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Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides

  • Applied genetics and molecular biotechnology
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Abstract

The oleaginous yeast Rhodosporidium toruloides, which belongs to the Pucciniomycotina subphylum in the Basidiomycota, has attracted strong interest in the biofuel community recently due to its ability to accumulate more than 60% of dry biomass as lipid under high-density fermentation. A 3,543-nucleotide (nt) DNA fragment of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD1) was isolated from R. toruloides ATCC 10657 and characterized in details. The 1,038-nt mRNA derived from seven exons encodes an open reading frame (ORF) of 345 amino acids that shows high identity (80%) to the Ustilago maydis homolog. Notably, the ORF is composed of codons strongly biased towards cytosine at the Wobble position. GPD1 is transcriptionally regulated by temperature shock, osmotic stress, and carbon source. Nested deletion analysis of the GPD1 promoter by GFP reporter assay revealed that two regions, −975 to −1,270 and −1,270 to −1,429, upstream from the translational start site of GPD1 were important for responses to various stress stimuli. Interestingly, a 176-bp short fragment maintained 42.2% promoter activity of the 795-bp version in U. maydis whereas it was reduced to 17.4% in R. toruloides. The GPD1 promoter drove strong expression of a codon-optimized enhanced green fluorescent protein gene (RtGFP) and a codon-optimized hygromycin phosphotransferase gene (hpt-3), which was critical for Agrobacterium tumefaciens-mediated transformation in R. toruloides.

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Acknowledgments

This work was financially supported by the Singapore Economic Development Board and Temasek Trust. We thank especially Prof. Dr. Ursula Kües, Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, Georg-August-University Göttingen, Göttingen, Germany, for many helpful suggestions for this manuscript, and Prof. Dr. Michael Feldbrügge, Institute for Microbiology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany, for the gift of plasmid pMF2-3c.

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  1. Biomaterials and Biocatalysts Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore

    Yanbin Liu, Chong Mei John Koh, Longhua Sun, Mya Myintzu Hlaing, Minge Du, Ni Peng & Lianghui Ji

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  1. Yanbin Liu
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  2. Chong Mei John Koh
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Correspondence to Yanbin Liu or Lianghui Ji.

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Liu, Y., Koh, C.M.J., Sun, L. et al. Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides . Appl Microbiol Biotechnol 97, 719–729 (2013). https://doi.org/10.1007/s00253-012-4223-9

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