Yeast strains with reduced fatty alcohol oxidase activity and method for the production of ?-hydroxy fatty acids and dicarboxylic acids
10093950 ยท 2018-10-09
Assignee
Inventors
Cpc classification
C12Y502/01008
CHEMISTRY; METALLURGY
C12P7/6427
CHEMISTRY; METALLURGY
International classification
C12P7/64
CHEMISTRY; METALLURGY
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 strain with reduced fatty alcohol oxidase activity as compared to that of a wild type Yarrowia lipolytica strain, wherein said genetically engineered Yarrowia lipolytica strain is engineered to eliminate the expression or activity of endogenous fatty alcohol oxidase encoded by the FAO1 YALI0B14014g gene, and wherein the strain is further engineered to eliminate the expression or activity of each of the following endogenous acyl-CoA oxidases: POX1, POX2, POX3, POX4, POX5, and POX6.
2. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to reduce or eliminate the expression or activity of one or more endogenous fatty alcohol dehydrogenases selected from the group consisting of FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7.
3. The genetically engineered Yarrowia lipolytica strain of claim 2, wherein the strain is engineered to reduce or eliminate the expression or activity of FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7.
4. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to increase the expression of one or more cytochrome P450 proteins selected from the group consisting of ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, ALK8, ALK9, ALK10, ALK11, and ALK12.
5. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to increase the expression of the cytochrome P450 reductase protein CPR1.
6. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to reduce or eliminate the expression or activity of an endogenous phosphatidic acid dephosphohydrolase.
7. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to reduce or eliminate the expression or activity of an endogenous glycerol-3-phosphate-acyltransferase.
8. A method for the production of ?-hydroxy fatty acids from a hydrophobic substrate, comprising: (a) providing a genetically engineered Yarrowia lipolytica strain according to claim 1; (b) cultivating the strain in a suitable cultivation medium; and (c) contacting the strain with the hydrophobic substrate to form one or more ?-hydroxy fatty acids.
9. The method of claim 8, further comprising step (d) of isolating the one or more (b-hydroxy fatty acids.
10. The method of claim 8, wherein the hydrophobic substrate is selected from the group consisting of n-alkanes having the general structure: H.sub.3C(CH.sub.2).sub.nCH.sub.3, alkenes having the general structure H.sub.3C(C.sub.nH.sub.2n-2)CH.sub.3, H.sub.3C(C.sub.nH.sub.2n-4)CH.sub.3 or H.sub.3C(C.sub.nH.sub.2n-6)CH.sub.3, fatty acids having the general structure: HOOC(CH.sub.2).sub.nCH.sub.3, HOOC(C.sub.nH.sub.2n-2)CH.sub.3, COOC(C.sub.nH.sub.2n-4)CH.sub.3 or HOOC(C.sub.nH.sub.2n-6)CH.sub.3, and fatty acid alkyl esters having the general structure ROOC(CH.sub.2).sub.nCH.sub.3, where n is in the range from 6 to 18.
11. The method of claim 8, wherein the hydrophobic substance is an n-alkane, an alkene or a fatty acid where n is in the range of 8 to 18.
12. The method of claim 8, wherein glucose is used as a carbon source.
13. The method of claim 8, wherein the pH value after step (c) of contacting is greater than 5.5.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXPLANATIONS ON THE GENETIC MODIFICATIONS
(9) ?P Deletion of the acyl-CoA oxidase genes (POX-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) ? C. Overexpression of the NADPH-cytochrome P450-reductase gene CPR1 ? F. 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.
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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 H222?P 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 Seguence(5 .fwdarw. 3) RS pPOX1_fw SEQIDNO7 TCCAGAAGCGCTACAAAGAG pPOX1_rv SEQIDNO8 attaccctgttatccctaTGAAGGTTGCAGTCGTAGTC I-SceI POX1t_fw SEQIDNO9 tagggataacagggtaatTGCGATCTCGATGAGTGATG I-SceI POX1t_rv SEQIDNO10 GCCCAGAAGATTGGAATGAC pPOX2_fw SEQIDNO11 atataccgcggGATTCCGCCAAGTGAGACTG Cfr42I pPOX2_rv SEQIDNO12 attaccctgttatccctaCGTCGAGGAAGTAGGTCATC I-SceI POX2t_fw SEQIDNO13 tagggataacagggtaatGCGAGCTTGATGAGGAATAG I-SceI POX2t_rv SEQIDNO14 atataccgcggCCTGACGCCAATTTGAAGAG Cfr42I pPOX3_fw SEQIDNO15 atataccgcggCTGGGCTGTTCGGTCGATAG Cfr42I pPOX3_2v SEQIDNO16 tagggataacagggtaatAGGACGCACAACGCCATCAC I-SceI POX3t_fw SEQIDNO17 attaccctgttatccctaCGCTCCCATTGGAAACTA I-SceI POX3t_ry SEQIDNO18 atataccgcggTCTCTTCGCTGTGGTCTAGG Cfr42I pPOX4_fw SEQIDNO19 atataccgcggTCCACCGTTCTCCTTCATAC Cfr42I pPOX4_2v SEQIDNO20 tagggataacagggtaatATGTCTCTAGGGTCGAAGTC I-SceI POX4t_fw SEQIDNO21 attaccctgttatccctaTGGCAAGCCTCACTACTACG 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 attaccctattatccctaACCCTCGACCTCCTTATTAC 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 BgII, 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, LaCroute 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 H222?P?A.
(31) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 was called H222?P?F.
(32) TABLE-US-00002 TABLE2 ontherecoveryanddetectionof(F)ADH-and FAOdeletioncassettes Name SEQID Sequence(5 .fwdarw. 3) RS pFADH_fw SEQID atataaagctTGCGGCT HindIII NO31 CGGCACGTGATCTG pFADH_rv SEQID atataggatccATCGTG BamHI NO32 CGTACGTCGCTAGTG FADHt_fw SEQID atataggatccCGACCG BamHI NO33 GCACGATCAATTGG FADHt_rv SEQID atatacatatgGGTGCA NdeI NO34 TCTCAGCCCGACCTC FADHt_rv2 SEQID TCCCGAAACACAGAACT NO35 TCC pADH1_fw SEQID atataaagctTGGTGGA HindIII NO36 CGTTCCGGCAGACAG pADR1_rv SEQID atataggatccCTCCCA BamHI NO37 GGCATCTCCACACTC ADH1t_fw SEQID atataggatccCACTTA BanHI NO38 CAGGCTTAGCAAGG ADH1t_rv SEQID atatagcggccgcGGAA NotI NO39 ATCACGCTTGATTCG ADH1t_rv2 SEQID TAGGCGCTGGTACAGAA NO40 GAG pADH2_fw SEQID atataaagctTGAGTAC HindIII NO41 AGTAGGTGGTACTC pADH2_rv SEQID atataggatccAGTGGT BamHI NO42 GGTGGTGGTGGTAG ADH2t_fw SEQID atataggatccTTTACG BamHI NO43 TGCAACAGGAGGAG ADH2t_rv SEQID atatacatatgGCCTGT NdeI NO44 CTTGAGTTCTTTGG ADH2t_rv2 SEQID AGGGTCGTAGATAACGA NO45 GTC pADH3_fw SEQID atataaagctTCACGTG HindIII NO46 GCTGCTGGGCCAACC pADH3_rv SEQID atataggatccCGCACG BamHI NO47 GTATCGGAGCATCG ADH3t_fw SEQID atataggatccCGCGGC BamHI NO48 TATTGACGCTGAGG ADH3t_rv SEQID atatacatatgCCCGTC NdeI NO49 AGCTCCATCGACGAGTG ADH3t_rv2 SEQID AGGTGTACTGTAGCCAC NO50 CCTGAC pADH4_fw SEQID atataaagctTCCGGCC HindIII NO51 AGCCGCTGGCAACG pADH4_rv SEQID atataggatccACACGA BamHI NO52 CAGCTGCACCTGAC ADH4t_fw SEQID atataggatccCAGCCA BamHI NO53 TGAGCCAGGCATTG ADH4t_rv SEQID atatacatatgGGCGCC NdeI NO54 AGCCACATTTGCCCTC ADH4t_rv2 SEQID AGCGATACAGCAGTTGA NO55 CTC pADH5_fw SEQID TCAGCCGTCTACTTGTA NO56 GAG pADH5_rv SEQID atataggatccGTGGCT BamHI NO57 CGGATACTCCTGAC ADH5t_fw SEQID atataggatccAGCCGG BamHI NO58 AGGTCAGATCAAGC ADH5t_rv SEQID atatacatatgGCGCAA NdeI NO59 TAGTTCGCCGQCCTG ADH5t_rv2 SEQID CTCGTGTTGTGCCTTTC NO60 TTG pADH6_fw SEQID atataaagctTGCGCGA HindIII NO61 CAACCCATAGCGATGGC pADH6_rv SEQID atataggatccGATAAG BamHI NO62 AGGGCGCTCTGACC ADH6t_fw SEQID atataggatccGGCGTG BamHI NO63 ACATCGAGTTTGG ADH6t_rv SEQID atatacatatgCTACGT NdeI NO64 CTCGCCGCAGAGGG ADH6t_rv2 SEQID AGCGAGAGGTTATACGG NO65 AAG pADH7_fw SEQID CTCCTACAGCCTCTCAA NO66 GAC ADH7t_rv SEQID GTCTACAAGACAGCCCA NO67 GAG pADH7_fw2 SEQID CCGCTTGAGAAGAGCAA NO68 TAC pFAO1_fw SEQID atataaagctTCGCCAC HindIII NO69 CTGTCCACGTCTCG pFAO1_rv SEQID atataggatccGCGAAG BamHI NO70 CGACGTGTGGTGAG FAO1t_fw SEQID atataggatccGCTGAG BamHI NO71 CACGCGAGTACACC FAO1t_rv SEQID atatagaattcGATCTG EcoRI NO72 TCGTACAACTAAGG FAO1t_rv2 SEQID CAGAAGTTACGACGCCA NO73 AGG
EXAMPLE 3
Comparative Cultivation of Various Production Strains in a Shaking Flask
(33) In order to check whether the initial strains produced Yarrowia lipolytica H222?P, H222?P?A and H222?P?F 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 l.sup.?1 CaCO.sub.3, 17.3 g l.sup.?1 KH.sub.2PO.sub.4, 1.35 g l.sup.?1 K.sub.2HPO.sub.4?3 H.sub.2O, 3 g l.sup.?1 (NH.sub.4).sub.2SO.sub.4, 0.7 g l.sup.?1 MgSO.sub.4?7 H.sub.2O, 0.5 g l.sup.?1 NaCl, 0.4 g l.sup.?1 Ca(NO.sub.3).sub.2?4 H.sub.2O, 0.5 mg l.sup.?1 H.sub.3BO.sub.3, 0.04 mg l.sup.?1 CuSO.sub.4?5 H.sub.2O, 0.1 mg l.sup.?1 KI, 0.4 mg l.sup.?1 MnSO.sub.4?4 H.sub.2O, 0.2 mg l.sup.?1 Na.sub.2MoO.sub.4?2 H.sub.2O, 0.4 mg l.sup.?1 ZnSO.sub.4?7 H.sub.2O, 6 mg l.sup.?1 FeCl.sub.3?6 H.sub.2O, 0.3 mg l.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 (
(34) Y. lipolytica H222?P 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 H222?P?A and also H222?P?F 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 H222?P?F is to be preferred to H222?P?A 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 H222?P and H222?P?F) 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 l.sup.?1 KH.sub.2PO.sub.4, 1.35 g l.sup.?1 K.sub.2HPO.sub.4?3 H.sub.2O, 3 g l.sup.?1 (NH.sub.4).sub.2SO.sub.4, 0.7 g l.sup.?1 MgSO.sub.4?7 H.sub.2O, 0.5 g l.sup.?1 NaCl, 0.4 g l.sup.?1 Ca(NO.sub.3).sub.2?4 H.sub.2O, 0.5 mg l.sup.?1 H.sub.3BO.sub.3, 0.04 mg l.sup.?1 CuSO.sub.4?5 H.sub.2O, 0.1 mg l.sup.?1 KI, 0.4 mg l.sup.?1 MnSO.sub.4?4 H.sub.2O, 0.2 mg l.sup.?1 Na.sub.2MoO.sub.4?2 H.sub.2O, 0.4 mg l.sup.?1 ZnSO.sub.4?7 H.sub.2O, 6 mg l.sup.?1 FeCl.sub.3?6 H.sub.2O, 0.3 mg l.sup.?1 thiamine hydrochloride) and glycerol (3% (v/v) glycerol, 1% (w/v) pentadecanoic acid, 17.3 g l.sup.?1 KH.sub.2PO.sub.4, 1.35 g l.sup.?1 K.sub.2HPO.sub.4?3 H.sub.2O, 3 g l.sup.?1 (NH.sub.4).sub.2SO.sub.4, 0.7 g l.sup.?1 MgSO.sub.4?7 H.sub.2O, 0.5 g l.sup.?1 NaCl, 0.4 g l.sup.?1 Ca(NO.sub.3).sub.2?4 H.sub.2O, 0.5 mg l.sup.?1 H.sub.3BO.sub.3, 0.04 mg l.sup.?1 CuSO.sub.4?5 H.sub.2O, 0.1 mg l.sup.?1 KI, 0.4 mg l.sup.?1 MnSO.sub.4?4 H.sub.2O, 0.2 mg l.sup.?1 Na.sub.2MoO.sub.4?2 H.sub.2O, 0.4 mg l.sup.?1 ZnSO.sub.4?7 H.sub.2O, 6 mg l.sup.?1 FeCl.sub.3?6 H.sub.2O, 0.3 mg l.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 H222?P, H222?P?A and H222?P?F) 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 l.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 l.sup.?1 KH.sub.2PO.sub.4, 1.35 g l.sup.?1 K.sub.2HPO.sub.4?3 H.sub.2O, 3 g l.sup.?1 (NH.sub.4).sub.2SO.sub.4, 0.7 g l.sup.?1 MgSO.sub.4?7 H.sub.2O, 0.5 g l.sup.?1 NaCl, 0.4 g l.sup.?1 Ca(NO.sub.3).sub.2?4 H.sub.2O, 0.5 mg l.sup.?1 H.sub.3BO.sub.3, 0.04 mg l.sup.?1 CuSO.sub.4?5 H.sub.2O, 0.1 mg 1.sup.?1 KI, 0.4 mg l.sup.?1 MnSO.sub.4?4 H.sub.2O, 0.2 mg l.sup.?1 Na.sub.2MoO.sub.4?2 H.sub.2O, 0.4 mg l.sup.?1 ZnSO.sub.4?7 H.sub.2O, 6 mg l.sup.?1 FeCl.sub.3?6 H.sub.2O, 0.3 mg l.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 H222?P?F after 10 days formed around 0.7 g l.sup.?1 ?-hydroxy pentadecanoic acid and 2.4 g l.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 l.sup.?1 KH.sub.2PO.sub.4, 1.35 g l.sup.?1 K.sub.2HPO.sub.4?3 H.sub.2O, 3 g l.sup.?1 (NH.sub.4).sub.2SO.sub.4, 0.7 g l.sup.?1 MgSO.sub.4?7 H.sub.2O, 0.5 g l.sup.?1 NaCl, 0.4 g l.sup.?1 Ca(NO.sub.3).sub.2?4 H.sub.2O, 0.5 mg l.sup.?1 H.sub.3BO.sub.3, 0.04 mg l.sup.?1 CuSO.sub.4?5 H.sub.2O, 0.1 mg l.sup.?1 KI, 0.4 mg l.sup.?1 MnSO.sub.4?4 H.sub.2O, 0.2 mg l.sup.?1 Na.sub.2MoO.sub.4?2 H.sub.2O, 0.4 mg l.sup.?1 ZnSO.sub.4?7 H.sub.2O, 6 mg l.sup.?1 FeCl.sub.3?6 H.sub.2O, 0.3 mg l.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 H222?P?F after 7 days formed around 3.9 g l.sup.?1 ?-hydroxy pentadecanoic acid and 1.3 g l.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 l.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.
(45) 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).
(46) Before a renewed transformation, the marker was recovered by FOA selection (Boeke J D, LaCroute F & Fink G R (1984) Mol Gen Genet 197: 345-346).
(47) 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.
(48) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also DGA1 was called H222?P?D.
(49) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also PAH1 was called H222?P?H.
(50) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also SCT1 was called H222?P?S.
(51) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and DGA1 was called H222?P?F?D.
(52) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and PAH1 was called H222?P?F?H.
(53) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5 and POX6 and also FAO1 and SCT1 was called H222?P?F?S.
(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 DGA1 was called H222?P?A?F?D.
(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 PAH1 was called H222?P?A?F?H.
(56) 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 H222?P?A?F?S.
(57) TABLE-US-00003 TABLE3 PrimersforrecoveryanddetectionofDGA1-, PAH1-andSCT1-deletioncassettes Name SEQID Sequene(5 .fwdarw. 3) RS pDGA1_fw SEQIDNO74 atataaagcttCCGTAA HindIII TAAATGCCCGTCTC pDGA1_rv SEQIDNO75 atataggatccAGGTCG BamHI ATTTCGGCGCTGTG DGA1t_fw SEQIDNO76 atataggatccGGTTAG BamHI GCAAATAGCTAATG DGA1t_rv SEQIDNO77 atatagaattcGCCTGG EcoRI AGCGAGTTTCTGAG DGA1t_rv2 SEQIDNO78 TCGCAAGGGCCATAGAG GTG pPAH1_fw SEQIDNO79 atataaagcttCTTGCA HindIII CATCTCCGATGAC pPAH1_rv SEQIDNO80 atataggatccGGTGTT BamHI ACGCCACCACGTTG PAH1t_fw SEQIDNO81 atataggatccGGGACC BamHI TGCGATACGAATGC PAH1t_rv SEQIDNO82 atatagaattcGCTTGC EcoRI GCAGCCGGTGTATC PAH1t_rv2 SEQIDNO83 GGCGTTGTGGAGCTATC ACC pSCT1_fw SEQIDNO84 atataaagcttGCGTGC HindIII GGTGCGTGCGTATG pSCT1_rv SEQIDNO85 atataggatccCAGCAC BamHI CACGAAATTATACG SCT1t_fw SEQIDNO86 atataggatccGTGCGC BamHI TTACATGTGGACCG SCT1t_rv SEQIDNO87 atatagaattcCAGGCA EcoRI GCTTCTTGCCAATG SCT1t_rv2 SEQIDNO88 GAGATAGGAGGTTCCCA TAC
(58) The strains Y. lipolytica H222?P?F?D, H222?P?F?H and H222?P?F?S as well as the initial strain Y. lipolytica H222?P?F were cultivated in minimal medium with glucose and pentadecanoic acid (3% (v/v) glucose, 1% (w/v) pentadecanoic acid, 17.3 g l.sup.?1 KH.sub.2PO.sub.4, 1.35 g l.sup.?1 K.sub.2HPO.sub.4?3 H.sub.2O, 3 g l.sup.?1 (NH.sub.4).sub.2SO.sub.4, 0.7 g l.sup.?1 MgSO.sub.4?7 H.sub.2O, 0.5 g l.sup.?1 NaCl, 0.4 g l.sup.?1 Ca(NO.sub.3).sub.2?4 H.sub.2O, 0.5 mg l.sup.?1 H.sub.3BO.sub.3, 0.04 mg l.sup.?1 CuSO.sub.4?5 H.sub.2O, 0.1 mg l.sup.?1 KI, 0.4 mg l.sup.?1 MnSO.sub.4?4 H.sub.2O, 0.2 mg l.sup.?1 Na.sub.2MoO.sub.4?2 H.sub.2O, 0.4 mg l.sup.?1 ZnSO.sub.4?7 H.sub.2O, 6 mg l.sup.?1 FeCl.sub.3?6 H.sub.2O, 0.3 mg l.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 (
(59) 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).
(60) 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 Yarrowla lipolytica H222?P
(61) 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.
(62) Furthermore, the FAO1-gene (YALI0B14014g) was amplified by means of PCR using the primers pTef_FAOo_ol_fw and FAOlo_SphI_rv (template: genomic DNA of Y. lipolytica H222).
(63) 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 FAOlo_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.
(64) 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.
(65) The plasmid pINTC-FAO1 was ultimately linearized with AscI and transformed into the desired Y. lipolytica recipient strain.
(66) 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.
(67) 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 H222?PoF and was used as initial strain for construction of further strains for the production of dicarboxylic acids.
(68) TABLE-US-00004 TABLE4 PrimersforoverexpressionofFAO1 Name SEQID Sequence(5.fwdarw.3) RS pTef_SpeI_fw3 SEQID CTACGCTTGTTCAGACTTTG NO89 pTef_FAOo_ol_ SEQID gtgtgcttgtcgtcagacatTTT rv NO90 GAATGATTCTTATACTCAGAAGG pTef_FAOO_ol_ SEQID ccttctgagtataagaatcattc fw NO91 aaaATGTCTGACGACAAGCACAC FAO1o_SphI_rv SEQID gcatgcTTAGATTCGAGGTCGGA SphI NO92 GAT INT_AscI_fw_ SEQID ggtacCACGCACGGATAGTTTAT KpnI KpnI NO93 CCA INT_AscI_rv_ SEQID ggtacCCAAAGTCAACTAATGTC KpnI KpnI NO94 AAGTAAAG INT_AscI_fw_ SEQID CCTCCAACGTGACTTTC out NO95 INT_AscI_rv_ SEQID AGAGACCTCCCACAAAG out NO96
EXAMPLE 7
Increased Expression of CPR1 in Yarrowia lipolytica H222?P and H222?P?A?F
(69) 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.
(70) 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).
(71) 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.
(72) The resulting plasmid was ultimately linearized with NotI and transformed into the desired Y. lipolytica recipient strain.
(73) 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.
(74) 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 H222?PoC and was used as initial strain for construction of further strains for the production of dicarboxylic acids.
(75) The strain which carried both the deletions of the genes POX1, POX2, POX3, POX4, POX5, POX6, FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, FAO1 and also the additional copy of CPR1 under control of the TEF1-promoter was called H222?P?A?FoC.
(76) TABLE-US-00005 TABLE5 PrimersforoverexpressionofCPR1 Name SEQID Sequence(5.fwdarw.3) RS pTef_SpeI_fw3 SEQID CTACGCTTGTTCAGACTTTG NO89 pTEF_CPR1_ol_ SEQID AGAGAGTCGAGTAGAGCCATTTT rv NO97 GAATGATTCTTATACTCAGAAGG pTEF_CPR1_ol_ SEQID CCTTCTGAGTATAAGAATCATTC fw NO98 AAAATGGCTCTACTCGACTCTCT CPR1_SphI_rv SEQID atatagcatgcCTACCACACATC SphI NO99 TTCCTGGTAGAC INTB_out_fw SEQID CTCAAGATACGGCATTGG NO100 INTB_out_rv SEQID TCCTTGGCTAGACGAATG NO101
EXAMPLE 8
Comparative Cultivation of Different Production Strains in Fermenter
(77) In order to check how far different genetic modifications affect the production of ?-hydroxy fatty acids and/or dicarboxylic acids, the strains Yarrowia lipolytica H222?P, H222?P?A, H222?P?F, H222?P?A?F, H222?PoF, H222?PoC, H222?P?D, H222?P?H, H222?P?S, H222?P?A?FoC, H222?P?A?F?D, H222?P?A?F?H and H222?P?A?F?S, 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 l.sup.?1 KH.sub.2PO.sub.4, 0.16 g l.sup.?1 K.sub.2HPO.sub.4?3 H.sub.2O, 3 g l.sup.?1 (NH.sub.4).sub.2SO.sub.4, 0.7 g l.sup.?1 MgSO.sub.4?7 H.sub.2O, 0.5 g l.sup.?1 NaCl, 0.4 g l.sup.?1 Ca(NO.sub.3).sub.2?4 H.sub.2O, 0.5 mg l.sup.?1 H.sub.3BO.sub.3, 0.04 mg l.sup.?1 CuSO.sub.4?5 H.sub.2O, 0.1 mg l.sup.?1 KI, 0.4 mg l.sup.?1 MnSO.sub.4?4 H.sub.2O, 0.2 mg l.sup.?1 Na.sub.2MoO.sub.4?2 H.sub.2O, 0.4 mg l.sup.?1 ZnSO.sub.4?7 H.sub.2O, 6 mg l.sup.?1 FeCl.sub.3?6 H.sub.2O, 0.3 mg l.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 l.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).
(78) Y. lipolytica H222?P 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 (
(79) Y. lipolytica H222?P?A and also H222?P?F 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 H222?P?F is to be preferred compared with H222?P?A (
(80) The overexpressions of FAO1 and CPR1 in the strain Y. lipolytica H222?P (resulting strains: Y. lipolytica H222?PoF and H222?PoC) in each case resulted in an increased production of dicarboxylic acids (
(81) The deletion of DGA1 in the strain Y. lipolytica H222?P (resulting strain: Y. lipolytica H222?P?D) resulted in an increased production of dicarboxylic acids whereas the deletions of PAH1 and SCT1 (resulting strains: Y. lipolytica H222?P?H and H222?P?S) did not significantly increase the production of dicarboxylic acids (
(82) The overexpression of CPR1 in the strain Y. lipolytica H222?P?A?F (resulting strain: Y. lipolytica H222?P?A?FoC) resulted in an increased production of dicarboxylic acids but not of ?-hydroxy fatty acids (
(83) The deletions of DGA1, PAH1 and SCT1 in the strain Y. lipolytica H222?P?A?F (resulting strains: Y. lipolytica H222?P?A?F?D, H222?P?A?F?H and H222?P?A?F?S) did not result in an increased production of ?-hydroxy fatty acids (
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 H222?P, H222?P?A, H222?P?F, H222?P?A?F and H222?PoF. 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 (?.sub.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.AB-TSox=18.4 mM.sup.?1 cm.sup.?1).
(88) Surprisingly no clear difference of the (fatty) alcohol dehydrogenase was determined between the strains (
(89) As was expected, the (fatty) alcohol oxidase activity could only be detected in the strains Y. lipolytica H222?P and H222?P?A and not in the FAO1-deletion strains Y. lipolytica H222?P?F and H222?P?A?F (
EXAMPLE 9
Cultivation of Y. lipolytica H222?P?F 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 H222?P?F 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 l.sup.?1 KH.sub.2PO.sub.4, 0.32 g l.sup.?1 K.sub.2HPO.sub.4?3 H.sub.2O, 6 g l.sup.?1 (NH.sub.4).sub.2SO.sub.4, 1.4 g l.sup.?1 MgSO.sub.4?7 H.sub.2O, 1 g l.sup.?1 NaCl, 0.8 g l.sup.?1 Ca(N03).sub.2?4 H.sub.2O, 1 mg l.sup.?1 H.sub.3BO.sub.3, 0.08 mg l.sup.?1 CuSO.sub.4?5 H.sub.2O, 0.2 mg l.sup.?1 KI, 0.8 mg l.sup.?1 MnSO.sub.4?4 H.sub.2O, 0.4 mg l.sup.?1 Na.sub.2MoO.sub.4?2 H.sub.2O, 0.8 mg l.sup.?1 ZnSO.sub.4?7 H.sub.2O, 12 mg l.sup.?1 FeCl.sub.3?6 H.sub.2O, 0.6 mg l.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 l.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 H222?P?F formed 29.5 g l.sup.?1 of ?-hydroxydodecanoic acid and 3.5 g l.sup.?1 of dodecanoic acid (