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Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1
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and development of genetic transformation method by dominant selection in
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oleaginous yeast Rhodosporidium toruloides
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Yanbin Liu*, Chong Mei John Koh, Longhua Sun, Mya Myintzu Hlaing, Minge Du,
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Ni Peng, Lianghui Ji*
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Biomaterials and Biocatalysts Group, Temasek Life Sciences Laboratory, 1 Research
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Link, National University of Singapore, Singapore 117604
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Running title: Rhodosporidium toruloides GPD1 promoter and its applications
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* Corresponding authors:
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Lianghui Ji:
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Tel: +65 6872 7483
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Fax: +65 6872 7007
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Email: jilh@tll.org.sg
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Yanbin Liu:
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Tel: +65 6872 7484
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Fax: +65 6872 7007
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Email: yanbin@tll.org.sg
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Supplementary materials
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Fig. S1. T-DNA organizations in constructs used in this study. All were inserted in
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the backbone of the binary vector pPZP200 (Lee and Gavin 2008). (a) pEX2. (b)
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pEC2. (c) pEC3. (d) pEC3GPD-EGFP. (e) pEC3Pxxx-EGFP. (f) pRH203. (g)
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pRH203Pxxx-RtGFP. (h) pEX2Pxxx-HPT. (i) pEC3Pxxx-HPT3. LB: left border of
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T-DNA; RB: right border of T-DNA; Pgpd: 595 bp promoter of Umgpd1; Pxxx:
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RtGPD1 promoter of various lengths; hpt: E. coli hygromycin B phosphotransferase
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encoding gene; hpt-3: codon-optimized hygromycin resistance gene based on the
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codon usage bias in R. toruloides; EGFP: gene for enhanced green fluorescence
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protein; RtGFP: codon-optimized gene for green fluorescent protein based on the
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codon usage bias in R. toruloides. Tnos: terminator of A. tumefaciens nopaline
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synthase gene; T35S: terminator of cauliflower mosaic virus 35S gene; cbx cassette:
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carboxin resistance gene expression cassette. All vectors have the same pPZP200
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backbone (Lee and Gelvin 2008).
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Fig. S2. Isolation of full-length sequence of GPD1 from R. toruloides ATCC 10657.
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(a) Degenerate PCR; (b) Inverse PCR; (c) 5’RACE and 3’RACE. (d) RT-PCR.
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Abbreviations: M, 1 kb molecular weight marker (New England Biolabs,
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Massachusetts, USA), from upper to lower size, 10, 8, 6, 5, 4, 3, 2, 1.5, 1.0, 0.5 kb; c,
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cDNA as the template; g, genomic DNA as the template; P, PstI; E, EcoRI; B,
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BamHI; 5', 5'RACE; 3', 3'RACE.
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Fig. S3. Schematic illustration of GPD1 from R. toruloides ATCC 10657. (a) Gene
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organization of R. toruloides GPD1. Exons are marked as solid boxes; (b) Genomic
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organization of R. toruloides GPD1 (3543 bp). Nucleotides are numbered from the
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translational initiation codon “ATG”. Exons and introns are labeled as capitals and
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italics, respectively. Abbreviations: ct box - pyrimidine-rich region; tsp -
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transcriptional starting site; PolyA site- polyadenylation site; putative CAAT box is
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underlined; three nucleotide substitutions (313G>A, 457T>C and 649T>C) in R.
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glutinis strain ATCC 90781 are indicated in italics and highlighted; putative substrate
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binding site (ASCTTNCL) and potential phosphorylation sites are double-underlined;
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TG repeats upstream of the polyA site are highlighted; residues of the NAD binding
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site in the protein sequence (D39 and N320) are in italics; catalytic residues (C156 and
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H183) are boxed and highlighted.
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Fig. S4. Morphology and fluorescent microscopy of transformants of EGFP and
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RtGFP constructs (vector series pEC3Pxxx-eGFP and pRH203Pxxx-RtGFP,
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respectively) driven by GPD1 promoter fragments in U. maydis and R. toruloides,
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respectively. DIC: Differentiation interference contrast; FL: Fluorescence with GFP
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filter set.
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Fig. S5. ATMT of U. maydis using truncated GPD1 promoters from R. toruloides
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(vector series pEC3Pxxx-HPT). Hygromycin resistance gene (hpt) was driven by the
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595 bp U. maydis gpd1 promoter (Um-595) and the 795 bp (795) and 176 bp (176) of
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R. toruloides GPD1 promoters.
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Fig. S6. Southern blot analysis of hpt-3-transformants. Genomic DNA samples (10
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g) of wild-type R. toruloides ATCC 10657 (WT) and six randomly selected
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transformants (pEC3Pxxx-HPT3 as binary vector) were digested with BamHI and the
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blot was probed with a 581 bp digoxigenin-labeled hpt-3 DNA fragment.
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Table S1. Comparison of introns in other basidiomycetous GPD1 genes
Length
Strains
Range
Number
(Mean ± SD) (nt)
Pycnoporus coccineus
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63 ± 4
58-67
Omphalotus olearius
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67 ± 24
51-121
Crinipellis perniciosa
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55 ± 5
49-65
Thanatephorus cucumeris
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49 ± 3
45-55
Xanthophyllomyces dendrorhous
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117 ± 44
77-200
Phaffia rhodozyma
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117 ± 42
78-195
Cryptococcus neoformans
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95 ± 43
59-143
Cryptococcus curvatus
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110 ± 62
66-153
Ustilago maydis
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407 ± 0
NA
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Note: Sequence information can be found in GenBank database under the following
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accession numbers: P. coccineus: AB194780; O. olearius: AJ439986; C. perniciosa:
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DQ099333; T. cucumeris: AF339929; X. dendrorhous: Y08366; P. rhodozyma:
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AF006483; C. neoformans: AE017349; C. curvatus: AF126158; U. maydis: X07879.
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NA: not applicable
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Reference
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Lee LY, Gelvin SB (2008) T-DNA binary vectors and systems. Plant Physiol
146:325-332
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