALI0C09141g) was obtained by means of PCR, where the primers pTef_SpeI_fw3 and pTef_FAOo_ol_rv were used. The plasmid pINTB_HMG1 (SEQ ID NO 5) was used as template here.

(61) Furthermore, the FAO1-gene (YALI0B14014g) was amplified by means of PCR using the primers pTef_FAOo_ol_fw and FAO1o_SphI_rv (template: genomic DNA of Y. lipolytica H222).

(62) By means of the primers pTef_FAOo_ol_rv and pTef_FAOo_ol_fw an overhang region was attached, with the aid of which both PCR fragments were linked by means of overlap PCR using the primers pTef_SpeI_fw3 and FAO1o_SphI_rv. The overlap fragment was then ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1), cut out from this using the restriction enzymes SpeI and SphI and ultimately ligated into the backbone of the plasmid pINTB_HMG1 (SEQ ID NO 5) cut with SpeI and SphI, where the plasmid pINTB-FAO1 was formed.

(63) Furthermore, an integration platform was obtained by means of PCT using the primers INT_AscI_fw KpnI and INT_AscI_rv_KpnI, cut with KpnI and ligated into the backbone of the vector pINTB-FAO1 obtained by restriction digestion with Kpnl, where the plasmid pINTC-FAO1 was formed.

(64) The plasmid pINTC-FAO1 was ultimately linearized with AscI and transformed into the desired Y. lipolytica recipient strain.

(65) Successful integration of the FAO1 gene was confirmed by PCR, where the primers INT_AscI_fw_out and INT_AscI_rv_out were used which bind in the genomic DNA outside the integrated construct.

(66) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also the additional copy of FAO1 under control of the TEF1-promoter was called H222PoF and was used as initial strain for construction of further strains for the production of dicarboxylic acids.

(67) TABLE-US-00004 TABLE4 PrimersforoverexpressionofFAO/ Name SEQID Sequence(5.fwdarw.3) RS pTef_SpeI_fw3 SEQIDNO89 CTACGCTTGTTCAGACTTTG pTef_FAOol_rv SEQIDNO90 gtgtgcttgtcgtcagacatTTTGAA TGATTCTTATACTCAGAAGG pTef_FAOol_fw SEQIDNO91 ccttctgagtataagaatcattcaaa ATGTCTGACGACAAGCACAC FAO1o_SphI_rv SEQIDNO92 gcatgcTTAGATTCGAGGTCGGAGAT SphI INT_AscI_fw_KpnI SEQIDNO93 ggtacCACGCACGGATAGTTTATCCA KpnI INT_AscI_rv_KpnI SEQIDNO94 ggtacCCAAAGTCAACTAATGTCAAG KpnI TAAAG INT_AscI_fw_out SEQIDNO95 CCTCCAACGTGACTTTC INT_AscI_rv_out SEQIDNO96 AGAGACCTCCCACAAAG

Example 7

Increased Expression of CPR1 in Yarrowia lipolytica H222P and H222PAF

(68) For construction of a vector for overexpression of FAO1 a part of the constitutive promoter of the translation elongation factor 1 alpha-gene (TEF1: YALI0C09141g) was obtained by means of PCR, where the primers pTef_SpeI_fw3 and pTEF_CPR1_ol_fw were used. The plasmid pINTB_HMG1 (SEQ ID NO 5) was used as template here.

(69) Furthermore, the CPR1-gene (YALI0D04422g) was amplified by means of PCR using the primers pTEF_CPR1_ol_fw and CPR1_SphI_rv (template: genomic DNA of Y. lipolytica H222).

(70) By means of the primers pTEF_CPR1_ol_rv and pTEF_CPR1_ol_fw an overhang region was attached, with the aid of which both PCR fragments were linked by means of overlap PCR using the primers pTef_SpeI_fw3 and CPR1_SphI_rv. The overlap fragment was then ligated into the vector pJET1.2/blunt (Fermentas; SEQ ID NO 1), cut out from this using the restriction enzymes SpeI and SphI and ultimately ligated into the backbone of the plasmid pINTB_HMG1 (SEQ ID NO 5) cut with SpeI and SphI, where the plasmid pINTB-CPR1 was formed.

(71) The resulting plasmid was ultimately linearized with NotI and transformed into the desired Y. lipolytica recipient strain.

(72) The successful integration of the CPR1-gene was confirmed by PCR, where the primers INTB_out_fw and INTB_out_rv were used, which bind in the genomic DNA outside the integrated construct.

(73) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also the additional copy of CPR1 under control of the TEF1-promoter was called H222PoC and was used as initial strain for construction of further strains for the production of dicarboxylic acids.

(74) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5, PPX6, FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and also the additional copy of CPR1 under control of the TEF1-promoter was called H222PAFoC.

(75) TABLE-US-00005 TABLE5 PrimersforoverexpressionofCPR1 Name SEQID Sequence(5.fwdarw.3) RS pTef_SpeI_fw3 SEQIDNO89 CTACGCTTGTTCAGACTTTG pTEF_CPR1_ol_rv SEQIDNO97 AGAGAGTCGAGTAGAGCCATTTT GAATGATTCTTATACTCAGAAGG pTEF_CPR1_ol_fw SEQIDNO98 CCTTCTGAGTATAAGAATCATTC AAAATGGCTCTACTCGACTCTCT CPR1_SphI_rv SEQIDNO99 atatagcatgcCTACCACACATC SphI TTCCTGGTAGAC INTB_out_fw SEQIDNO100 CTCAAGATACGGCATTGG INTB_out_rv SEQIDNO101 TCCTTGGCTAGACGAATG

Example 8

Comparative Cultivation of Different Production Strains in Fermenter

(76) In order to check how far different genetic modifications affect the production of -hydroxy fatty acids and/or dicarboxylic acids, the strains Yarrowia lipolytica H222P, H222PA, H222PF, H222PAF, H222PoF, H222PoC, H222PD, H222PH, H222PS, H222PAFoC, H222PAFD, H222PAFH and H222PAFS, whose construction was described in Examples 1, 2, 5, 6 and 7 were cultivated in the fermenter. The cultivation medium in this case was minimal medium with glucose (5% (w/v) glucose, 1 g 1.sup.1 KH.sub.2PO.sub.4, 0.16 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 3 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 0.7 g 1.sup.1 MgSO.sub.47 H.sub.2O, 0.5 g 1.sup.1 NaCl, 0.4 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 0.5 mg 1.sup.1 H.sub.3BO.sub.3, 0.04 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.1 mg 1.sup.1 KI, 0.4 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.2 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.4 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 6 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.3 mg 1.sup.1 thiamine hydrochloride). Cultivation was carried out in the fermenter at 28 C. The oxygen saturation was set at 55%. The cells were inoculated with an OD.sub.600 of 1 and incubated for 24 h at pH 5.5 (automated titration of HCl and NaOH). Then 15 g 1.sup.1 DD was added and the pH was set to 8.0. Glucose was added every 24 h to adjust a final concentration of 5% (w/v).

(77) Y. lipolytica H222P here formed relatively large quantities of dicarboxylic acids and can thus be used as initial strain for the construction of a corresponding production strain for dicarboxylic acids (FIG. 5A).

(78) Y. lipolytica H222PA and also H222PF formed increased quantities of -hydroxy fatty acids and can thus be used as initial strains for the construction of corresponding production strains for -hydroxy fatty acids. Here Y. lipolytica H222PF is to be preferred compared with H222PA (FIG. 5A).

(79) Y. lipolytica H222PAF formed only small quantities of dicarboxylic acids and is thus the preferred initial strain for the construction of production strains for -hydroxy fatty acids (FIG. 5A).

(80) The overexpressions of FAO1 and CPR1 in the strain Y. lipolytica H222P (resulting strains: Y. lipolytica H222PoF and H222PoC) in each case resulted in an increased production of dicarboxylic acids (FIG. 5B).

(81) The deletion of DGA1 in the strain Y. lipolytica H222P (resulting strain: Y. lipolytica H222PD) resulted in an increased production of dicarboxylic acids whereas the deletions of PAH1 and SCT1 (resulting strains: Y. lipolytica H222PH and H222PS) did not significantly increase the production of dicarboxylic acids (FIG. 5B).

(82) The overexpression of CPR1 in the strain Y. lipolytica H222PAF (resulting strain: Y. lipolytica H222PAFoC) resulted in an increased production of dicarboxylic acids but not of -hydroxy fatty acids (FIG. 5C).

(83) The deletions of DGA1, PAH1 and SCT1 in the strain Y. lipolytica H222PAF (resulting strains: Y. lipolytica H222PAFD, H222PAFH and H222PAFS) did not result in an increased production of -hydroxy fatty acids (FIG. 5B).

Example 9

Enzyme Test to Determine the (Fatty) Alcohol Dehydrogenase and Oxidase Activity

(84) The enzyme activities of the (fatty) alcohol dehydrogenases and oxidase were determined in cell lysates of the strains Yarrowia lipolytica H222P, H222PA, H222PF, H222PAF and H222PoF. The cells were cultivated as described in Example 8 and harvested and macerated after three days.

(85) The (fatty) alcohol dehydrogenase and oxidase activities were performed as described in Matatiele (2005) (Matatiele P R (2005) PhD thesis, University of the Free State, Republic of South Africa).

(86) The (fatty) alcohol dehydrogenase assay (50 mM Tris-HCl pH 8.5, 1.3 mM dodecan-1-ol in DMSO, 2 mM NAD.sup.+, 2 mM NAP.sup.+, 1.5% (v/v) cell extract) was measured using a recording spectrophotometer at 30 C. and =340 nm and the enzyme activity was calculated with the aid of the determined increase in extinction (NAD(P)H=6.3 mM.sup.1 cm.sup.1).

(87) The (fatty) alcohol oxidase assay (50 mM glycine NaOH pH 9.0, 0.35 mM dodecan-1-ol in DMSO, 0.013% (w/v) peroxidase (150 U/mg), 0.044% (w/v) ABTS, 6 mM sodium azide, 1-5% (v/v) cell extract) was measured using a recording spectrophotometer at =405 nm and the enzyme activity was calculated with the aid of the determined increase in extinction (.sub.ABTSox=18.4 mM.sup.1 cm.sup.1).

(88) Surprisingly no clear difference of the (fatty) alcohol dehydrogenase was determined between the strains (FIG. 6A). This could be due to the remaining alcohol dehydrogenase activity. Furthermore, it is possible that the measured conversion of dodecan-1-ol is also catalyzed by cytochrome P450 and ultimately the activity of aldehyde dehydrogenases is determined.

(89) As was expected, the (fatty) alcohol oxidase activity could only be detected in the strains Y. lipolytica H222P and H222PA and not in the FAO1-deletion strains Y. lipolytica H222PF and H222PAF (FIG. 6B). The measured (fatty) alcohol oxidase activity was increased more than ten times by the overexpression of FAO1 (strain: Y. lipolytica H222PoF).

Example 9

Cultivation of Y. lipolytica H222PF in the Fermenter

(90) In order to check whether the production strains are capable of producing larger quantities of dicarboxylic acids and/or -hydroxy fatty acids and accumulating in the culture medium, the strain Y. lipolytica H222PF was cultivated for 7 d in the fermenter. The cultivation medium here was minimal medium with glucose and an increased quantity of mineral salts, trace elements and vitamins (5% (w/v) glucose, 2 g 1.sup.1 KH.sub.2PO.sub.4, 0.32 g 1.sup.1 K.sub.2HPO.sub.43 H.sub.2O, 6 g 1.sup.1 (NH.sub.4).sub.2SO.sub.4, 1.4 g 1.sup.1 MgSO.sub.47 H.sub.2O, 1 g 1.sup.1 NaCl, 0.8 g 1.sup.1 Ca(NO.sub.3).sub.24 H.sub.2O, 1 mg 1.sup.1 H.sub.3BO.sub.3, 0.08 mg 1.sup.1 CuSO.sub.45 H.sub.2O, 0.2 mg 1.sup.1 KI, 0.8 mg 1.sup.1 MnSO.sub.44 H.sub.2O, 0.4 mg 1.sup.1 Na.sub.2MoO.sub.42 H.sub.2O, 0.8 mg 1.sup.1 ZnSO.sub.47 H.sub.2O, 12 mg 1.sup.1 FeCl.sub.36 H.sub.2O, 0.6 mg 1.sup.1 thiamine hydrochloride). Cultivation was carried out in a fermenter at 28 C. The oxygen saturation was adjusted to 55%. The cells were inoculated with an OD.sub.600 of 1 and incubated for 2 d at pH 5.5 (automated titration of HCl and NaOH). After 2 d the pH was adjusted to 8.0 and 15 g 1.sup.1 of dodecane was added after 2 d and 3 d. Glucose was added every 24 h in order to adjust a final concentration of 5-10% (w/v).

(91) After cultivation for 6 d (i.e. after 4 d in the production phase), Y. lipolytica H222PF formed 29.5 g 1.sup.1 of -hydroxydodecanoic acid and 3.5 g 1.sup.1 of dodecanoic acid (FIG. 7).