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Knockout of the DNA ligase IV homolog gene in the sphingoid base producing yeast Pichia ciferrii significantly increases gene targeting efficiency

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Abstract

The yeast Pichia ciferrii produces large quantities of the sphingoid base tetraacetyl phytosphingosine (TAPS) and is an interesting platform organism for the biotechnological production of sphingolipids and ceramides. Ceramides have attracted great attention as a specialty ingredient for moisture retention and protection of the skin in the cosmetics industry. First attempts have been started to metabolically engineer P. ciferrii for improved production of TAPS and other sphingoid bases. However, rational metabolic engineering of P. ciferrii is difficult due to a low gene targeting efficiency. In eukaryotes, two major pathways coexist, which are responsible for genomic DNA integration, homologous recombination (HR) and non-homologous end joining (NHEJ). Integration via HR is targeted, while NHEJ involves ectopic (non-targeted) integration depending on a ligation step mediated by DNA ligase IV (Lig4). Here, we demonstrate a dramatical increase in gene targeting efficiency in a P. ciferrii lig4 knockout strain, deficient in NHEJ. Furthermore, a quick and easy to use freeze–thaw method was developed to transform P. ciferrii with high efficiency. Owing to the ability of targeting genomic DNA integration our results pave the way for further genetic and metabolic engineering approaches with P. ciferrii by means of knocking out or overexpressing predestinated genes.

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References

  • Adachi N, Ishino T, Ishii Y, Takeda S, Koyama H (2001) DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: implications for DNA double-strand break repair. Proc Natl Acad Sci USA 98:12109–12113

    Article  PubMed  CAS  Google Scholar 

  • Bae JH, Sohn JH, Park CS, Rhee JS, Choi ES (2003) Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast Pichia ciferrii. Appl Environ Microbiol 69:812–819

    Article  PubMed  CAS  Google Scholar 

  • Baudin A, Ozier-Kalogeropoulos O, Denouel A, Lacroute F, Cullin C (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:3329–3330

    Article  PubMed  CAS  Google Scholar 

  • Boles E, Lehnert W, Zimmermann FK (1993) The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant. Eur J Biochem 217:469–477

    Article  PubMed  CAS  Google Scholar 

  • Boulton SJ, Jackson SP (1996a) Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double-strand break rejoining and in telomeric maintenance. Nucleic Acids Res 24:4639–4648

    Article  PubMed  CAS  Google Scholar 

  • Boulton SJ, Jackson SP (1996b) Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J 15:5093–5103

    PubMed  CAS  Google Scholar 

  • Byrne KP, Wolfe KH (2005) The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Res 15:1456–1461

    Article  PubMed  CAS  Google Scholar 

  • Cho S, Kim HH, Lee MJ, Lee S, Park CS, Nam SJ, Han JJ, Kim JW, Chung JH (2008) Phosphatidylserine prevents UV-induced decrease of type I procollagen and increase of MMP-1 in dermal fibroblasts and human skin in vivo. J Lipid Res 49:1235–1245

    Google Scholar 

  • Critchlow SE, Bowater RP, Jackson SP (1997) Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol 7:588–598

    Article  PubMed  CAS  Google Scholar 

  • Daley JM, Palmbos PL, Wu D, Wilson TE (2005) Nonhomologous end joining in yeast. Annu Rev Genet 39:431–451

    Article  PubMed  CAS  Google Scholar 

  • Dohmen RJ, Strasser AW, Honer CB, Hollenberg CP (1991) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7:691–692

    Article  PubMed  CAS  Google Scholar 

  • Dore AS, Furnham N, Davies OR, Sibanda BL, Chirgadze DY, Jackson SP, Pellegrini L, Blundell TL (2006) Structure of an Xrcc4-DNA ligase IV yeast ortholog complex reveals a novel BRCT interaction mode. DNA Repair (Amst) 5:362–368

    Article  CAS  Google Scholar 

  • Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145

    Article  PubMed  CAS  Google Scholar 

  • Fisher TS, Taggart AK, Zakian VA (2004) Cell cycle-dependent regulation of yeast telomerase by Ku. Nat Struct Mol Biol 11:1198–1205

    Article  PubMed  CAS  Google Scholar 

  • Giaever G, Chu AM, Ni L, Connelly C, Riles L, Veronneau S, Dow S, Lucau-Danila A, Anderson K, Andre B, Arkin AP, Astromoff A, El-Bakkoury M, Bangham R, Benito R, Brachat S, Campanaro S, Curtiss M, Davis K, Deutschbauer A, Entian KD, Flaherty P, Foury F, Garfinkel DJ, Gerstein M, Gotte D, Guldener U, Hegemann JH, Hempel S, Herman Z, Jaramillo DF, Kelly DE, Kelly SL, Kotter P, LaBonte D, Lamb DC, Lan N, Liang H, Liao H, Liu L, Luo C, Lussier M, Mao R, Menard P, Ooi SL, Revuelta JL, Roberts CJ, Rose M, Ross-Macdonald P, Scherens B, Schimmack G, Shafer B, Shoemaker DD, Sookhai-Mahadeo S, Storms RK, Strathern JN, Valle G, Voet M, Volckaert G, Wang CY, Ward TR, Wilhelmy J, Winzeler EA, Yang Y, Yen G, Youngman E, Yu K, Bussey H, Boeke JD, Snyder M, Philippsen P, Davis RW, Johnston M (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391

    Article  PubMed  CAS  Google Scholar 

  • Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96

    Article  PubMed  CAS  Google Scholar 

  • Grawunder U, Wilm M, Wu X, Kulesza P, Wilson TE, Mann M, Lieber MR (1997) Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 388:492–495

    Article  PubMed  CAS  Google Scholar 

  • Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524

    Article  PubMed  CAS  Google Scholar 

  • Hamacher T, Becker J, Gardonyi M, Hahn-Hagerdal B, Boles E (2002) Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 148:2783–2788

    PubMed  CAS  Google Scholar 

  • Hastings PJ, McGill C, Shafer B, Strathern JN (1993) Ends-in vs. ends-out recombination in yeast. Genetics 135:973–980

    PubMed  CAS  Google Scholar 

  • Imokawa G, Akasaki S, Hattori M, Yoshizuka N (1986) Selective recovery of deranged water-holding properties by stratum corneum lipids. J Invest Dermatol 87:758–761

    Article  PubMed  CAS  Google Scholar 

  • Ishibashi K, Suzuki K, Ando Y, Takakura C, Inoue H (2006) Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci U S A 103:14871–14876

    Article  PubMed  CAS  Google Scholar 

  • Kooistra R, Hooykaas PJ, Steensma HY (2004) Efficient gene targeting in Kluyveromyces lactis. Yeast 21:781–792

    Article  PubMed  CAS  Google Scholar 

  • Kramer KM, Brock JA, Bloom K, Moore JK, Haber JE (1994) Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events. Mol Cell Biol 14:1293–1301

    PubMed  CAS  Google Scholar 

  • Leung W, Malkova A, Haber JE (1997) Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction. Proc Natl Acad Sci USA 94:6851–6856

    Article  PubMed  CAS  Google Scholar 

  • Liang L, Deng L, Nguyen SC, Zhao X, Maulion CD, Shao C, Tischfield JA (2008) Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks. Nucleic Acids Res 36:3297–3310

    Google Scholar 

  • Lin Cereghino GP, Lin Cereghino J, Sunga AJ, Johnson MA, Lim M, Gleeson MA, Cregg JM (2001) New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris. Gene 263:159–169

    Article  PubMed  CAS  Google Scholar 

  • Ma JL, Kim EM, Haber JE, Lee SE (2003) Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences. Mol Cell Biol 23:8820–8828

    Article  PubMed  CAS  Google Scholar 

  • Maassen N, Freese S, Schruff B, Passoth V, Klinner U (2008) Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis. FEMS Yeast Res 8:735–743

    Article  PubMed  CAS  Google Scholar 

  • Morrow DM, Connelly C, Hieter P (1997) “Break copy” duplication: a model for chromosome fragment formation in Saccharomyces cerevisiae. Genetics 147:371–382

    PubMed  CAS  Google Scholar 

  • Myung K, Ghosh G, Fattah FJ, Li G, Kim H, Dutia A, Pak E, Smith S, Hendrickson EA (2004) Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86. Mol Cell Biol 24:5050–5059

    Article  PubMed  Google Scholar 

  • Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci USA 101:12248–12253

    Article  PubMed  CAS  Google Scholar 

  • Ohkuma M, Muraoka S, Hwang CW, Ohta A, Takagi M (1993) Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering of Candida maltosa. Curr Genet 23:205–210

    Article  PubMed  CAS  Google Scholar 

  • Paques F, Haber JE (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63:349–404

    PubMed  CAS  Google Scholar 

  • Philippsen P, Stotz A, Scherf C (1991) DNA of Saccharomyces cerevisiae. Methods Enzymol 194:169–182

    Article  PubMed  CAS  Google Scholar 

  • Porter SE, Greenwell PW, Ritchie KB, Petes TD (1996) The DNA-binding protein Hdf1p (a putative Ku homologue) is required for maintaining normal telomere length in Saccharomyces cerevisiae. Nucleic Acids Res 24:582–585

    Article  PubMed  CAS  Google Scholar 

  • Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Schaffer S, Van Den BMA, Boergel D, Hueller T (2007) Improved production of sphingoid bases using genetically engineered microbial strains. Patent WO2007131720.PCT

  • Schiestl RH, Dominska M, Petes TD (1993) Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences. Mol Cell Biol 13:2697–2705

    PubMed  CAS  Google Scholar 

  • Stellwagen AE, Haimberger ZW, Veatch JR, Gottschling DE (2003) Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes Dev 17:2384–2395

    Article  PubMed  CAS  Google Scholar 

  • Suzuki C, Yoshida N, Okano E, Kawasumi T, Kashiwagi Y (2003) Cloning and chromosomal mapping of URA3 genes of Pichia farinosa and P. sorbitophila encoding orotidine-5’-phosphate decarboxylase. Yeast 20:905–912

    Article  PubMed  CAS  Google Scholar 

  • Symington LS (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 66:630–670 Table of contents

    Article  PubMed  CAS  Google Scholar 

  • Teo SH, Jackson SP (1997) Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair. EMBO J 16:4788–4795

    Article  PubMed  CAS  Google Scholar 

  • Ueno K, Uno J, Nakayama H, Sasamoto K, Mikami Y, Chibana H (2007) Development of a highly efficient gene targeting system induced by transient repression of YKU80 expression in Candida glabrata. Eukaryot Cell 6:1239–1247

    Article  PubMed  CAS  Google Scholar 

  • Umezu K, Amaya T, Yoshimoto A, Tomita K (1971) Purification and properties of orotidine-5’-phosphate pyrophosphorylase and orotidine-5’-phosphate decarboxylase from baker’s yeast. J Biochem 70:249–262

    PubMed  CAS  Google Scholar 

  • Van Den BMA, Schaffer S (2006) Microbial strains producing sphingoid bases or derivatives thereof. Patent WO2006048458.PCT

  • van Maris AJ, Geertman JM, Vermeulen A, Groothuizen MK, Winkler AA, Piper MD, van Dijken JP, Pronk JT (2004) Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyper producing yeast. Appl Environ Microbiol 70:159–166

    Article  PubMed  Google Scholar 

  • Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808

    Article  PubMed  CAS  Google Scholar 

  • Wickerham LJ, Burton KA (1962) Phylogeny and biochemistry of the genus Hansenula. Bacteriol Rev 26:382–397

    PubMed  CAS  Google Scholar 

  • Wickerham LJ, Stodola FH (1960) Formation of extracellular sphingolipides by microorganisms. I. Tetraacetylphyto-sphingosine from Hansenula ciferri. J Bacteriol 80:484–491

    PubMed  CAS  Google Scholar 

  • Wilson TE, Grawunder U, Lieber MR (1997) Yeast DNA ligase IV mediates non-homologous DNA end joining. Nature 388:495–498

    Article  PubMed  CAS  Google Scholar 

  • Zimmermann FK (1975) Procedures used in the induction of mitotic recombination and mutation in the yeast Saccharomyces cerevisiae. Mutat Res 31:71–86

    PubMed  CAS  Google Scholar 

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Acknowledgment

We would like to thank Evonik Industries, the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF; Bioindustrie 2021, “FerDi”) who supported this work, and Dr. Steffen Schaffer (Evonik Industries, Germany) for critically reading the manuscript and kindly providing the codon-optimized clonNAT cassette, the URA3 sequence, and the P. ciferrii codon usage table. Furthermore, we would like to thank Thomas E. Wilson (University of Michigan Medical School) and Stefan Åström (Stockholm University) for insightful discussions and comments regarding this work.

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Authors and Affiliations

  1. Institute of Molecular Biosciences, Goethe-University Frankfurt am Main, 60438, Frankfurt, Germany

    Christoph Schorsch & Eckhard Boles

  2. Evonik Industries, 45772, Marl, Germany

    Tim Köhler

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  1. Christoph Schorsch
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  2. Tim Köhler
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Correspondence to Christoph Schorsch.

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Communicated by K. Breunig.

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Schorsch, C., Köhler, T. & Boles, E. Knockout of the DNA ligase IV homolog gene in the sphingoid base producing yeast Pichia ciferrii significantly increases gene targeting efficiency. Curr Genet 55, 381–389 (2009). https://doi.org/10.1007/s00294-009-0252-z

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