Reassessment of IRES Element Activity in Komagataella phaffii Using a Standardized Bicistronic System: Cryptic Promoter Activity Mimics Cap-Independent Translation

Figures

• 

  Figure 1. Schematic of the genetic constructs with expression cassettes under the control of the PAOX1 promoter. The upper part of the figure shows the bicistronic construct EGFP-IRES-mKate2, in which IRES is one of the IRES elements analyzed, namely those of the TEV, PVY, EMCV, HCV, and RhPV viruses and the S. cerevisiae GPR1 mRNA. The lower part of the figure shows the control expression cassettes under the control of the PAOX1 promoter: K1, a bicistronic cassette isogenic to EGFP-IRES-mKate2 but carrying a − 1 frameshift mutation that inactivates the mKate2 ORF, so that only EGFP is functionally expressed (the single-nucleotide deletion is indicated by a vertical arrowhead); and K2, a monocistronic cassette with the mKate2 ORF.

• 

  Figure 2. Time course of EGFP and mKate2 expression from mono- and bicistronic expression cassettes under the control of the PAOX1 promoter in K. phaffii. Each of the first seven panels shows the mean fluorescence intensity (relative fluorescence units, RFU; logarithmic scale) of the EGFP (green) and mKate2 (red) reporters at 0, 24, 48, 72, and 120 h of induction for one construct: K1, the EGFP-expression control; AOX1-EMCV, AOX1-HCV, AOX1-GPR1, AOX1-PVY, AOX1-TEV and AOX1-RhPV, bicistronic EGFP–IRES–mKate2 cassettes carrying the EMCV, HCV, GPR1, PVY, TEV and RhPV IRES elements, respectively. The lower-right panel (“mKate2, 120 h”) summarizes the mKate2 signal at 120 h for the same constructs together with the monocistronic mKate2 control K2, included as a positive reference (Supplementary File S4, Table S6). In all panels, the dashed and dotted lines mark the limit of detection (LOD, 2163 RFU) and the limit of quantification (LOQ, 4207 RFU) of the mKate2 signal, respectively (Section 4; Supplementary File S4, Table S5). Values are means ± SD (n = 3); host-strain (GS115) autofluorescence was subtracted from all values.

• 

  Figure 3. Production of the mKate2 protein and expression of EGFP in K. phaffii cells carrying mono- and bicistronic expression cassettes under the control of the PAOX1 promoter. (A) Immunoblot analysis of lysates of induced cells with anti-mKate2 antibodies (Section 4). (B) In-gel fluorescence of EGFP after separation of lysates of the same cells by non-denaturing PAGE, recorded on a Typhoon FLA 9500 imager (Section 4). Lanes (for A and B): K2, monocistronic mKate2; K1, EGFP-expression control (with a frameshift in the mKate2 ORF); T, P, R, G, E, H, bicistronic constructs carrying the TEV, PVY, RhPV, GPR1, EMCV, and HCV IRES elements, respectively. Arrows indicate the positions of molecular weight markers in kDa. In panel B, the marker positions are shown for orientation only, since electrophoretic mobility under non-denaturing conditions does not reflect molecular mass.

• 

  Figure 4. Scheme for the generation of the genetic constructs with expression cassettes from which the PAOX1 promoter has been deleted. Plasmids pHik-GTK, pHik-GPK, and pHik-GRK were derived from p9KAox-GTK, p9KAox-GPK, and p9KAox-GRK, respectively, by deletion of the PAOX1 promoter; in all other respects, the cassettes are isogenic to the parental ones.

• 

  Figure 5. Analysis of EGFP and mKate2 ORF expression in K. phaffii within bicistronic cassettes in constructs from which the PAOX1 promoter has been deleted. Flow cytometry data are shown for the EGFP and mKate2 channels of recombinant K. phaffii cells transformed with the plasmids: RhPV, pHik-GRK; TEV, pHik-GTK; and PVY, pHik-GPK. The histogram shows fluorescence intensity on a logarithmic scale. RFU, relative fluorescence units. Green bars correspond to the EGFP signal and red bars to the mKate2 signal: light green and light red, 0 h; dark green and dark red, 120 h after transfer to methanol-containing medium. The dashed and dotted lines mark the limit of detection (LOD, 2163 RFU) and the limit of quantification (LOQ, 4207 RFU) of the mKate2 signal, respectively (Section 4; Supplementary File S4, Table S5). Values are means ± SD (n = 3); host-strain (GS115) autofluorescence was subtracted from all values.

• 

  Figure 6. Schematic of the genetic constructs with expression cassettes under the control of the PTHI11 promoter. The bicistronic construct EGFP-IRES-mKate2 is shown, in which IRES is one of the IRES elements analyzed, that of the TEV (plasmid p9KTh11-GTK) or PVY (plasmid p9KTh11-GPK) virus.

• 

  Figure 7. Dynamics of EGFP and mKate2 ORF expression in K. phaffii within bicistronic cassettes in constructs under the control of the PTHI11 promoter. Flow cytometry data are shown for the EGFP and mKate2 channels of recombinant K. phaffii cells transformed with the plasmids: TEV, p9KTh11-GTK; and PVY, p9KTh11-GPK. The histogram shows fluorescence intensity in relative fluorescence units (RFU). Green bars correspond to the EGFP signal and red bars to the mKate2 signal. Values are means ± SD (n = 3); host-strain (GS115) autofluorescence was subtracted from all values.