Pichia ciferrii cells and uses thereof

Abstract

The invention relates to genetically modified Pichia ciferrii cells, to the use thereof and to a method of producing sphingoid bases and sphingolipids.

Claims

1. A genetically modified Pichia ciferrii cell, wherein said genetically modified Pichia ciferrii cell comprises, compared to its wild type, a reduced activity of an E.sub.1 enzyme encoded by any one of the intron-free nucleic acid sequences selected from the groups consisting of: A) SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11; and B) a sequence which is at least 90% identical to any one of the sequences SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, wherein said activity of said E.sub.1 enzyme is the catalysis of the reaction 5,10-methylenetetrahydrofolate+L-glycine+H.sub.2O to tetrahydrofolate+L-serine.

2. The genetically modified Pichia ciferrii cell according to claim 1, characterized in that wherein said reduction of the enzymatic activity is achieved by modifying a gene comprising any one of the nucleic acid sequences specified in claim 1, wherein the modification is selected from the group consisting of: insertion of foreign DNA into the gene, deletion of at least a portion of the gene, point mutations in the gene sequence, exposing the gene to the influence of RNA interference, and replacement of a portion of the gene with foreign DNA.

3. The genetically modified Pichia ciferrii cell according to claim 2, wherein said foreign DNA is a selection marker gene that can be removed without leaving a trace and which leaves a deletion in the target gene.

4. The genetically modified Pichia ciferrii cell according to claim 1, wherein the Pichia ciferrii cell is obtained from strains selected from the group consisting of Pichia ciferrii NRRL Y-1031 F-60-10; and Pichia ciferrii CS.PCPro2.

5. The genetically modified Pichia ciferrii cell according to claim 1, characterized in that the cell further comprises, compared to its wild type, an increased enzymatic activity of an enzyme E.sub.2, wherein the enzyme E.sub.2 catalyses the reaction of sphinganine to phytosphingosine.

6. A method for producing a genetically modified Pichia ciferri cell comprising: I) providing a Pichia ciferrii cell, and II) modifying at least one gene comprising any one of the nucleic acid sequences set forth in groups A) and B) in claim 1 by: insertion of foreign DNA, wherein said insertion is DNA coding for a selection marker gene; deletion of at least a portion of the gene; insertion of a point mutation in the gene sequence; exposing the gene to the influence of RNA interference; or replacement of a portion of the gene with foreign DNA.

7. A method for producing sphingoid bases and sphingolipids comprising the steps of a) contacting a genetically modified Pichia ciferrii cell according to claim 1 with a medium including a carbon source, b) culturing the cell from step a) under conditions which enable the cell to produce sphingoid bases and sphingolipids from said carbon source, and c) optionally isolating the sphingoid bases and sphingolipids produced.

8. The genetically modified Pichia ciferrii cell according to claim 5, wherein the enzyme E.sub.2 is a sphinganine C4-hydroxylase.

9. The genetically modified Pichia ciferrii cell according to claim 5, wherein the E.sub.2 enzyme is encoded by the nucleic acid set forth in SEQ ID NO: 17.

Description

(1) The following figures are part of the Examples:

(2) FIG. 1: Design principle of gene deletion cassettes

(3) FIG. 2: Design principle of overexpression cassettes

EXAMPLES

Construction of Gene Deletion Cassettes

(4) Unless stated otherwise, gene deletions were carried out by means of classical one-step gene replacement, as described in Rothstein 1983, Methods Enzymol 101: 202-211.

(5) Deletion cassettes were constructed by in vivo cloning, ultimately resulting in plasmids which were used as templates for PCR-based amplification of the deletion cassettes. These PCR products were then transformed into P. ciferrii with the aim of deleting a particular gene.

(6) The deletion cassettes were constructed by employing the plasmid p426HXT7-6HIS (Hamacher et al., 2000; Microbiology 148, 2783-8) as shuttle vector. p426HXT7-6HIS was first cleaved with BamHI and EcoRI, resulting in a 5.69 kb fragment which was used as backbone for the subsequent cloning steps. Initially, three overlapping DNA fragments were generated by PCR for each P. ciferrii deletion cassette: a dominant clonNAT marker, which could later be eliminated again, as the central part (nat1 resistance cassette) (cf. Schorsch et al., Curr Genet. 2009 August; 55(4):381-9), a second fragment of about 500 bp in length, representing the 5-untranslated region of the ORF to be deleted (promoter region, PR) and with overlap to the start of the clonNAT-marker fragment, and a third fragment of about 500 bp in length, representing the 3-untranslated region (terminator region, TR) of the ORF to be deleted and with overlap to the end of the clonNAT-marker fragment.

(7) Each deletion cassette was constructed by amplifying by means of PCR the promoter region (PR) and the terminator region (TR) of the gene to be deleted from genomic P. ciferrii wild-type DNA, in each case employing gene-specific primers. To this end, primer pairs, P1/P2 for PR and P3/P4 for TR, were used in each case. The primers were chosen so as to have at the 5 end regions of about 30-35 bps in length which were overlapping with the DNA elements to be fused:

(8) TABLE-US-00001 Primer 5 End overlapping with: P1 Cloning vector p426HXT7-6HIS P2 nat1 Resistance cassette (PCR amplicon of plasmid pCS.LoxP.nat1 with primers LPNTL.fw and LPNTL.rv) P3 nat1 Resistance cassette P4 Cloning vector p426HXT7-6HIS

(9) The central fragment (nat1 resistance cassette, Seq ID No 19) was amplified using in each case the primer pair LPNTL.fw (TGGCGCTTCGTACCACTGGGTAAC) and LPNTL.rv (GAAATTAATACGACTCACTATAGG), with plasmid pCS.LoxP.nat1 (Schorsch et al., 2009; Curr. Genet. 55, 381-9) being employed as template (all primer sequences are given in 5.fwdarw.3 orientation).

(10) The PCR products of primer pairs P1/P2, P3/P4 and LPNTL.fw/LPNTL.rv, together with the p426HXT7-6HIS plasmid previously linearized by digestion with BamHI and EcoRI, were transformed into S. cerevisiae strain K26. The PCR products and the linearized vector were joined together in vivo by homologous recombination, causing the linearized vector to be re-circularized and able to be propagated in S. cerevisiae. Transformants obtained were selected by means of the marker gene (nat1) on YEPD plates with clonNAT, their DNA was isolated and transformed into E. coli, and the plasmids re-isolated therefrom were verified by restriction mapping or sequencing.

(11) The deletion cassettes were amplified using the primer pairs 426L.fw (GCTTCCGGCTCCTATGTTG, Seq ID No 23) and 426R.rv (ACCCTATGCGGTGTGAAATAC, Seq ID No 24) or HXT7 (GCCAATACTTCACAATGTTCGAATC, Seq ID No 25) and CYC (CGTGAATGTAAGCGTGACATAAC, Seq ID No 26), unless stated otherwise. See FIG. 1 for clarification.

(12) To successively delete multiple genes, a marker rescue was performed after each deletion. This was accomplished by transformation with plasmid pCS.opt.Cre (Seq ID. No 20) as described previously (Schorsch et al., Curr Genet. 2009 August; 55(4):381-9). The gene deletions were verified by PCR analyses using genomic DNA of the transformants as template.

(13) The particular gene deletion cassettes of the genes with sequences Seq ID No 1, Seq ID No 3, Seq ID No 5, Seq ID No 7, Seq ID No 9 and Seq ID No 11 were constructed using the primers listed in the table below. For each of the Seq IDs, the first two primers listed (SH11 and SH12 or SH21 and SH22 or C1 and C2 or HXT7-LCB4.fw and LCB4.HXT7.rv or HXT7-DPL1.fw and DPL1.rv2 or ORM-426L.fw and ORM-LPNTL.rv) were used in each case for amplification of PR, with the next two primers listed (SH13 and SH14 or SH23 and SH24 or C3 and C4 or LCB4.rv and LCB4.fw or DPL1.fw2 and CYC-DPL1.rv or ORM-LPNTL.fw2 and ORM-426R.rv) being used for amplification of TR. The last two primers listed in each case (SHMT1.pop-in.fw and SHMT1.veri.rv or SHMT2.pop-in.fw and SHMT2.veri.rv or CHA1.pop-in.fw and CHA1.veri.rv or LCB4.pop-in.fw and LCB4.veri.rv or DPL1.pop-in.fw and DPL1.veri.rv or ORM1.pop-in.fw and ORM.veri.rv) are used for detecting integration or the wild-type allele.

(14) TABLE-US-00002 Gene Primername Sequence(5 -> 3) SeqIDNo1 SH11 CAAAAAGTTAACATGCATCACCATCACCATCACA SeqIDNo27 CTAACCCAACTAGGCTCATTAAC SH12 GTTATCTGCAGGTTACCCAGTGGTACGAAGCGC SeqIDNo28 CATCAGCCATTTCTGGATCAATTTC SH13 TGCCGGTCTCCCTATAGTGAGTCGTATTAATTTC SeqIDNo29 ATCCAGTTCCAGGTGAATTATAAG SH14 TAACTAATTACATGACTCGAGGTCGACGGTATC SeqIDNo30 CCATACTATGCTTGGCATCTTAAAC SHMT1.pop-in.fw TTGATAGGGCAAATTCTCCAAC SeqIDNo31 SHMT1.veri.rv TTCACCTGGATAACCTTCTG SeqIDNo32 SeqIDNo3 SH21 CAAAAAGTTAACATGCATCACCATCACCATCACA SeqIDNo33 TGTCCTTGCAGGTGGTATTC SH22 TTATCTGCAGGTTACCCAGTGGTACGAAGCGCC SeqIDNo34 AGGTAAAGCGTATGGCATGTTG SH23 CTGCCGGTCTCCCTATAGTGAGTCGTATTAATTT SeqIDNo35 CGCTGGTGAATTCCCATTATCTG SH24 TAACTAATTACATGACTCGAGGTCGACGGTATC SeqIDNo36 CATAACCATCTAAAGCATTATAGTC SHMT2.pop-in.fw AAGTTTCAGCAAATGGTTTGAC SeqIDNo37 SHMT2.veri.rv TATCTTGCACCTGGATAACC SeqIDNo38 SeqIDNo5 C1 CAAAAAGTTAACATGCATCACCATCACCATCACA SeqIDNo39 ATCTAAGAGGTAAAGTTCAACATTC C2 GTTATCTGCAGGTTACCCAGTGGTACGAAGCGC SeqIDNo40 CATTGGTTTGCCGTGTGGATTG C3 CTGCCGGTCTCCCTATAGTGAGTCGTATTAATTT SeqIDNo41 CGGAGTTCAACAACCGTTCAAG C4 TAACTAATTACATGACTCGAGGTCGACGGTATC SeqIDNo42 ATGAAGTTGATGCTGCTTTGG CHA1.pop-in.fw ATTTAGAAGCTAGAGGTTCAGAAAG SeqIDNo43 CHA1.veri.rv TAGAAGAATGACCATGCCATATAG SeqIDNo44 SeqIDNo7 HXT7-LCB4.fw TTTTAATTTTAATCAAAAAGTTAACATGCATCACC SeqIDNo45 ATCACCATCACACTCACAGAGTCAACTCCTGTAT ATTC LCB4.HXT7.rv TGAATGTAAGCGTGACATAACTAATTACATGACT SeqIDNo46 CGAGGTCGACGGTATCTCTGGCGGTATTGAACT TTGTGGAG LCB4.rv GTTATCTGCAGGTTACCCAGTGGTAAAGTGTAT SeqIDNo47 GGATGGGTTGAAGTATGTCTTTATATC LCB4.fw ACGAAGTTATGAGCTCGAATTCATCGATGCTAC SeqIDNo48 CCGGTGCTGCAAAGACTTTACTAAG LCB4.pop-in.fw GTGAATGGTTAATAGTGCGCTATG SeqIDNo49 LCB4.veri.rv CTAACAAATACCACTTCGACATCAG SeqIDNo50 SeqIDNo9 HXT7-DPL1.fw TTTTAATTTTAATCAAAAAGTTAACATGCATCACC SeqIDNo51 ATCACCATCACACCTTCCGTGAGATTTCCCTTGT TTAC DPL1.rv2 TATACGAAGTTATCTGCAGGTTACCCAGTGGTA SeqIDNo52 TAACCCATAACCAGTGATGTTAACC DPL1.fw2 GAAGTTATGAGCTCGAATTCATCGATGACCACT SeqIDNo53 GGTGTTGTTGATCG CYC-DPL1.rv TGAATGTAAGCGTGACATAACTAATTACATGACT SeqIDNo54 CGAGGTCGACGGTATCCGACGGTAATGAGGAT GTAAATGAG DPL1.pop-in.fw AAACAAGAGCAGCATGCAACTTGAG SeqIDNo55 DPL1.veri.rv AGTGACACCAGGAACTCTAAAG SeqIDNo56 SeqIDNo11 ORM-426L.fw GCTTTACACTTTATGCTTCCGGCTCCTATGTTGA SeqIDNo57 ACTATGTCAATATCGATCGTATG ORM-LPNTL.rv TATCTGCAGGTTACCCAGTGGTACGAAGCGCCA SeqIDNo58 AACAGAAATTGGTTCATGTGTTG ORM-LPNTL.fw2 GCCGGTCTCCCTATAGTGAGTCGTATTAATTTCT SeqIDNo59 GGTGTACCAATTTGGTTATTTC ORM-426R.rv ATATCAGTTATTACCCTATGCGGTGTGAAATACA SeqIDNo60 CAAGTACAACAACAACAGATTTAG ORM1.pop-in.fw TACCCACCTTTGACATAATCAG SeqIDNo61 ORM.veri.rv ATTCAAATGGCGTACCTTTAAC SeqIDNo62
Construction of Overexpression Cassettes

(15) Overexpression cassettes were constructed in principle in the same way as the method used for the deletion cassettes. In the case of overexpression cassettes, however, an additional fourth PCR product was generated (promoter fragment, PF), representing a fragment of the PcTDH3 or the PcENO1 promoter. This was later linked in vivo to the nat1 resistance cassette and the third PCR fragment which in this case had an overlap with the start of the ORF to be overexpressed (see Figure. 2).

(16) For overexpression of the gene product Seq ID No 13, the native promoter in P. ciferrii was replaced with the PcENO1.sup.584-1 (Seq ID No 21) promoter fragment. In contrast, the particular native promoter in P. ciferrii was replaced with the PcTDH3.sup.420-1 promoter fragment (Seq ID No 22) for overexpression of the gene products Seq ID No 15 and Seq ID No 17.

(17) In principle, three different gene-specific primer pairs were used for constructing the particular overexpression cassettes. The primers were chosen so as to have at the 5 end regions of about 30-35 bps in length which were overlapping with the DNA elements to be fused.

(18) TABLE-US-00003 Primer 5 End overlapping with: P5 Cloning vector p426HXT7-6HIS P6 nat1 Resistance cassette (PCR amplicon of plasmid pCS.LoxP.nat1 with primers LPNTL.fw and LPNTL.rv) P9 nat1 Resistance cassette P10 5-End of the ORF to be overexpressed P7 3-End of the PcENO1.sup.-584-1 or PcTDH3.sup.-420-1 promoter fragment P8 Cloning vector p426HXT7-6HIS

(19) The nat1 resistance cassette was amplified using in each case the primer pair LPNTL.fw and LPNTL.rv, with plasmid pCS.LoxP.nat1 (Schorsch et al., 2009; Curr. Genet. 55, 381-9) being employed as template. The PCR products of primer pairs P5/P6, P7/P8, P9/P10 and LPNTL.fw/LPNTL.rv, together with the p426HXT7-6HIS plasmid previously linearized by digestion with Hpal and NgoMIV, were transformed into S. cerevisiae strain K26. The PCR products and the linearized vector were joined together in vivo by homologous recombination, causing the linearized vector to be re-circularized and able to be propagated in S. cerevisiae. Transformants obtained were selected by means of the marker gene (nat1) on YEPD plates with clonNAT, their DNA was isolated and transformed into E. coli, and the plasmids re-isolated therefrom were verified by restriction mapping or sequencing. The overexpression cassettes were amplified using the primer pair 426L.fw & 426R.rv in each case.

(20) Cf. FIG. 2 for Clarification.

(21) For combined overexpression of multiple genes, or for combining overexpressions of one or more target genes with one or more gene deletions, a marker rescue was performed after each step (deletion of a target gene or chromosomal integration of an overexpression cassette). This was accomplished by transformation with plasmid pCS.opt.Cre as described previously (Schorsch et al., Curr Genet. 2009 August; 55(4):381-9). Integration of the overexpression cassettes was verified by PCR analyses using genomic DNA of the transformants as template.

(22) The particular overexpression cassettes for enzymes encoded by the sequences Seq ID No 13, Seq ID No 15 and Seq ID No 17 were constructed using the primers listed in the table below. For each of the Seq IDs, the first two primers listed (LCB1.426L.fw and LCB1.LPNTL.rv or LCB2-426L.fw and LCB2-LPNTL.rv or SYR2oe.426L and SYR2oe.LPNTL.rv) were used in each case for amplification of PR. The next two primers listed (P-ENO.LPNTL.fw and LCB1.P-ENO.rv or TDH3-LPNTL.fw and P-TDH3.rv or TDH3-LPNTL.fw and P-TDH3.rv) were used for amplification of the particular PcENO1.sup.584-1 or PcTDH3.sup.420-1 promoter fragment. The next two primers listed (P-ENO.LCB1.fw and LCB1.426R.rv or LCB2.P-TDH3.fw and LCB2-426R.rv or SYR2oe.P-TDH3.fw and SYR2oe.426R) were used for amplification of the 5-ORF fragments of the target genes to be overexpressed in each case. The last two primers listed in each case (P-ENO.veri.rv and LCB1e.verisv or P-TDH3.pop.fw and LCB2e.verisv or P-TDH3.pop.fw and SYR2oe.veri.rv) are used for detecting integration or the wild-type allele.

(23) TABLE-US-00004 Gene Primername Sequence(5 -> 3) SeqIDNo13 LCB1.426L.fw GCTTTACACTTTATGCTTCCGGCTCCTATGTTGGGACT SeqIDNo63 GCTACACTCCAAATATG LCBI.LPNTL.rv TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCATAA SeqIDNo64 TAGAAGAAACACGTCAAATACC P-ENO.LPNTL.fw GCCGGTCTCCCTATAGTGAGTCGTATTAATTTCCAGAT SeqIDNo65 CAAACCACATCATGAG LCB1.P-ENO.rv GTAGCAGTGACGTTCATTGTGTAATGTGTATATGTTTT SeqIDNo66 ATC P-ENO.LCB1.fw CATATACACATTACACAATGAACGTCACTGCTACAAC SeqIDNo67 LCB1.426R.rv ATATCAGTTATTACCCTATGCGGTGTGAAATACACAAG SeqIDNo68 CACCAACACCATTAC P-ENO.veri.rv GTTGTGCGTGGCTTGAC SeqIDNo69 LCB1e.veri.rv ATAATACAGCACCACCAACTTC SeqIDNo70 SeqIDNo15 LCB2-426L.fw GCTTTACACTTTATGCTTCCGGCTCCTATGTTGGGCC SeqIDNo71 ATGAGATGACTTTGTACG LCB2-LPNTL.rv TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCAGTT SeqIDNo72 CTTGTTTGAATTCGCGTTTG TDH3-LPNTL.fw GTTATGAGCTCGAATTCATCGATGATATCAGGGACCG SeqIDNo73 TTAATTACCAACAATCTC P-TDH3.rv TGTTAATTAATTATTTGTTTGTTTG SeqIDNo74 LCB2.P-TDH3.fw ACAAACAAACAAACAAATAATTAATTAACAATGTCATT SeqIDNo75 GGTAATACCTCAAATAG LCB2-426R.rv ATATCAGTTATTACCCTATGCGGTGTGAAATACAAAGC SeqIDNo76 GGCTTGAGTACATGC P-TDH3.pop.fw AACTGACGTTTCAAGAACATC SeqIDNo77 LCB2e.veri.rv ATAAACTTGCATTTGTTGCATACC SeqIDNo78 SeqIDNo17 SYR2oe.426L GCTTTACACTTTATGCTTCCGGCTCCTATGTTGAAAGT SeqIDNo79 GTAAATAGACGTCATGAG SYR2oe.LPNTL.rv TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCACTG SeqIDNo80 TGTACTAAACGTGATAAATCC TDH3-LPNTL.fw GTTATGAGCTCGAATTCATCGATGATATCAGGGACCG SeqIDNo81 TTAATTACCAACAATCTC P-TDH3.rv TGTTAATTAATTATTTGTTTGTTTG SeqIDNo82 SYR2oe.P-TDH3.fw AAAACAAACAAACAAACAAATAATTAATTAACAATGAG SeqIDNo83 CTCTCATCAGTTTTTG SYR2oe.426R ATATCAGTTATTACCCTATGCGGTGTGAAATACAAGAC SeqIDNo84 GATGATGTCTTGAATG P-TDH3.pop.fw AACTGACGTTTCAAGAACATC SeqIDNo85 SYR2oe.veri.rv AGTAACAATTGCAGCAATACC SeqIDNo86
Production of Acetylated Sphingoid Bases by the Genetically Modified Strains

(24) Increased titres of acetylated sphingoid bases were achieved by the following genetic modifications:

(25) The tables below depict the titres of acetylated sphingoid bases (tetraacetylphytosphingosine, TAPS and optionally triacetylsphinganine, TriASa) of the different recombinant P. ciferrii strains after growth to the stationary phase in a shaker flask.

(26) Details (media used, growth conditions, extraction, quantification by HPLC analysis) are described in Schorsch et al., Curr Genet. 2009 August; 55(4):381-9. The strain employed in the present application corresponds to Pichia ciferri CS.PCPro2 designated in the above reference, which is also referred to for short as CS hereinbelow.

(27) First, the influence of deletions of various genes on the production of acetylated sphingoid bases was investigated. The results are depicted in the table below. Individually, deletion of PcSHM2 in particular was shown to markedly increase production of acetylated sphingoid bases. This effect was further enhanced by the combination with a PcSHM1 deletion. Further enhancement was achieved by an additional deletion of PcCHA1. This strain, with the relevant genotype of chat shm1 shm2, yielded by far the highest titre of 64 mg of TAPS * g-1 (CDW) plus 3 mg of TriASa * g-1 (CDW).

(28) Influence of Deletions of Various Genes on Production of Acetylated Sphingoid Bases:

(29) TABLE-US-00005 Relevant mg of TAPS * mg of TriASa * Strain genotype.sup.1 g.sup.1 (CDW) g.sup.1 (CDW) .sup.2 CS 21 CS.S1 shm1 20 CS.S2 shm2 26 CS.SS shm1shm2 42 CS.C cha1 23 CS.CS1 cha1 shm1 23 CS.CS2 cha1 shm2 29 CS.CSS cha1 shm1 shm2 65 3 .sup.1Relationship with SEQ-IDs: shm1, SEQ-ID No 1; shm2, SEQ-ID No 3 cha1, SEQ-ID No 5 .sup.2 Titres below 2 mg/g of cell dry mass are not shown.

(30) Next, the influence of various genetic modifications for enhancing enzyme activities were investigated, in the background of strain CS.CSS (cha1 shm1 shm2). For this purpose, the following genetic modifications, both individual and by way of selected combinations, were carried out in the strain CS.CSS:

(31) deletion of PcLCB4, Seq ID No 7

(32) deletion of PcDPL1, Seq ID No 9

(33) deletion of PcORM12, Seq ID No 11

(34) overexpression of PcLCB1 Seq ID No 13

(35) overexpression of PcLCB2 Seq ID No 15

(36) overexpression of PcSYR2 Seq ID No 17.

(37) Moreover, the effects of the deletions of PcLCB4 and PcDPL1 were also addressed alone, that is without combination with the cha1 shm1 shm2 genotype.

(38) To achieve additive or synergistic effects, a multiplicity of the genetic modifications promoting sphingoid base production were combined in different ways in a single strain. The strain with the following genotype turned out to be the best here: cha1 shm1 shm2 Icb4 orm12 TDH3p:LCB2 ENO1p:LCB1 TDH3p:SYR2.

(39) This strain produced in a shaker flask a titre of 199 mg of TAPS * g.sup.1 (CDW) (plus 12 mg of triacetylsphinganine (TriASa) * g.sup.1 (CDW), while the CS reference strain produced only 21 mg of TAPS * g.sup.1 (CDW).

(40) The results are depicted in the table below.

(41) Influence of Genetic Modifications of Sphingolipid Metabolism on Production of Acetylated Sphingoid Bases

(42) TABLE-US-00006 Relevant mg of TAPS * mg of TriASa * Strain genotype.sup.1; 2 g.sup.1 (CDW) g.sup.1 (CDW) .sup.3 CS 21 CS.L4 lcb4 54 CS.DPL1 dpl1 28 CS.S2 shm2 26 CS.SS shm1 shm2 42 CS.CSS cha1 shm1 shm2 65 3 CSS.L1 cha1 shm1 shm2 74 2 NO1p:LCB1 CSS.L2 cha1 shm1 shm2 82 4 TDH3p:LCB2 cha1 shm1 shm2 CSS.L1.L2 ENO1p:LCB1 102 8 TDH3p:LCB2 CSS.L4 cha1 shm1 shm2 116 8 lcb4 CSS.O cha1 shm1 shm2 104 6 orm12 CSS.D cha1 shm1 shm2 84 3 dpl1 CSS.L4.O cha1 shm1 shm2 172 8 lcb4 orm12 cha1 shm1 shm2 CSS.L4.O.L2 lcb4 orm12 182 16 TDH3p:LCB2 cha1 shm1 shm2 CSS.L4.O.L2.L1 lcb4 orm12 178 44 TDH3p:LCB2 ENO1p:LCB1 cha1 shm1 shm2 lcb4 orm12 CSS.L4.O.L2.L1.S2 TDH3p:LCB2 199 12 ENO1p:LCB1 TDH3p:SYR2 .sup.1Relationship with SEQ-IDs: shm1, SEQ-ID No 1; shm2, SEQ-ID No 3; chat, SEQ-ID No lcb4, SEQ-ID No 7; dpl1, SEQ-ID No 9; orm12, SEQ-ID No 11; LCB1, SEQ-ID No 13; LCB2, SEQ-ID No 15; SYR2, SEQ-ID No 17 .sup.2 Inactivated genes are listed in lower-case letters. Overexpressed genes are listed in capital letters and with the particular promoter (abbreviation p), under the control of which they are. .sup.3 Titres below 2 mg/g of cell dry mass are not shown.