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A site-directed integration system for the nonuniversal CUGSer codon usage species Pichia farinosa by electroporation

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Abstract

Halotolerant yeast, Pichia farinosa, is a valuable yeast strain in fermentation industry because it produces high yield of glycerol and xylitol, and can tolerate both contamination and high-density growth during fermentation. However, the lack of genetic manipulation tools makes it less popular as a gene engineering strain. Expression systems commonly used in other yeast systems, such as Saccharomyces cerevisiae and Pichia pastoris cannot be used in P. farinosa because it translates universal Leu codon CUG as Ser. Here we reported a modified expression vector and a transformation system with enhanced efficiency in P. farinosa. The results showed that cells of OD600 0.8–1.0 with DTT treatment can obtain high transformation efficiency. The optimized electroporation condition was 900 V, 25 μF, and 200 Ω. The DNA concentration did not influence the transformation. Our system provides the potential not only for applying P. farinosa as an industrial strain of gene engineering, but also for studying gene function in its native host.

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Reference

  • Bisping B, Baumann U, Rehm HJ (1990) Production of glycerol by immobilised Pichia farinosa. Appl Microbiol Biotechnol 32:380–386

    Article  CAS  Google Scholar 

  • Brzobohaty B, Kovac L (1986) Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity. J Gen Microbiol 132:3089–3093

    PubMed  CAS  Google Scholar 

  • Burke D, Dawson D, Stearns T (2000) Methods in yeast Genetics. A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, New York, pp 109–114

  • Chise S, Nao Y, Etsuko O, Toshiyuki K, Yutaka K (2003) Cloning and chromosomal mapping of URA3 genes of Pichia farnosa and P. sorbitophila encoding orotidine-5′-phosphate decarboxylase. Yeast 20:905–912

    Article  Google Scholar 

  • Fabienne F, Florence CL, Jean V, Thierry N (2004) Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker. Yeast 21:95–106

    Article  Google Scholar 

  • Gerald K, Stephanus GK, Bernard AP (2001) Conservation and release of osmolytes by yeasts during hypo-osmotic stress. Arch Microbiol 177:29–35

    Article  Google Scholar 

  • Lenka P, Hana S (2003) Efficient transformation of the osmotolerant yeast Zygosaccharomyces rouxii by electroporation. J Microbio Methods 55:481–484

    Article  Google Scholar 

  • Hiroshi O, Toshiyuki S (1969) Microbial production of xylitol from glucose Appl Microbiol 18:1031–1035

    Google Scholar 

  • Luis R, Francisco PC, Liliana B, Tanilo R, Julio MD (1998) Development of an integrative DNA transformation system for the yeast Candida utilis. FEMS Microbiol Lett 165:335–340

    Google Scholar 

  • Nakamura Y, Gojobori T, Ikemura T (2000) Codon usage tabulated from the international DNA sequence databases: status for the year. Nucl Acids Res 28:292

    Article  PubMed  CAS  Google Scholar 

  • Shye-Jye T, Kuang-Hui S, Guang-Huan S, Terng-Yuan C, Wan-Lin W, Guan-Chiun L (2003) A transformation system for the nonuniversal CUGSer codon usage species Candida rugosa. J Microbio Methods 52:231–238

    Article  Google Scholar 

  • Suzuki C, Kashiwagi T, Hirayama K (2002) Alternative CUG codon usage (Ser for Leu) in Pichia farinosa and the effect of a mutated killer gene in Saccharomyces cerevisiae. Protein Eng 15:251–255

    Article  PubMed  CAS  Google Scholar 

  • Takeshi O, Tsutomu S, Miki M et al (1993) Non-universal decoding of the leucine codon CUG in several Candida species. Nucleic Acids Res 21:4039–4045

    Article  Google Scholar 

  • Tomita M, Iwatan S, Yamamoto S (1996) Alteration in cell wall chitin of Zygosaccharomyces rouxii. J Ferment. Technol 66:481–488

    Google Scholar 

  • Toshikuni T, Takeshi S, Hiromichi O (2001) Microbial asymmetric reduction of α-hydroxyketones in the anti-prelog selectivity tetrahedron. Asymmetry 12:2543–2549

    Article  Google Scholar 

  • Vijaikishore P, Karanth NG (1986) Factors affecting glycerol production by Pichia farinosa under alkaline conditions. Appl Biochem Biotechnol 13:189–205

    Article  CAS  Google Scholar 

  • Vina WY, Jere AM, Brian PD, Thomas WJ (1994) High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2. Appl Environ Microbiol 60:4245–4254

    Google Scholar 

  • Waterham HR et al (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186:37–44

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biology, Sichuan University, Wangjiang Street No.29, Chengdu, Sichuan, China

    Xiaoxing Wang & Fang Chen

  2. Department of Microbial Fermentation, Sichuan Chuanda Guangyao biotechnology Ltd, Chengdu, Sichuan, China

    Gang LI, Yuntao Deng & Xuanwei Yu

Authors
  1. Xiaoxing Wang
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  2. Gang LI
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  3. Yuntao Deng
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  4. Xuanwei Yu
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  5. Fang Chen
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Correspondence to Fang Chen.

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Wang, X., LI, G., Deng, Y. et al. A site-directed integration system for the nonuniversal CUGSer codon usage species Pichia farinosa by electroporation. Arch Microbiol 184, 419–424 (2006). https://doi.org/10.1007/s00203-005-0062-8

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  • DOI: https://doi.org/10.1007/s00203-005-0062-8

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