An innovative protein expression system using RNA polymerase I for large‐scale screening of high‐nucleic‐acid content Saccharomyces cerevisiae strains

Fig. 1. The physical maps of the vectors. A. The elements used in the Pol I‐mediated protein expression cassettes: 578 nt upstream of rDNA gene, containing the transcription initiation site, was used as the rDNA promoter (rDNAp); the type IV IRES from CrPV (192 nt) with the just following 12 nt sequence (called IRES12 in this study), containing GCU initiation codon (NCBI Reference Sequence: NC_003924.1) (Hodgman and Jewett, 2014), was used for inducing the 5′ cap‐independent translation initiation process; the genes with no original initiation codon ATG was used as reporter genes, that is, ATGΔ URA3, ATGΔ yEGFP3 and ATGΔ URA3‐FLAG with the FLAG tagged at the 3′‐end. Meanwhile, normal URA3, with no IRES12, was used as negative control; 50 nt synthetic polythymine marked as oligo‐dT; 284 nt downstream of the rDNA gene was used as rDNA terminator (rDNAt). B. The normal URA3 expression cassette controlled by Pol II recognized TEF1p was used as a positive control. All the expression cassettes were ligated in the skeleton derived from the episomal plasmid pMA91 (Mellor et al., 1983, Fig. S1). The genotype properties of all plasmids were also listed in Table 1.

Fig. 2. The Pol I‐mediated URA3 expression system worked well. The spot growth assay on SC medium supplemented without (left) or with (right) uracil. The cells were serially 10‐fold diluted from an initial OD600 1.0, then spotted on the plates and incubated for 2 days at 30°C. The cell lysates were detected by western blot analysis. A and H. cells with empty plasmid, thus devoid of URA3. B. cells harbouring plasmid with Pol II‐mediated URA3 expression cassette. C. cells harbouring plasmid (i.e. pMIU in Table 1, Line 4) with intact elements for Pol I‐mediated expression cassette. D. cells harbouring plasmid with the Pol I‐mediated cassette but no IRES12. E. cells with pMIU lacking eIF5B (fun12Δ). F. cells with pMIU lacking Met‐tRNAi Met (imt3Δ imt4Δ). G and I. cells harbouring pMIUF (in Table 1, Line 5) that the FLAG was tagged at the 3′‐end of URA3 in pMIU also showed growth phenotype and the correct size of the Ura3p in its cell lysate detected by western blot analysis.

Fig. 3. Flow cytometry analysis of the Pol I‐mediated GFP expression system. The fluorescence histogram plot of the cells with (A) empty plasmid, (B) plasmid harbouring Pol I‐mediated GFP expression cassette, and (C) after mutagenesis. The dashed line marks the fluorescence intensity of 40 arbitrary units (a.u.). The cells with fluorescence intensity > 40 a.u. are represented in green, while those with fluorescence intensity > 100 a.u. are represented in purple. MFI: mean fluorescence intensity. (D) The number of cells with fluorescence intensity > 40 a.u. The data from the five independent cultivations were compared using the Mann–Whitney U test. *P < 0.05 was considered statistically significant. A total of 2 × 105 cells were analysed by the flow cytometer with Kaluza Analysis 2.1 software.

Fig. 4. The cells with higher mean fluorescence intensity (A) also exhibited higher total RNA content (B). The mutants BXZ08m1‐3 were single cells with higher fluorescence intensity sorted from the parental strain BXZ08 (ATGΔ yEGFP3 IRES12, Pol I) after mutagenesis. The black columns stand for the first generation cells and the grey columns stand for the cells continuously transferred to fresh medium after 10 days. The coefficients of the 1.0 OD600 to dry cell weight (DCW) were ± 1.38 × 10−4 g for all the tested strains. The data from the independent triplicate cultivations were compared using one‐way ANOVA followed by LSD post hoc test. *P < 0.05 was considered statistically significant. **P < 0.01 was considered highly significant.