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

Saccharomyces cerevisiae is the preferred source of RNA derivatives, which are widely used as supplements for foods and pharmaceuticals. As the most abundant RNAs, the ribosomal RNAs (rRNAs) transcribed by RNA polymerase I (Pol I) have no 5' caps, thus cannot be translated to proteins. To screen high-nucleic-acid content yeasts more efficiently, a cap-independent protein expression system mediated by Pol I has been designed and established to monitor the regulatory changes of rRNA synthesis by observing the variation in the reporter genes expression. The elements including Pol I-recognized rDNA promoter, the internal ribosome entry site from cricket paralytic virus which can recruit ribosomes internally, reporter genes (URA3 and yEGFP3), oligo-dT and an rDNA terminator were ligated to a yeast episomal plasmid. This system based on the URA3 gene worked well by observing the growth phenotype and did not require the disruption of cap-dependent initiation factors. The fluorescence intensity of strains expressing the yEGFP3 gene increased and drifted after mutagenesis. Combined with flow cytometry, cells with higher GFP level were sorted out. A strain showed 58% improvement in RNA content and exhibited no sequence alteration in the whole expression cassette introduced. This study provides a novel strategy for breeding high-nucleic-acid content yeasts.

Introduction

Saccharomyces cerevisiae is a Generally Regarded As Safe (GRAS) microorganism and the preferred industrial source of RNA (Nagodawithana, 1992). RNA degradation products and their derivatives have many beneficial uses, especially in food and medical industry, and have important implications for improving the overall economic productivity of the yeast industry. For example, 5′‐inosine monophosphate (5′‐IMP) and 5′‐guanine monophosphate (5′‐GMP) can be safely used as food additives (Olmedo et al., 1994; Zhao and Fleet, 2005; Cairoli et al., 2008), while the derivatives of ribonucleotides (e.g. pyrimidines and purines) can be used as raw materials for food and pharmaceutical intermediates and so on (Rudolph et al., 1990).

About 80% of total RNAs in yeast cells are ribosomal RNAs (rRNAs) (Warner, 1999), therefore, an effective way to increase RNA content is to enhance rRNA biosynthesis. The transcription of rRNAs (the primary transcript is 35S pre‐rRNA) is efficiently and tightly regulated by RNA polymerase I (Pol I) and other factors through the ribosomal DNA promoter (rDNAp) (Planta, 1997; Hannan et al., 1998). The rDNA genes are located on chromosome XII and consist of 150 to 200 repeated copies of a 9.1‐kb unit in S. cerevisiae (Petes, 1979). For breeding higher RNA content strains, the screening model of the poor growth mutagenized cells on higher concentration (1.5 mol l−1) KCl was used in Candida yeast in the early patent (U.S. Pat. No. 3,909,352, Akiyama et al., 1975). However, the mechanism of such screening model and the association between KCl and rRNA synthesis was not mentioned. Rational disruption of some functional genes can change rRNA synthesis and was thus used to set up the screening models. In Prof. Harashima’s study, the non‐essential gene RRN10, encoding a subunit of the upstream activation factor for rRNA transcription initiation, was disrupted in diploid cells. The tiny colonies grown from single cell by the tetrad analysis from asci were picked up as the parental strain with poor growth phenotype. After mutagenesis, one of the growth‐recovering suppressors showed RNA content twofold and 1.3‐fold higher than the Δrrn10 and the parental strain, respectively. The reason could be the detected upregulation of the RPL40A and RPL40B genes, which encode the components of the 60S large ribosomal subunit. When the RRN10 was integrated into the suppressor genome, the total RNA content became twofold higher than in the parental strain (Chuwattanakul et al., 2011; Khatun et al., 2013). Actually, RRN10 is an important gene for promoting a high level of transcription of rDNA, but based on its deletion (Δrrn10), cells with higher RNA content were still screened out (Chuwattanakul et al., 2011), indicating that to explore more efficient elements for enhancing RNA content and to understand rRNA synthesis mechanism is still challenging. On the other hand, such depending on cell poor growth screening model seems difficult to pick up the tiny colonies, since the better growth cells normally gave strong background on the plates. Meanwhile, in Candida tropicalis, the two heterologous genes were introduced for perturbing the redox force. The RNA content was increased 5.5% by the membrane‐bound transhydrogenase gene pntAB but decreased 10.6% by the NADH oxidase gene noxE (Li et al., 2019). Fermentation parameters can also affect RNA yield to some extent: for example, pH 4.0 favoured the RNA accumulation in Candida yeast (Li et al., 2012).

This handful of research data showed that there are still limitations in both breeding of high RNA content yeasts and screening out such yeast cells in large scale. The screening model based on the Pol I‐mediated expression system might be powerful for these purposes, since the major components rRNAs were synthesized by Pol I. Generally, observing the variation in a reporter gene expression is one of the effective strategies to detect changes in regulatory mechanism. Unlike Pol II, which transcribes and processes mRNA by adding a 5′ cap and a 3′ poly‐A tail, Pol I cannot mediate such post‐transcriptional modifications (Sims et al., 2004; Buratowski, 2009; Heidemann et al., 2013), the resulting rRNAs cannot be translated to proteins, even they play roles in the process of translation. This provides great challenges for establishment of a Pol I‐mediated protein expression system, as well as observing changes in regulation of rRNA synthesis.

In eukaryotes, the mRNA 5′ cap (m7GpppN) is normally necessary for the initial stage of protein synthesis. It is recognized by a series of translation initiation factors (eIFs) together with Met‐tRNAi Met to recruit 40S small ribosomal subunits and form a preinitiation complex (Aitken and Lorsch, 2012). This is called a 5' cap‐dependent translation initiation process. On the other hand, the internal ribosome entry sites (IRESs) can mediate cap‐independent translation initiation, since their secondary structure can recruit ribosomes internally (Pfingsten et al., 2006). IRESs were discovered in picornavirus first (Pelletier and Sonenberg, 1988), then in other eukaryotic viruses (Reynolds et al., 1995; Ohlmann, 2000). IRESs were also found in some mRNAs of cellular organisms and functioned when cells are under special stress conditions, such as heat shock, hypoxia, nutrient deprivation and so on (Holcik et al., 1999; Holcik and Sonenberg, 2005; Gilbert et al., 2007; Reineke et al., 2011). The specific conserved IRES sequence in genome of hepatitis C virus (HCV) was studied to screen anti‐HCV drugs (Kikuchi et al., 2005). An IRES sequence from encephalomyocarditis virus (EMCV) together with the following reporter gene were ligated under the control of rDNA promoter to let the Pol I‐mediated transcripts can translated into proteins in mammalian cells. The rDNA promoters from human and mouse were functioned in this system, and the human rDNA promoter led to an increase of neomycin phosphotransferase activity up to 70% of the Pol II‐mediated protein expression system in the same plasmid (Palmer et al., 1993). IRESs were also used to establish the artificial bicistronic expression system. Pol II‐dependent promoter drove the two reporter genes transcription. Before the second cistron, IRES was inserted as an element for inducing the cap‐independent translation initiation process in animal cells to co‐express two proteins for researching bivalent drugs, vaccines and so on (Pizzato et al., 1998; Guerbois et al., 2013; Renaud‐Gabardos et al., 2015; Al‐Allaf et al., 2019).

Based on different secondary structures, the requirements for translation initiation factors and the manner of recruiting the ribosome, IRESs can be divided into four types. Among them, type IV IRESs, characterized by having three pseudoknots, and requiring no known translation initiation factors and initiator tRNA (Met‐tRNAi Met), can recruit directly 40S small ribosomal subunits to the non‐AUG start codon and can work in S. cerevisiae. The CrPV‐IRES – IRES in the intergenic region (IGR) of the cricket paralysis virus (CrPV) – is a typical type IV example (Jan and Sarnow, 2002; Pestova et al., 2004; Deniz et al., 2009; Hertz and Thompson, 2011a, 2011b,2011a, 2011b; Thompson, 2012; Hodgman and Jewett, 2014). CrPV‐IRES was also used for establishing the artificial bicistronic expression system in S. cerevisiae cells to overcome the challenge of only monocistronic messages in eukaryotes. Similar as that in animal cells, a Pol II‐dependent promoter (e.g. promoters of CUP1, PGK1 and so on) drove the transcription of two reporter genes, and the fragment of the native CrPV‐IRES was inserted just before the second one. When the second reporter gene was URA3, which encodes orotidine‐5′‐phosphate decarboxylase and allows cell growth in the absence of uracil (‐Ura), the strain showed no growth phenotype on ‐Ura plate, until certain genes encoding cap‐dependent translation initiation factors (e.g. FUN12‐coding eIF5B; IMT3‐, IMT4‐coding Met‐tRNAi Met) were deleted (Thompson et al., 2001; Makelainen and Makinen, 2007; Deniz et al., 2009; Mardanova et al., 2009; Edwards and Wandless, 2010; Hertz and Thompson, 2011b). IRES was also tested in vitro: in the yeast cell‐free protein synthesis system, the native CrPV‐IRES allowed the translation of the reporter protein under the control of the T7 promoter with 0.92 μg ml−1 luciferase. Furthermore, combining the native CrPV‐IRES with the just following 12 nucleotide (nt) sequence (GCTACATTTCAA) which contains the IRES‐preferred initiation codon GCU, pushed luciferase translation up to 4.3 μg ml−1, indicating that this 12 nt sequence was very important for the function of IRES (Martinez‐Salas et al., 2008; Hertz and Thompson, 2011b; Thompson, 2012; Hodgman and Jewett, 2014).

It can be imagined if there is an efficient protein expression system mediated by Pol I, the changes in regulation of rRNAs synthesis might be got indirectly but conveniently by observing the variation in a reporter gene expression. That also gives a possibility to develop a high‐throughput screening model for breeding high‐nucleic‐acid content yeasts, when combined with flow cytometry. Therefore, in this study, the Pol I‐mediated protein expression system with an IRES element was constructed in an episomal plasmid to create a monocistron of 5′ cap‐independent translation initiation process in S. cerevisiae. The URA3 gene was first expressed to evaluate the expression system by observing the growth phenotype in uracil deficient medium. Based on the variation of GFP fluorescence, the system was also tested for high‐nucleic‐acid content yeast cells in large‐scale screening by flow cytometry sorting.

Results

Design statement of Pol I‐mediated protein expression system

Pol I catalyses rRNA synthesis and first recognizes the transcription initiation site (TIS), therefore, the region including the TIS within a 578 nt sequence upstream of the rDNA gene was selected as the promoter (rDNAp). The key for setting up the Pol I‐mediated protein expression system is the element for translation initiation, while the internal ribosome entry site (IRES) can induce the 5′ cap‐independent translation initiation. Since the 12 nt sequence (GCTACATTTCAA) containing GCU initiation codon that follow the native CrPV‐IRES (192 nt) (NCBI Reference Sequence: NC_003924.1) favoured translation was confirmed in vitro (Hodgman and Jewett, 2014), total 204 nt fragment (named IRES12 in this study) was arranged behind rDNAp to induce cap‐independent translation initiation. The original initiation codon ATG in the following reporter gene was therefore removed. In order to observe clearly if the Pol I‐mediated expression system worked or not, the growth‐based marker URA3 was first tested. Meanwhile, the FLAG was also tagged at the 3′‐end of URA3 for western blot analysis. The 50 nt oligo‐dT was artificially synthesized and introduced in the expression cassettes in order to transcribe directly into a 3′ poly‐A tail for translation termination (Gan and Jewett, 2014; Hodgman and Jewett, 2014). Finally, the 284 nt fragment downstream of the rDNA gene was used as the terminator (Peyresaubes et al., 2017). The designed Pol I‐mediated URA3 expression cassettes with intact elements are the rDNAp‐IRES12‐ATGΔ URA3 (or ATGΔ URA3‐FLAG)‐oligo‐dT‐rDNAt. The expression cassette with no IRES12, but with the original initiation codon ATG (rDNAp URA3 oligo‐dT rDNAt), was used for the negative control. Meanwhile, the URA3 under the control of the translational elongation factor EF‐1 alpha promoter (TEF1p) transcribed by Pol II was also constructed (TEF1p URA3 PGK1t) as a positive control. All the URA3 expression cassettes were ligated into the skeleton of the yeast episomal plasmid derived from pMA91 (Mellor et al., 1983) (Table 1, Line 2, 3, 4 and 5; Fig. 1).

Table 1

Plasmids and strains used in this study.

Plasmids/strains	Genotype/properties	Source/reference
Plasmids
(1) pMA91	Episomal plasmid; LEU2, ura3	Mellor et al. (1983)
(2) pMU	pMA91 derivative; LEU2, TEF1p ‐ URA3 ‐ PGK1t (Pol II mediated)	This study
(3) pMNU	pMA91 derivative; LEU2, rDNAp ‐ URA3 ‐ oligo‐dT ‐ rDNAt (Pol I mediated)	This study
(4) pMIU	pMA91 derivative; LEU2, rDNAp‐IRES12‐ATGΔ URA3‐oligo‐dT‐rDNAt (Pol I mediated)	This study
(5) pMIUF	pMA91 derivative; LEU2, rDNAp‐IRES12‐ATGΔ URA3 ‐ FLAG‐oligo‐dT‐rDNAt (Pol I mediated)	This study
(6) pMIG	pMA91 derivative; LEU2, rDNAp ‐IRES12‐ ATGΔ yEGFP3‐oligo‐dT ‐ rDNAt (Pol I mediated)	This study
Strains
(7) BY4741	MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0	EUROSCARF
(8) BXZ01	BY4741 derivative; empty plasmid {pMA91}/ (LEU2, ura3)	This study
(9) BXZ02	BY4741 derivative; {pMU}/ (LEU2, URA3 Pol II)	This study
(10) BXZ03	BY4741 derivative; {pMNU}/ (LEU2, URA3 iresΔ, Pol I)	This study
(11) BXZ04	BY4741 derivative; {pMIU}/ (LEU2, ATGΔ URA3 IRES12, Pol I)	This study
(12) BXZ05	BY4741 derivative; {pMIUF}/ (LEU2, ATGΔ URA3‐FLAG IRES12, Pol I)	This study
(13) BXZ06	BXZ04 derivative; imt3Δ imt4Δ::loxP‐KanMX‐loxP/ (LEU2, ATGΔ URA3 IRES12, Pol I, imt3Δ imt4Δ)	This study
(14) BXZ07	BXZ04 derivative; fun12Δ::loxP‐KanMX‐loxP/ (LEU2, ATGΔ URA3 IRES12, Pol I, fun12Δ)	This study
(15) BXZ08	BY4741 derivative; {pMIG}/ (LEU2, ATGΔ yEGFP3 IRES12, Pol I)	This study
(16) BXZ08m1	BXZ08 derivative; after ARTP mutagenesis	This study
(17) BXZ08m2	BXZ08 derivative; after ARTP mutagenesis	This study
(18) BXZ08m3	BXZ08 derivative; after ARTP mutagenesis	This study

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 (); 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, , and with the FLAG tagged at the 3′‐end. Meanwhile, normal , with no IRES12, was used as negative control; 50 nt synthetic polythymine marked as ; 284 nt downstream of the rDNA gene was used as rDNA terminator ().

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.

Availability of the Pol I‐mediated URA3 expression system proof by growth phenotype

The pMA91 plasmid (Fig. S1) and its derivatives (Table 1; Fig. 1) were transformed into strain BY4741 (leu2, ura3) and the recombinants were selected by the original LEU2 selection marker. The transformants were then used for the spot dilution growth assay. All the strains grew well on the plates containing uracil (+Ura). On the plates without uracil (−Ura), as the negative and positive controls, the cells with no URA3 showed no growth phenotype, while, the Pol II driven cassette (TEF1p URA3 PGK1t) induced normal cell growth (Fig. 2A and B). The Pol I‐mediated URA3 expression cassette including the intact elements we designed also guided good cell growth, as we expected, but the strain without IRES12 exhibited no growth phenotype on the −Ura plate (Fig. 2C and D). Since disruption of some genes encoding cap‐dependent translation initiation factors can improve IRES‐mediated cap‐independent translation initiation process (Thompson et al., 2001; Deniz et al., 2009), the plasmid with the intact elements for Pol I‐mediated URA3 expression cassette (rDNAp‐IRES12‐ATGΔ URA3‐oligo‐dT‐rDNAt) (Table 1, Line 4; Fig. 1) was also introduced into the strains with eIF5B and Met‐tRNAi Met coding gene deletions, fun12Δ and imt3Δ imt4Δ, respectively (Table 1, Line 13, 14). The fun12Δ encouraged cell growth on ‐Ura plate more than the imt3Δ imt4Δ did (Fig. 2E and F) compared with the wild type (Fig. 2C). On the other hand, the strain with intact elements for Pol I‐mediated URA3‐FLAG expression cassette also showed the growth phenotype on −Ura plate (Fig. 2G), and the URA3 coding protein (Ura3p) at its correct size (30 kDa), was directly detected from its cell lysates by western blot analysis (Fig. 2I). In contrast, the Ura3p produced by the bicistronic expression system, in which IRES guided the expression of the second cistron, can not be detected by western blot, until disruption of some genes encoding cap‐dependent translation initiation factors, such as fun12Δ (Deniz et al., 2009). These results confirmed that the innovative monocistronic expression system mediated by Pol I and IRES has been successfully established in S. cerevisiae. Furthermore, IRES12 played a key role in the functionality of the innovative protein expression system.

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.

Enhancement of GFP fluorescence intensity by the Pol I‐mediated expression system

Based on the positive results described above, to set up a convenient large‐scale screening model for high‐nucleic‐acid content yeast combined with flow cytometry, the reporter gene in this Pol I‐mediated expression system was replaced by the GFP gene without the original initiation codon ATG (ATGΔ yEGFP3). The recombinant plasmid (Table 1, Line 6; Fig. 1A) was also transformed into the strain BY4741 and the transformants were selected by the original LEU2 selection marker in pMA91 (Fig. S1). A total of 2 × 105 cells were monitored by the flow cytometer and the cells were analysed with Kaluza Analysis 2.1 software. Compared with the strain with empty plasmid, the resulting strain’s fluorescence histogram plot drifted to higher direction (right in Fig. 3A and B). The mean fluorescence intensity (MFI) increased from 4.6 to 14.0 arbitrary units (a.u.) in average (Fig. 3B). Meanwhile, the number of cells with fluorescence intensity > 40 a.u. increased from 9 to 3540 (Fig. 3B green part, 3d). These results indicate that the GFP was also successfully expressed by the innovative Pol I‐mediated system.

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.

The cells with higher GFP expression mediated by Pol I after mutagenesis were sorted through flow cytometry and exhibited higher RNA content

Since the intracellular rRNA synthesis forms tight regulation homoeostasis by Pol I and other transcription factors, the ARTP system was used to mutagenize the strain harbouring the Pol I‐mediated GFP expression cassette (LEU2, ATGΔ yEGFP3 IRES12, Pol I) to globally perturb the rRNA synthesis. The working dose with 90% lethality rate (Fig. S2) was used for treating the 104–105 cells by ARTP mutagenesis system. After mutagenesis, the cells were incubated in an SC liquid medium without leucine (−Leu) to recover the cell growth. The fresh cultures at mid‐exponential phase were analysed by flow cytometry. The fluorescence histogram plot of 2 × 105 cells further drifted to higher direction (right in Fig. 3C), and the MFI increased from 14.0 to 22.4 a. u., which was about 1.6‐fold higher than that of the strain before mutagenesis (Fig. 3C). The number of cells with fluorescence intensity > 40 a.u. reached 28,260 (Fig. 3C green and purple parts, 3D). These results indicate that mutagenesis perturbed the Pol I‐mediated GFP expression even in the episomal plasmid.

Since the rDNAp drove the GFP expression in this monocistronic expression system, we therefore speculated that the cells with higher fluorescence intensity could also have higher RNA content. Hence the mutagenized cells with fluorescence intensity > 2000 a. u. were sorted out as high‐nucleic‐acid content yeast candidates. Only about 100 out of 2 × 105 cells showed such a feature and were dropped separately onto the plates. Among several dozens of the grown colonies, 12 colonies were randomly selected to detect the MFI, and three (BXZ08m1‐3) of them, especially BXZ08m3, showed higher MFI than the parental strain BXZ08 (ATGΔ yEGFP3 IRES12, Pol I) (Fig. 4A, black columns). Not unexpectedly, their total RNA content had increased significantly, especially that of BXZ08m3 reached 176.8 mg g−1 DCW, with a 58% improvement compared to the BXZ08 (Fig. 4B, black columns). Meanwhile, three mutants showed almost the same MFI and total RNA content before and after 10 days of subculture (Fig. 4). The sequence variation of all elements in the expression cassette, especially rDNAp and IRES12, could be one of the reasons for the enhanced GFP expression level. However, the DNA sequencing results showed no changes in the Pol I‐mediated yEGFP3 expression cassettes in plasmids rescued from the mutants BXZ08m1‐3 and their parental strain BXZ08 (Fig. S3). These results indicate that mutagenesis could probably induce some changes in other factors and thus alter rRNA synthesis regulation homoeostasis. Coupling the innovative Pol I and IRES‐mediated monocistronic GFP expression system with flow cytometry, a large‐scale screening model for S. cerevisiae with high‐nucleic‐acid content worked well.

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.

Discussion

IRESs are the 5′ cap‐independent translation initiation elements. In particular, the native CrPV‐IRES, found in the IGR of CrPV, led the translation of the second reporter gene in the Pol II‐mediated dicistronic expression system in S. cerevisiae. However, its emerging validity should cooperate with the disruption of some cap‐dependent translation initiation factors (Thompson et al., 2001; Deniz et al., 2009). A more effective element called IRES12 (the native CrPV‐IRES with the just following12 nt sequence) already tested in vitro in yeast cell‐free protein synthesis system (Hodgman and Jewett, 2014), was used for inducing the translation in the IRES and Pol I‐mediated monocistronic expression system in this study. Normally, Pol I can only transcribe rRNA, but by using the IRES and Pol I‐mediated monocistronic expression system, we showed that proteins could be smoothly synthesized (Fig. 2I), and we highlighted the unnecessary disruption of some cap‐dependent translation initiation factors (Fig. 2C). Nevertheless, deletion of eIF5B (fun12Δ) indeed enhanced cell growth phenotypes (Fig. 2E), indicating that the cap‐dependent translation initiation factors also have a negative impact on such cap‐independent translation induced by IRES12, the more effective IRES element. In fact, it was demonstrated that, in the dicistronic expression system, the efficiency reduction of cap‐dependent initiation can also enhance the activity of CrPV‐IRES, even if it does not require any known translation initiation factors. Cap‐dependent and cap‐independent translation initiation competed with each other due to the limited number of unemployed ribosomes in vivo (Thompson et al., 2001; Jan and Sarnow, 2002; Deniz et al., 2009). On the other hand, it was reported that IRES‐mediated cap‐independent translation initiation in eukaryotes generally functions when cells encounter some stress such as heat shock, hypoxia and nutrient deprivation (Holcik et al., 1999; Holcik and Sonenberg, 2005; Gilbert et al., 2007; Reineke et al., 2011). We suggest that if the stress‐related gene is used as a selective marker, the expression system might work better under stress conditions.

By combining the innovative IRES and Pol I‐mediated monocistronic expression system with flow cytometry, a large‐scale screening model for high‐nucleic‐acid content yeasts based on the change of GFP green fluorescence intensity has been successfully established. After ARTP mutagenesis, about 100 yeast cells with fluorescence intensity > 2000 a. u. caught our attention; among them, the BXZ08m3 strain with 176.8 mg g−1 DCW total RNA content was selected through simple fluorescence intensity test and RNA content measurement, which was with 58% improvement compared to the parental strain BXZ08. Meanwhile, the data kept steady at least after 10 days of subculture (Fig. 4). Importantly, since there are strong or weak promoters in single‐nucleotide polymorphism, the rDNAp sequence controlling the yEGFP3 in these high‐nucleic‐acid content yeast episomal plasmids did not change (Fig. S3). Besides, the sequences of other introduced elements also showed no variation. These results indicated that mutagenesis might perturb the rRNA biosynthesis homoeostasis.

Conclusions

In this study, an innovative IRES and Pol I‐mediated monocistronic expression system for large‐scale screening of high‐nucleic‐acid content yeasts has been designed and established, in which the elements included rDNAp, IRES12 from CrPV carrying its preferred initiation codon GCU, reporter genes without their original initiation codon ATG, oligo‐dT and rDNAt. The regulatory changes of rRNA synthesis perturbed by mutagenesis were monitored by observing the variation in the expression status of GFP. In combination with flow cytometry, the BXZ08m3 strain which showed a 58% improvement in total RNA content has been efficiently sorted out and exhibited no sequence alteration in the Pol I‐mediated yEGFP3 expression cassette in the episomal plasmid. This provides at least two meaningful enlightenment. First, the episomal plasmid containing yEGFP3 (Table 1, Line 6) can be easily lost under non‐selective pressure condition continuously (Dani and Zakian, 1983) to get a non‐transgenic high‐nucleic acid industrial strain (wild type, polyploidy), which might be very beneficial in food industry for health so far. Second, these high‐nucleic‐acid content yeasts could be the useful research materials in the study of the rRNA synthesis regulation mechanism, as in omics analysis.

Experimental procedures

Plasmid/strain construction and media

In this study, the skeleton of the yeast episomal plasmid was pMA91 with a LEU2 marker (Mellor et al., 1983), but without the PGK1 promoter (PGK1p), which was removed by partial digestion with BglII and HindIII (Fig. S1). The fragments of URA3 and yEGFP3 without the original initiation codon ATG (ATGΔ URA3 and ATGΔ yEGFP3) were respectively amplified from plasmids pJFE3 (Shen et al., 2012) and pJFE3‐yEGFP3 (Wang et al., 2013). The different Pol I‐mediated gene expression cassettes (Fig. 1A) were ligated to the skeleton between the BglII and HindIII sites in three steps. (i) First, the fragments containing rDNAp, IRES12, reporter genes (ATGΔ URA3 and ATGΔ yEGFP3), but devoid of oligo‐dT and rDNAt were obtained by overlap extension polymerase chain reaction (OE‐PCR), in which XhoI, SdaI and BamHI restriction sites were respectively introduced to the 3'‐end of the reporter genes, and ligated to the skeleton between the BglII and HindIII sites (Fig. S1). (ii) Subsequently, to reduce undesired mutations occurred in oligo‐dT, the sequence was artificially synthesized and constructed in pUC19, and the rDNAt fragment was amplified and ligated to the oligo‐dT containing plasmid between XbaI and SdaI sites. (iii) Finally, the XhoI/SdaI‐digested fragment of oligo‐dT‐rDNAt was inserted between XhoI and SdaI sites which located downstream of the reporter genes, resulting in the individual completed expression plasmids (Fig. 1A). Meanwhile, a normal URA3 expression cassette controlled by Pol II recognized TEF1p (Translational Elongation Factor 1‐alpha promoter) was also ligated between the BglII and HindIII sites (Fig. 1B). The plasmids constructed in this study are listed in Table 1, and the primers used in this study are listed in Table S1.

The S. cerevisiae strain BY4741 (MATa, his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) (EUROSCARF, Frankfurt am Main, Germany) was used as the host. Following methods described in our previous work (Peng et al., 2012), the three disruption cassettes FUN12F‐loxP‐KanMX‐loxP‐FUN12R, IMT3F‐loxP‐KanMX‐loxP‐IMT3R and IMT4F‐loxP‐KanMX‐loxP‐IMT4R were obtained from PCR by using the three pairs of primers containing the upstream and downstream sequences of the relevant genes as recombinant arms. The disruption cassettes were then transformed into BY4741 by the dominant selection marker KanMX (Güldener et al., 1996) to disrupt the genes FUN12 (encoding the translation initiation factor eIF5B) and IMT3/IMT4 (encoding the Met‐tRNAi Met) (Thompson et al., 2001; Deniz et al., 2009). The empty and recombinant plasmids were transformed into BY4741 and its derivatives, the fun12Δ and imt3Δ/4Δ deletion strains, and were selected by the original LEU2 selection marker in pMA91. All S. cerevisiae strains used in this study are listed in Table 1, and the primers used in this study are listed in Table S1.

Yeast extract peptone dextrose (YPD) (20 g l−1 glucose, 10 g l−1 yeast extract, 20 g l−1 peptone) and synthetic complete (SC) (20 g l−1 glucose, 1.7 g l−1 yeast nitrogen base, 5 g l−1 ammonium sulfate, supplemented respectively 0.77 g l−1 CSM‐Ura or CSM‐Leu (Sunrise Science Products, USA) media were used to cultivate the yeast cells.

Spot dilution growth assay

The overnight cultures were inoculated in fresh liquid medium and cultured to the mid‐exponential phase (OD600 0.8–1.0), centrifuged, washed and resuspended in 1.0 ml sterile water for 9 h to consume the intracellular nutrients. The cell density was normalized to OD600 1.0. The 4 µl of serial 10‐fold dilutions were spotted onto the appropriate plates and incubated at 30 °C.

Cell disruption and western blot analysis

Cells were cultured to the mid‐exponential phase, then disrupted by vortexing with glass beads in a 4°C cold breaking buffer (20 mmol l−1 Tris‐HCl, pH 7.9, 10 mmol l−1 MgCl2, 1 mmol l−1 EDTA, 1 mmol l−1 PMSF, 1 mmol l−1 DTT, 0.3 mmol l−1 (NH4)2SO4, 5% glycerol phosphate). In total, the cell lysates containing 100 μg proteins were analysed by SDS‐PAGE and transferred to a polyvinylidene fluoride membrane, using an anti‐FLAG antibody at 1:2500 dilution, antibody IgG (H + L) HRP at 1:10,000 dilution (both from ABclonal Biotechnology, China), and finally detected with the Amersham Imager 600 imaging system (GE Healthcare, USA) (MacPhee, 2010).

Cell fluorescence intensity analysis and cell sorting via flow cytometry

Cells were cultured in SC medium minus leucine (−Leu) and harvested at mid‐exponential phase, washed twice with ice‐cold 10 mmol l−1 phosphate buffer (PBS, pH 7.0) and resus pended in PBS. Samples of 2 × 105 cells were monitored through the FITC (Fluorescein isothiocyanate) channel (excitation and emission wavelengths of GFP were 488 and 507 nm, respectively) (Zhang et al., 2015) via flow cytometer (MoFlo™ XDP High‐Performance Cell Sorter, Beckman Coulter, USA) and analysed with the Kaluza Analysis 2.1 software. The mean fluorescence intensity (MFI) was the sum of fluorescence intensity for each single cell divided by the 2 × 105 analysed cells. Meanwhile, the cells with fluorescence intensity > 2000 a.u. were sorted out as high‐nucleic‐acid content yeast candidates.

ARTP mutagenesis process

ARTP (ARTP‐IIS Wuxi Tmaxtree Biotechnology, Wuxi, China) is an effective and now widely used physical mutagenic system (Zhang et al., 2014; Guo et al., 2019). The 10 SLM (standard litre per minute) high‐purity helium as the plasma working gas, 90W of the radio frequency power input, and 3.5 mm distance between the plasma torch nozzle and the treated sample plates were used in this study. Briefly, cells were cultured to mid‐exponential phase and harvested. About 104–105 cells suspended in 5% glycerol were spread on a sterilized sample plate and treated with the ARTP system. The lethality rate was calculated as untreated colonies (total cells) minus treated colonies (survived cells), divided by the total cells which were grown on YPD plates (Chen et al., 2010). An exposure time of 20 s with 90% lethality rate (Fig. S2) was used as the working dose. After treatment, the samples were washed with 1 ml sterile water and inoculated into a SC liquid medium minus leucine (−Leu), then incubated for 2 days at 30°C, after which the cells were transferred to the medium again, the fresh culture in mid‐exponential phase were ready for flow cytometry analysis.

Meanwhile, the high‐nucleic‐acid content mutants were continuously subcultured. Once the cells reached the stationary phase, a new batch was started by transferring the culture into fresh medium. After 10 days, the fresh cultures in mid‐exponential phase were also harvested for the MFI test and total RNA content measurement.

Extraction and measurement of total RNAs

Total intracellular RNAs were extracted with perchloric acid (PCA) (Herbert et al., 1971; Chuwattanakul et al., 2011). Briefly, 1.0 OD600 cells were suspended in 1 ml of 0.25 mol l−1 ice‐cold PCA for 30 min and centrifuged. The cell pellets were then resuspended in 1.0 ml of 0.5 mol l−1 PCA at 70°C for 20 min. After centrifugation at 12,000 r.p.m. for 2 min, the absorbance at 260 nm of the supernatant, multiplied by a 0.04 coefficient (Eppendorf BioPhotometer D30), was the determined RNA content (mg) of 1.0 OD600 cells. The dry cell weight (DCW) of strains were determined according to a previously reported method (Xu et al., 2014). The RNA content value was then divided by the DCW to get the RNA content (mg g−1 DCW).

Statistical analysis

Statistical significance was determined by Mann–Whitney U test for the differences between two groups and by one‐way ANOVA and post hoc LSD test (homogeneity of variance) for differences between multiple groups using the software SPSS (SPSS Inc, Chicago, USA). Error bars denote mean ± standard deviation. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01).

Conflict of interest

None declared.

Fig. S1. The skeleton of the yeast episomal plasmid in this study. Physical maps of plasmid (a) pMA91 (Mellor et al., 1983), and (b) the skeleton derived from pMA91 with no PGK1 promoter.

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Fig. S2. The lethality curve of the ARTP treatment. The ARTP‐IIS Wuxi Tmaxtree Biotechnology, Wuxi, China mutagenesis system was used to deal with 104‐105 cells harbouring the Pol I‐mediated GFP expression cassette (Table 1, Line 15), with the distance of 3.5 mm between plasma nozzle and the sample plates.

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Fig. S3. The 1850 bp DNA sequences from promoter to terminator in the Pol I‐mediated yEGFP3 expression system in mutants BXZ08m1‐3 did not generate any changes after mutagenesis, compared with their parental strain BXZ08 (ATGΔ yEGFP3 IRES12, Pol I).

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Table S1. List of primers used in this study.

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