# Patent

## Title
Pichia ciferrii cells and use 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.

## Background
FIELD OF THE INVENTION
The invention relates to genetically modified Pichia ciferrii cells, to the use thereof and to a method of producing sphingoid bases and sphingolipids.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The Sequence Listing in an ASCII text file, named 30707_SequenceListing.txt of 111 KB, created on Feb. 10, 2014, and submitted to the United States Patent and Trademark Office via EFS-Web, is incorporated herein by reference.
PRIOR ART
Since the beginning of the 1960s, Pichia ciferrii has been used in the production of sphingoid bases and sphingolipids, cf. Wickerham et al. 1960, J Bacteriol. 80, 484-91.
It is always worth improving the wild-type strain yields of sphingoid bases and sphingolipids.
It was the object of the invention to make available Pichia ciferrii cells which have increased productivity regarding sphingoid bases and sphingolipids.
DESCRIPTION OF THE INVENTION
Surprisingly, we found that the cells described hereinbelow having reduced, specific enzyme activities are capable of achieving the object of the invention.
The present invention therefore describes genetically modified Pichia ciferrii cells having, in comparison with their wild type, reduced activities of the enzymes as described in the present Claim 1.
The invention further relates to the use of the cells mentioned above and to a method of producing sphingoid bases and sphingolipids.
One advantage of the present invention is that of the cells according to the invention being able to grow to high cell densities.
Another advantage of the present invention is that of the cells producing markedly increased titres of acetylated sphingoid bases when grown in appropriate nutrient media.
A further advantage of the present invention is the high genetic stability of the strains which rules out reversion to the original genotype. Said high genetic stability moreover allows culturing in the absence of antibiotics, since there is no selection pressure to be maintained.
A further advantage of the present invention is the possibility of employing the cells in the biotechnological, environmentally friendly production of sphingoid bases from inexpensive and renewable raw materials.
The present invention relates to a Pichia ciferrii cell which is characterized in that the cell has, compared to its wild type, a reduced activity of at least one of the enzymes which are encoded by the intron-free nucleic acid sequences selected from the two groups A) and B) consisting of
A) Seq ID No 1, Seq ID No 3, Seq ID No 5, Seq ID No 7, Seq ID No 9, Seq ID No 11,
B) a sequence which is at least 80%, particularly preferably at least 90%, additionally preferably at least 95%, and most preferably at least 99%, identical to any of the sequences Seq ID No 1, Seq ID No 3, Seq ID No 5, Seq ID No 7, Seq ID No 9, Seq ID No 11.
In this context, group A) is the nucleic acid sequence group preferred according to the invention.
A “wild type” of a cell means in the context of the present invention preferably the parent strain from which the cell according to the invention has evolved through manipulation of the elements (for example the genes comprising the specified nucleic acid sequences coding for corresponding enzymes or the promoters present in corresponding genes and functionally linked to the nucleic acid sequences specified) that influence the activities of the enzymes encoded by the nucleic acid Seq ID No specified.
The term “activity of an enzyme” in connection with the enzyme encoded by Seq ID No 1 or 3 or by a sequence at least 80%, particularly preferably at least 90%, additionally preferably at least 95%, and most preferably at least 99%, identical to Seq ID No 1 or 3 is always understood as meaning the enzymic activity which catalyses the reaction 5,10-methylenetetrahydrofolate+L-glycine+H2O<=>tetrahydrofolate+L-serine. This activity is preferably determined by the method described in Schlüpen, 2003.
The term “activity of an enzyme” in connection with the enzyme encoded by Seq ID No 5 or by a sequence at least 80%, particularly preferably at least 90%, additionally preferably at least 95%, and most preferably at least 99%, identical to Seq ID No 5 is always understood as meaning the enzymic activity which catalyses the reaction L-serine<=>pyruvate+NH3.
This activity is preferably determined by the method described in Ramos and Wiame, Eur J Biochem. 1982 April; 123(3):571-6.
The term “activity of an enzyme” in connection with the enzyme encoded by Seq ID No 7 or by a sequence at least 80%, particularly preferably at least 90%, additionally preferably at least 95%, and most preferably at least 99%, identical to Seq ID No 7 is always understood as meaning the enzymic activity which catalyses the reaction ATP+sphinganine<=>ADP+sphinganine 1-phosphate.
This activity is preferably determined by the method described in Lanterman and Saba, Biochem J. 1998 Jun. 1; 332 (Pt 2):525-31.
The term “activity of an enzyme” in connection with the enzyme encoded by Seq ID No 9 or by a sequence at least 80%, particularly preferably at least 90%, additionally preferably at least 95%, and most preferably at least 99%, identical to Seq ID No 9 is always understood as meaning the enzymic activity which catalyses the reaction sphinganine 1-phosphate<=>phosphoethanolamine+palmitaldehyde.
This activity is preferably determined by the method described in Van Veldhoven and Mannaerts, J Biol Chem. 1991 Jul. 5; 266(19):12502-7.
The term “activity of an enzyme” in connection with the enzyme encoded by Seq ID No 11 or by a sequence at least 80%, particularly preferably at least 90%, additionally preferably at least 95%, and most preferably at least 99%, identical to Seq ID No 11 is understood as meaning the level of the rate of expression of the enzyme in question, in particular the intracellular concentration. This is determined by 2-D gel technology or Western-blot methods described below.
The wording “reduced activity compared to its wild type” means preferably an activity reduced by at least 50%, particularly preferably by at least 90%, additionally preferably by at least 99.9%, additionally even more preferably by at least 99.99% and most preferably by at least 99.999%, based on the wild-type activity.
Reduction of the particular activities of the cell according to the invention compared to its wild type is determined by above-described methods of determining the activity by employing, where possible, equal cell numbers/concentrations, the cells having been grown under identical conditions such as medium, gassing, agitation, for example. “Nucleotide identity” in relation to the sequences stated may be determined with the aid of known methods. In general, special computer programs with algorithms are used which take into account special requirements.
Preferred methods of determining identity firstly generate the highest agreement between the sequences to be compared. Computer programs for determining identity include but are not limited to the GCG program package including
GAP (Deveroy, J. et al., Nucleic Acid Research 12 (1984), page 387, Genetics Computer Group University of Wisconsin, Medicine (Wi), and
BLASTP, BLASTN and FASTA (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410. The BLAST program may be obtained from the National Center For Biotechnology Information (NCBI) and from other sources (BLAST manual, Altschul S. et al., NCBI NLM NIH Bethesda N. Dak. 22894; Altschul S. et al., supra).
The known Smith-Waterman algorithm may also be used for determining nucleotide identity.
Preferred parameters for determining “nucleotide identity” when using the BLASTN program (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410) are:
















Expect Threshold:
10



Word size:
28



Match Score:
1



Mismatch Score:
−2



Gap costs:
linear










The above parameters are the default parameters in nucleotide sequence comparison.
The GAP program can also be used with the above parameters.
An identity of 80% according to the above algorithm means in the context of the present invention 80% identity. The same applies to higher identities.
The term “which are encoded by the intron-free nucleic acid sequences” clearly sets out that a sequence comparison involving the sequences stated herein requires the nucleic acid sequences to be compared to be cleared of any introns beforehand.
All percentages (%) are percentages by mass, unless stated otherwise.
Cells preferred according to the invention are characterized in that reduction of the enzymic activity is achieved by modifying at least one gene comprising any of the sequences selected from the nucleic acid sequence groups A) and B) specified hereinabove, the modification being selected from the group comprising, preferably consisting of, insertion of foreign DNA into the gene, deletion of at least parts of the gene, point mutations in the gene sequence, and exposing the gene to the influence of RNA interference, or replacement of parts of the gene with foreign DNA, in particular of the promoter region.
Foreign DNA is understood in this connection as meaning any DNA sequence which is “foreign” to the gene (and not to the organism), i.e. even Pichia ciferrii endogenous DNA sequences may act as “foreign DNA” in this connection.
In this context, particular preference is given to the gene being disrupted by insertion of a selection marker gene, thus the foreign DNA being a selection marker gene, in particular one comprising a sequence coding for the Streptomyces noursei nat1 gene, which sequence is preferably flanked by the sequence of the Pichia PDA1 promoter and the sequence of the Pichia TEF terminator, as described in Schorsch et al., 2009; Current Genetics (2009), 55(4), 381-389, for example, the sequence coding for the Streptomyces noursei nat1 gene preferably being codon-optimized for P. ciferrii, with said insertion preferably having been accomplished by homologous recombination into the gene locus.
In this connection, it may be advantageous for the selection marker gene to be extended by further functionalities which in turn make subsequent removal from the gene possible, which can be achieved, for example, by recombination systems foreign to the organism, such as a Cre/loxP system or FRT (flippase recognition target) system, or the organism's own homologous recombination system.
Preference is given according to the invention to the cell having, compared to its wild type, a combination of reduced activities of the enzymes which are encoded by the intron-free nucleic acid sequences:
Seq ID No 1 or its group B analogue;
Seq ID No 3 or its group B analogue;
Seq ID No 5 or its group B analogue;
Seq ID No 7 or its group B analogue;
Seq ID No 9 or its group B analogue;
Seq ID No 11 or its group B analogue;
Seq ID No 1 or its group B analogue and Seq ID No 3 or its group B analogue;
Seq ID No 1 or its group B analogue and Seq ID No 5 or its group B analogue;
Seq ID No 3 or its group B analogue and Seq ID No 5 or its group B analogue;
Seq ID No 1 or its group B analogue and Seq ID No 3 or its group B analogue and Seq ID No 5 or its group B analogue;
Seq ID No 1 or its group B analogue and Seq ID No 3 or its group B analogue and Seq ID No 5 or its group B analogue and Seq ID No 7 or its group B analogue;
Seq ID No 1 or its group B analogue and Seq ID No 3 or its group B analogue and Seq ID No 5 or its group B analogue and Seq ID No 11 or its group B analogue;
Seq ID No 1 or its group B analogue and Seq ID No 3 or its group B analogue and Seq ID No 5 or its group B analogue and Seq ID No 7 or its group B analogue and Seq ID No 11 or its group B analogue;
In connection with the combinations listed above, preference is given to reducing the enzyme activities encoded by the members of group A.
Cells preferred according to the invention are characterized in that the Pichia ciferrii cell derives from strains selected from the group consisting of Pichia ciferrii NRRL Y-1031 F-60-10, the Pichia ciferrii strains disclosed in the examples of WO 95/12683, and the strain Pichia ciferrii CS.PCΔPro2, described in Schorsch et al., 2009, Curr Genet. 55, 381-9.
Cells preferred according to the invention are characterized in that the cell has, compared to its wild type, an increased enzymic activity of at least one of the enzymes selected from
an enzyme E1, which catalyses the reaction of serine and palmitoyl-CoA to give 3-ketosphinganine, in particular a serine palmitoyl transferase, in particular those encoded by Seq ID No 13 and/or Seq ID No 15,
an enzyme E2, which catalyses the reaction of sphinganine to phytosphingosine, in particular a sphinganine C4-hydroxylase, in particular that encoded by Seq ID No 17.
The term “activity of an enzyme” in connection with the enzyme E1 is always understood as meaning the enzymic activity which catalyses the reactions of palmitoyl-CoA+L-serine<=>CoA+3-dehydro-D-sphinganine+CO2.
This activity is preferably determined by the method described in Zweerink et al., J Biol Chem. 1992 Dec. 15; 267(35):25032-8.
The term “activity of an enzyme” in connection with the enzyme E2 is always understood as meaning the enzymic activity which catalyses the reaction sphinganine+NADPH+H++O2<=>phytosphingosine+NADP++H2O.
This activity is preferably determined by the method described in Grilley et al., J Biol Chem. 1998 May 1; 273(18):11062-8.
The term “increased activity of an enzyme” as used hereinabove and in the comments below in the context of the present invention is preferably understood as meaning increased intracellular activity.
The following comments regarding the increase in enzyme activity in cells apply both to the increase in activity of the enzymes E1 to 2 and to all enzymes specified hereinbelow, whose activity may be increased where appropriate.
In principle, an increase in enzymic activity can be achieved by increasing the copy number of the gene sequence(s) coding for the enzyme, by using a strong promoter, by altering the codon usage of the gene, by increasing in various ways the half life of the mRNA or of the enzyme, by modifying the regulation of expression of the gene, or by utilizing a gene or allele coding for a corresponding enzyme with increased activity, and by combining these measures where appropriate. Cells genetically modified according to the invention are generated, for example, by transformation, transduction, conjugation or a combination of these methods with a vector containing the desired gene, an allele of this gene or parts thereof and a promoter enabling the gene to be expressed. Heterologous expression is achieved in particular by integrating the gene or alleles into the chromosome of the cell or an extrachromosomally replicating vector.
An overview of the options for increasing enzyme activity in cells is given for pyruvate carboxylase by way of example in DE-A-100 31 999 which is hereby incorporated by way of reference and whose disclosure forms part of the disclosure of the present invention regarding the options for increasing enzyme activity in cells.
Expression of the enzymes or genes specified hereinabove and all enzymes or genes specified below is detectable with the aid of one- and two-dimensional protein gel fractionation and subsequent optical identification of protein concentration in the gel using appropriate evaluation software.
If the increase in an enzyme activity is based exclusively on an increase in expression of the corresponding gene, the increase in said enzyme activity can be quantified simply by comparing the one- or two-dimensional protein fractionations of wild-type and genetically modified cells. A customary method of preparing the protein gels in the case of bacteria and of identifying the proteins is the procedure described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001). The protein concentration may likewise be analysed by Western-blot hybridization with an antibody specific to the protein to be detected (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989) and subsequent optical evaluation using appropriate software for concentration determination (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647).
Preference is given according to the invention to the cell which, compared to its wild type, has an increased enzymic activity of enzyme E1 having, in comparison with its wild type, a combination of reduced activities of the enzymes encoded by the intron-free nucleic acid sequences:
in the combination of Seq ID No 1 or its group B analogue and Seq ID No 3 or its group B analogue and Seq ID No 5 or its group B analogue,
or in the combination of Seq ID No 1 or its group B analogue and Seq ID No 3 or its group B analogue and Seq ID No 5 or its group B analogue and Seq ID No 7 or its group B analogue and Seq ID No 11 or its group B analogue.
Preference is given according to the invention to the cell which, compared to its wild type, has an increased enzymic activity of enzymes E1 and E2 having, in comparison with its wild type, a combination of reduced activities of the enzymes encoded by the intron-free nucleic acid sequences:
Seq ID No 1 or its group B analogue and Seq ID No 3 or its group B analogue and Seq ID No 5 or its group B analogue and Seq ID No 7 or its group B analogue and Seq ID No 11 or its group B analogue.
In one variant embodiment, P. ciferrii cells according to the invention are such as those described in WO2006048458 and WO2007131720 and additionally having the changes in enzymic activities described above in connection with the present cells according to the invention.
A further contribution to achieving the object of the invention is made by using the cells according to the invention for producing sphingoid bases and sphingolipids.
The term “sphingoid bases” in the context of the present invention is understood as meaning phytosphingosine, sphingosine, sphingadienine, 6-hydroxysphingosine and sphinganine (dihydrosphingosine), also in the acetylated form, such as for example tetraacetylphytosphingosine, triacetylphytosphingosine, diacetylphytosphingosine, O-acetylphytosphingosine, triacetylsphinganine, diacetylsphinganine, O-acetylsphinganine, triacetylsphingosine, diacetylsphingosine, O-acetylsphingosine, tetraacetyl-6-hydroxysphingosine, triacetyl-6-hydroxysphingosine, diacetyl-6-hydroxysphingosine, O-acetyl-6-hydroxysphingosine, triacetylsphingadienine, diacetylsphingadienine, O-acetylsphingadienine.
The term “sphingolipids” in the context of the present invention is understood as meaning compounds which comprise sphingoid bases covalently linked via an amide bond to a fatty acid. The fatty acid may be saturated or mono- or polyunsaturated. The fatty acid side chain may vary in length. The fatty acid side chain may also have functional groups such as hydroxy groups. Sphingolipids include, for example, phytoceramides, ceramides and dihydroceramides, and the more complex glucosylceramides (cerebrosides) and the inositol phosphorylceramides, mannosylinositol phosphorylceramides and mannosyldiinositol phosphorylceramides. The sphingolipids here also include sphingoid bases linked via an amide bond to an acetyl radical, such as for example N-acetylphytosphingosine, N-acetylsphinganine, N-acetylsphingosine, N-acetyl-6-hydroxysphingosine. These compounds are also known by the term of short-chain ceramides.
The use of the cells according to the invention for producing sphingoid bases and sphingolipids selected from the group consisting of phytosphingosine, sphingosine, sphingadienine, 6-hydroxysphingosine, sphinganine (dihydrosphingosine), tetraacetylphytosphingosine (TAPS), triacetylphytosphingosine, diacetylphytosphingosine, O-acetylphytosphingosine, N-acetylphytosphingosine, triacetylsphinganine (TriASa), diacetylsphinganine, O-acetylsphinganine, N-acetylsphinganine, triacetylsphingosine (TriASo), diacetylsphingosine, O-acetylsphingosine, N-acetylsphingosine, tetraacetyl-6-hydroxysphingosine, triacetyl-6-hydroxysphingosine, diacetyl-6-hydroxysphingosine, O-acetyl-6-hydroxysphingosine, N-acetyl-6-hydroxysphingosine, triacetylsphingadienine, diacetylsphingadienine, O-acetylsphingadienine is particularly advantageous. Very particular preference is given to the use of the cells according to the invention for producing tetraacetylphytosphingosine (TAPS).
A use which is preferred according to the invention is characterized according to the invention in that cells preferred according to the invention, as described above, are used.
P. ciferrii cells which are used in particular for producing the above-described sphingosine and sphinganine derivatives are such as those described in WO2006048458 and WO2007131720 and additionally having the changes in enzymic activities described above in connection with the present cells according to the invention.
A further contribution to achieving the object of the invention is made by a method of producing the previously described cell according to the invention, said method comprising the steps of:
I) providing a Pichia ciferrii cell, and
II) modifying at least one gene comprising any of the sequences selected from the nucleic acid sequence groups A) and B) specified in Claim 1 by
insertion of foreign DNA, in particular DNA coding for a selection marker gene, preferably one which can be removed without leaving a trace and which leaves a deletion in the target gene, into the gene,
deletion of at least parts of the gene,
point mutations in the gene sequence,
exposing the gene to the influence of RNA interference, and
replacement of parts of the gene with foreign DNA, in particular of the promoter region.
A further contribution to achieving the object of the invention is made by a method of producing sphingoid bases and sphingolipids, said method comprising the steps of
a) contacting the cell according to the invention with a medium including a carbon source,
b) culturing the cell 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.
Methods preferred according to the invention employ cells specified above as preferred according to the invention.
Carbon sources which may be employed are carbohydrates, such as for example glucose, fructose, glycerol, sucrose, maltose, molasses, or else alcohols, such as for example ethanol, and organic acids, such as for example acetate. Nitrogen sources which may be employed are for example ammonia, ammonium sulphate, ammonium nitrate, ammonium chloride, organic nitrogen compounds (such as yeast extract, malt extract, peptone, corn steep liquor). Inorganic compounds, such as for example phosphate salts, magnesium salts, potassium salts, zinc salts, iron salts and others, may also be employed.
Suitable culturing conditions for Pichia ciferrii are known to the skilled worker from WO2006048458 and WO2007131720, for example.
The method according to the invention is particularly suitable for producing tetraacetylphytosphingosines (TAPS).
The Examples listed hereinbelow describe the present invention by way of example, but the embodiments specified in said examples are not intended to limit the invention, the scope of use of which ensues from the entire description and the claims.



The following figures are part of the Examples:
FIG. 1: Design principle of gene deletion cassettes
FIG. 2: Design principle of overexpression cassettes



EXAMPLES
Construction of Gene Deletion Cassettes
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.
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.
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.
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:











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









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′→3′ orientation).
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. 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.
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.
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.












Gene
Primer name
Sequence (5′->3′)







Seq ID No 1
SH11
CAAAAAGTTAACATGCATCACCATCACCATCACACT



Seq ID No 27
AACCCAACTAGGCTCATTAAC



SH12
GTTATCTGCAGGTTACCCAGTGGTACGAAGCGCCA



Seq ID No 28
TCAGCCATTTCTGGATCAATTTC



SH13
TGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCAT



Seq ID No 29
CCAGTTCCAGGTGAATTATAAG



SH14
TAACTAATTACATGACTCGAGGTCGACGGTATCCCA



Seq ID No 30
TACTATGCTTGGCATCTTAAAC



SHMT1.pop-in.fw
TTGATAGGGCAAATTCTCCAAC



Seq ID No 31




SHMT1.veri.rv
TTCACCTGGATAACCTTCTG



Seq ID No 32



 


Seq ID No 3
SH21
CAAAAAGTTAACATGCATCACCATCACCATCACATG



Seq ID No 33
TCCTTGCAGGTGGTATTC



SH22
TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCAG



Seq ID No 34
GTAAAGCGTATGGCATGTTG



SH23
CTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCG



Seq ID No 35
CTGGTGAATTCCCATTATCTG



SH24
TAACTAATTACATGACTCGAGGTCGACGGTATCCAT



Seq ID No 36
AACCATCTAAAGCATTATAGTC



SHMT2.pop-in.fw
AAGTTTCAGCAAATGGTTTGAC



Seq ID No 37




SHMT2.veri.rv
TATCTTGCACCTGGATAACC



Seq ID No 38



 


Seq ID No 5
C1
CAAAAAGTTAACATGCATCACCATCACCATCACAAT



Seq ID No 39
CTAAGAGGTAAAGTTCAACATTC



C2
GTTATCTGCAGGTTACCCAGTGGTACGAAGCGCCA



Seq ID No 40
TTGGTTTGCCGTGTGGATTG



C3
CTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCG



Seq ID No 41 
GAGTTCAACAACCGTTCAAG



C4
TAACTAATTACATGACTCGAGGTCGACGGTATCATG



Seq ID No 42
AAGTTGATGCTGCTTTGG



CHA1.pop-in.fw
ATTTAGAAGCTAGAGGTTCAGAAAG



Seq ID No 43




CHA1.veri.rv
TAGAAGAATGACCATGCCATATAG



Seq ID No 44



 


Seq ID No 7
HXT7-LCB4.fw
TTTTAATTTTAATCAAAAAGTTAACATGCATCACCAT



Seq ID No 45
CACCATCACACTCACAGAGTCAACTCCTGTATATTC



LCB4.HXT7.rv
TGAATGTAAGCGTGACATAACTAATTACATGACTCG



Seq ID No 46
AGGTCGACGGTATCTCTGGCGGTATTGAACTTTGT




GGAG



LCB4. rv
GTTATCTGCAGGTTACCCAGTGGTAAAGTGTATGGA



Seq ID No 47
TGGGTTGAAGTATGTCTTTATATC



LCB4.fw
ACGAAGTTATGAGCTCGAATTCATCGATGCTACCCG



Seq ID No 48
GTGCTGCAAAGACTTTACTAAG



LCB4.pop-in.fw
GTGAATGGTTAATAGTGCGCTATG



Seq ID No 49




LCB4.veri.rv
CTAACAAATACCACTTCGACATCAG



Seq ID No 50



 


Seq ID No 9
HXT7-DPL1.fw
TTTTAATTTTAATCAAAAAGTTAACATGCATCACCAT



Seq ID No 51
CACCATCACACCTTCCGTGAGATTTCCCTTGTTTAC



DPL1.rv2
TATACGAAGTTATCTGCAGGTTACCCAGTGGTATAA



Seq ID No 52
CCCATAACCAGTGATGTTAACC



DPL1.fw2
GAAGTTATGAGCTCGAATTCATCGATGACCACTGGT



Seq ID No 53
GTTGTTGATCG



CYC-DPL1.rv
TGAATGTAAGCGTGACATAACTAATTACATGACTCG



Seq ID No 54
AGGTCGACGGTATCCGACGGTAATGAGGATGTAAA




TGAG



DPL1.pop-in.fw
AAACAAGAGCAGCATGCAACTTGAG



Seq ID No 55




DPL1.veri.rv
AGTGACACCAGGAACTCTAAAG



Seq ID No 56



 


Seq ID No 11
ORM-426L.fw
GCTTTACACTTTATGCTTCCGGCTCCTATGTTGAAC



Seq ID No 57
TATGTCAATATCGATCGTATG



ORM-LPNTL.rv
TATCTGCAGGTTACCCAGTGGTACGAAGCGCCAAA



Seq ID No 58
CAGAAATTGGTTCATGTGTTG



ORM-LPNTL.fw2
GCCGGTCTCCCTATAGTGAGTCGTATTAATTTCTGG



Seq ID No 59
TGTACCAATTTGGTTATTTC



ORM-426R.rv
ATATCAGTTATTACCCTATGCGGTGTGAAATACACA



Seq ID No 60
AGTACAACAACAACAGATTTAG



ORM1.pop-in.fw
TACCCACCTTTGACATAATCAG



Seq ID No 61




ORM.veri.rv
ATTCAAATGGCGTACCTTTAAC



Seq ID No 62









Construction of Overexpression Cassettes
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 FIG. 2).
For overexpression of the gene product Seq ID No 13, the native promoter in P. ciferrii was replaced with the PcENO1−584-1 (Seq ID No 21) promoter fragment. In contrast, the particular native promoter in P. ciferrii was replaced with the PcTDH3−420-1 promoter fragment (Seq ID No 22) for overexpression of the gene products Seq ID No 15 and Seq ID No 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.











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 PcENO1-584-1 or PcTDH3-420-1 promoter fragment


P8
Cloning vector p426HXT7-6HIS









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 HpaI 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.
Cf. FIG. 2 for clarification.
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.
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 PcENO1−584-1 or PcTDH3−420-1 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 LCB1üe.veri.rv or P-TDH3.pop.fw and LCB2üe.veri.rv or P-TDH3.pop.fw and SYR2oe.veri.rv) are used for detecting integration or the wild-type allele.












Gene
Primer name
Sequence (5′->3′)







Seq ID No 13
LCB1.426L.fw
GCTTTACACTTTATGCTTCCGGCTCCTATGTTGGGACTGCT



Seq ID No 63
ACACTCCAAATATG



LCB1.LPNTL.rv
TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCATAATAG



Seq ID No 64
AAGAAACACGTCAAATACC



P-ENO.LPNTL.fw
GCCGGTCTCCCTATAGTGAGTCGTATTAATTTCCAGATCAA



Seq ID No 65
ACCACATCATGAG



LCB1.P-ENO.rv
GTAGCAGTGACGTTCATTGTGTAATGTGTATATGTTTTATC



Seq ID No 66




P-ENO.LCB1.fw
CATATACACATTACACAATGAACGTCACTGCTACAAC



Seq ID No 67




LCB1.426R.rv
ATATCAGTTATTACCCTATGCGGTGTGAAATACACAAGCAC



Seq ID No 68
CAACACCATTAC



P-ENO.veri.rv
GTTGTGCGTGGCTTGAC



Seq ID No 69




LCB1üe.verisv
ATAATACAGCACCACCAACTTC



Seq ID No 70



 


Seq ID No 15
LCB2-426L.fw
GCTTTACACTTTATGCTTCCGGCTCCTATGTTGGGCCATGA



Seq ID No 71
GATGACTTTGTACG



LCB2-LPNTL.rv
TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCAGTTCTT



Seq ID No 72
GTTTGAATTCGCGTTTG



TDH3-LPNTL.fw
GTTATGAGCTCGAATTCATCGATGATATCAGGGACCGTTAA



Seq ID No 73
TTACCAACAATCTC



P-TDH3.rv
TGTTAATTAATTATTTGTTTGTTTG



Seq ID No 74




LCB2.P-TDH3.fw
ACAAACAAACAAACAAATAATTAATTAACAATGTCATTGGTA



Seq ID No 75
ATACCTCAAATAG



LCB2-426R.rv
ATATCAGTTATTACCCTATGCGGTGTGAAATACAAAGCGGC



Seq ID No 76
TTGAGTACATGC



P-TDH3.pop.fw
AACTGACGTTTCAAGAACATC



Seq ID No 77




LCB2üe.veri.rv
ATAAACTTGCATTTGTTGCATACC



Seq ID No 78



 


Seq ID No 17
SYR2oe.426L
GCTTTACACTTTATGCTTCCGGCTCCTATGTTGAAAGTGTA



Seq ID No 79
AATAGACGTCATGAG



SYR2oe.LPNTL.rv
TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCACTGTGT



Seq ID No 80
ACTAAACGTGATAAATCC



TDH3-LPNTL.fw
GTTATGAGCTCGAATTCATCGATGATATCAGGGACCGTTAA



Seq ID No 81
TTACCAACAATCTC



P-TDH3.rv




Seq ID No 82
TGTTAATTAATTATTTGTTTGTTTG



SYR2oe.P-TDH3.fw
AAAACAAACAAACAAACAAATAATTAATTAACAATGAGCTCT



Seq ID No 83
CATCAGTTTTTG



SYR2oe.426R
ATATCAGTTATTACCCTATGCGGTGTGAAATACAAGACGAT



Seq ID No 84
GATGTCTTGAATG



P-TDH3.pop.fw
AACTGACGTTTCAAGAACATC



Seq ID No 85




SYR2oe.veri.rv
AGTAACAATTGCAGCAATACC



Seq ID No 86









Production of Acetylated Sphingoid Bases by the Genetically Modified Strains
Increased titres of acetylated sphingoid bases were achieved by the following genetic modifications:
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.
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 ciferrii CS.PCΔPro2 designated in the above reference, which is also referred to for short as “CS” hereinbelow.
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 cha1 shm1 shm2, yielded by far the highest titre of 64 mg of TAPS*g−1 (CDW) plus 3 mg of TriASa*g−1 (CDW).
Influence of deletions of various genes on production of acetylated sphingoid bases:














Relevant
mg of TAPS *
mg of TriASa *


Strain
genotype1
g−1 (CDW)
g−1 (CDW)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





1Relationship with SEQ-IDs: shm1, SEQ-ID No 1; shm2, SEQ-ID No 3; cha1, SEQ-ID No 5


2Titres below 2 mg/g of cell dry mass are not shown.






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:
deletion of PcLCB4, Seq ID No 7
deletion of PcDPL1, Seq ID No 9
deletion of PcORM12, Seq ID No 11
overexpression of PcLCB1 Seq ID No 13
overexpression of PcLCB2 Seq ID No 15
overexpression of PcSYR2 Seq ID No 17.
Moreover, the effects of the deletions of PcLCB4 and PcDPL1 were also addressed alone, that is without combination with the cha1 shm1 shm2 genotype.
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 lcb4 orm12 TDH3p:LCB2 ENO1p:LCB1 TDH3p:SYR2. This strain produced in a shaker flask a titre of 199 mg of TAPS*g−1 (CDW) (plus 12 mg of triacetylsphinganine (TriASa)*g−1 (CDW), while the CS reference strain produced only 21 mg of TAPS*g−1 (CDW).
The results are depicted in the table below.
Influence of Genetic Modifications of Sphingolipid Metabolism on Production of Acetylated Sphingoid Bases














Relevant
mg of TAPS *
mg of TriASa *


Strain
genotype1; 2
g−1 (CDW)
g−1 (CDW)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


CSS.L1.L2
cha1 shm1 shm2
102
8



ENO1p:LCB1



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


CSS.L4.O.L2
cha1 shm1 shm2
182
16



lcb4 orm12



TDH3p:LCB2


CSS.L4.O.L2.L1
cha1 shm1 shm2
178
44



lcb4 orm12



TDH3p:LCB2



ENO1p:LCB1


CSS.L4.O.L2.L1.S2
cha1 shm1 shm2
199
12



lcb4 orm12



TDH3p:LCB2



ENO1p:LCB1



TDH3p:SYR2





1Relationship with SEQ-IDs: shm1, SEQ-ID No 1; shm2, SEQ-ID No 3; cha1, 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


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.


3Titres below 2 mg/g of cell dry mass are not shown.

## Claims

1. An genetically modified Pichia ciferrii cell, wherein said genetically modified Pichia ciferrii cell has, compared to its wild type, a reduced activity of an E1 enzyme encoded by the intron-free nucleic acid sequences selected from the group consisting of
A) SEQ ID NO: 1, and
B) a sequence which is at least 90% identical to SEQ ID NO: 1, wherein said activity of said E1 enzyme is the catalysis of the reaction 5,10-methylenetetrahydrofolate+L-glycine+H2O to tetrahydrofolate+L-serine.

2. The genetically modified Pichia ciferrii cell according to claim 1, wherein said reduction of enzymatic activity is achieved by modifying a gene comprising the nucleic acid sequence set forth in SEQ ID NO: 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.PCΔPro2.

5. The genetically modified Pichia ciferrii cell according to claim 1, characterized in that the cell further has, compared to its wild type, an increased enzymatic activity of an enzyme E2, wherein said enzyme E2 catalyzes the reaction of sphinganine to phytosphingosine.

6. A method of producing an isolated Pichia ciferrii cell according to claim 1, said method comprising the steps of:
I) providing an isolated Pichia ciferrii cell, and
II) modifying at least one gene comprising any of the sequences selected from the nucleic acid sequence groups A) and B) specified in claim 1 by insertion of foreign DNA into the gene, deletion of at least parts of the gene, point mutations in the gene sequence, exposing the gene to the influence of RNA interference, and replacement of parts of the gene with foreign DNA.

7. A method of producing sphingoid bases and sphingolipids, said method comprising the steps of
acting the cell according to claim 1 with a medium including a carbon source,
b) culturing the cell 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 2, wherein said replacement of a portion of the gene with foreign DNA is of the promoter region.

9. The genetically modified Pichia ciferrii cell according to claim 5, wherein said enzyme E2 is a sphinganine C4-hydroxylase.

10. The genetically modified Pichia ciferrii cell according to claim 9, wherein said sphinganine C4-hydroxylase is encoded by SEQ ID NO: 17.

11. The method according to claim 6, wherein said foreign DNA is DNA coding for a selection marker gene.

12. The method according to claim 6, wherein said replacement of parts of the gene with foreign DNA is of the promoter region.

13. The genetically modified Pichia ciferrii cell of claim 1, wherein said intron-free nucleic acid sequences consists of a sequence that is at least 95% identical to SEQ ID NO: 1.

14. The genetically modified Pichia ciferrii cell of claim 13, wherein said intron-free nucleic acid sequences consists of a sequence that is at least 99% identical to SEQ ID NO: 1.