Characterization of five terminator regions that increase the protein yield of a transgene in Saccharomyces cerevisiae

Journal of Biotechnology 168 (2013) 486–492

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Characterization of five terminator regions that increase the protein yield of a transgene in Saccharomyces cerevisiae Yoichiro Ito a , Mamoru Yamanishi a , Akinori Ikeuchi b , Chie Imamura b , Kenro Tokuhiro b , Takao Kitagawa c , Takashi Matsuyama a,∗ a

Matsuyama Research Group, Toyota Central Research and Development Laboratories, Incorporated, Japan Biotechnology Laboratory, Toyota Central Research and Development Laboratories, Incorporated, Japan c Department of Biochemistry and Functional Proteomics, Yamaguchi University, Japan b

a r t i c l e

i n f o

Article history: Received 14 June 2013 Received in revised form 30 August 2013 Accepted 30 September 2013 Available online 12 October 2013 Keywords: Metabolic engineering Terminator region 3 -UTR activity Fluorescent protein Cellulase

a b s t r a c t Strong terminator regions could be used to improve metabolically engineered yeasts by increasing the target enzyme protein yields above those achieved with traditional terminator regions. We recently identified five strong terminator regions (RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t) in a comprehensive analysis of Saccharomyces cerevisiae. The effect of the terminator regions was analyzed by measuring the protein production of a linked transgene, and was shown to be twice that of a traditional terminator region (PGK1t). Here, we investigated whether the activity of the terminator regions is affected by exchange of a strong promoter or reporter in the linked transgene, carbon source for cell growth, stress factors, host yeast strain, or stage of the growth phase. Our results indicate that the activities of all five terminator regions were twice that of PGK1t in all conditions tested. In addition, we demonstrated that the strong activity of these terminator regions could be used to improve secretory production of endoglucanase II derived from Tricoderma ressei, and that the DIT1t strain was the best of the five strains for this purpose. We therefore propose that DIT1t, and the four other terminator regions, could be applied to the development of improved metabolically engineered yeasts. © 2013 Elsevier B.V. All rights reserved.

1. Introduction To develop transgenic microorganisms that produce biocatalytic enzymes needed for the manufacture of bio-plastics and bio-fuels (Jarboe et al., 2010; Kung et al., 2012; Luengo et al., 2003; PeraltaYahya and Keasling, 2010; Siddiqui et al., 2012; Stephanopoulos, 1998), exogenous genes encoding the target proteins must be strongly expressed (Da Silva and Srikrishnan, 2012). In genetically engineered yeast, a strong constitutive promoter, TDH3 promotor (TDH3pro) or ADH1pro, is primarily used for this purpose; but, in some cases, inducible or strong conditional promoters such as GAL1pro and PDC1pro (Ishida et al., 2005) are selected. In contrast to promoters, only a small subset of terminator regions, comprising PGK1t, ADH1t, and CYC1t, are used routinely. Terminator regions are primarily associated with two types of complex events. One of these events is “transcriptional termina-

Abbreviations: opGFP, codon-optimized green fluorescent protein; FI, fluorescence intensity; SS, side-scatter intensity; RP, ribosomal protein. ∗ Corresponding author at: Toyota Central Research and Development Laboratories, Incorporated, Aichi 480-1192, Japan. Tel.: +81 561 71 7424; fax: +81 561 63 6498. E-mail address: [email protected] (T. Matsuyama). 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.09.024

tion”, in which the cleavage of 3 -mRNA and poly(A) addition occurs (Kuehner et al., 2011; Richard and Manley, 2009). The other is “post-transcriptional regulation”, in which the 3 -UTR of mRNA determines stability, translational efficiency, and localization of mRNA (Kuersten and Goodwin, 2003). In S. cerevisiae, the 3 -UTR of mating pheromone a-factor (MFA2) has been investigated in detail. MFA2 mRNA has a short half-life (t1/2 ) of 3.5 min (Decker and Parker, 1993; Herrick et al., 1990; Muhlrad and Parker, 1992). When the 3 -UTR of PGK1 (t1/2 = 45 min) was substituted for that of MFA2, the stability of the chimeric PGK1-MFA2 transcript was about half that of the parental PGK1 transcript, demonstrating that the 3 -UTR sequence of MFA2 can destabilize its mRNA. Thus, replacement of the 3 -UTR potentially modulates the protein yield of transgenes. If terminator regions increase expression of the target enzymes, use of a strong terminator region together with a strong promoter would be expected to synergistically increase transgene production. Recently, we performed a comprehensive analysis of yeast terminator region activity (5302 out of a total of 5880 genes) by measuring the expression of a linked GFP reporter gene under the control of TDH3pro (Yamanishi et al., 2013). The activities of the terminator regions ranged from 0.036 to 2.52 relative to that of the standard, PGK1t, which is one of the most routinely used terminator regions. Twenty-one of the terminator regions exhibited

Y. Ito et al. / Journal of Biotechnology 168 (2013) 486–492

BglII,ClaI

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Fig. 1. Schematic diagram of the genome-integrated gene constructs. Each terminator region (PGK1t, RPL41Bt, RPL15At, DIT1t, RPL3t, or IDP1t) was inserted as a module after a promoter (TDH3pro or ADH1pro) and a fluorescent reporter protein gene (opGFP or mKO2). Restriction sites are indicated. PDC6-5 and PDC6-3 denote the 5 - and 3 -regions of PDC6, respectively. A marker gene (TRP1 or URA3) was inserted for selection purposes.

strong activity that was more than twice that of PGK1t. We proposed that the terminator regions modulate the protein production of the transgene as a genetic component, as does the ribosome binding site in Escherichia coli. In this study, we characterized the activity of the top 5ranked terminator regions (RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t) under various growth conditions, under two different strong promoters, and in several yeast strains, because 3 -UTR activity depends on the growth conditions (Lind and Norbeck, 2009) as well as promoter activity (Partow et al., 2010). To increase the efficiency of ethanol production from biomass feedstock in the sustainable society to come, we require improved techniques for display of functional cellulosomes or cellulases on the yeast surface, based on secretory production of exogenous enzymes (Fujita et al., 2002; Tsai et al., 2013; Wen et al., 2010; Yamada et al., 2011). Here, we show that the strong terminator regions improved the secretory production of endoglucanase II derived from Tricoderma ressei, TrEG2, and the secretory production of cellulase, as shown by the production of the fluorescent protein. Taken together, our results suggest that these terminator regions will be useful for metabolic engineering in S. cerevisiae. 2. Materials and methods 2.1. Strains and media Six S. cerevisiae strains were used as wild-type strains in this study: A451 (MAT␣ can1 leu2 trp1 ura3 aro7); BY4741 (MATa his31 leu20 met150 ura30); TDO2 (MATa/␣ trp1/trp1 ura3/ura3) made from OC-2T (Saitoh et al., 1996) with the use of 5-Fluoroorotic acid (5-FOA); W303-1a (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15); YPH499 (MATa ura3-52 lys2-801 ade2-101 trp163 his3-200 leu2-1); and BJ5465 (MATa ura3–52 trp1 leu2D1 his3D200 pep4::HIS3 prb1D1.6R can1 GAL). Yeast strains were grown in synthetic complete medium (SC medium), which contains a 0.67% Yeast Nitrogen Base without amino acids (Difco, Detroit, MI), 0.082% Complete Supplement Mixture without TRP or URA (both from ForMedium, Norfolk, UK), and adenine (40 mg/L), supplemented with various carbon sources, or in YPD medium, which contains 1% yeast extract, 2% peptone, 2% glucose, and adenine (40 mg/L).

(−12 to +481), and IDP1t (+1 to +462). These regions are the same as those used in the previous genome-wide analysis (Yamanishi et al., 2013). A TRP1 or URA3 marker gene was inserted between the BamHI and PstI sites downstream of the terminator region. The genome of the W303-1a strain was used as the template for PCR amplification of the terminator sequences. All cloning procedures were performed with In-Fusion Advantage PCR Cloning Kit (Clontech, Mountain View, CA). The genome integration cassettes were linearized by ClaI/ApaI digestion and then introduced into the yeast strains at PDC6 locus by using a Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA). The genome-integrated strains were grown on suitable selection media. The integration of each DNA fragment into genomic DNA was confirmed by applying the colony PCR technique. The genotypes of the 48 genome-integrated strains used in this study are summarized in Table S1. 2.3. Promoter and reporter swapping experiment The promoter and reporter swap strains were grown in SD medium (i.e., SC medium containing 2% glucose) at 30 ◦ C in a test tube shaken at 70 rpm. Overnight cultures were diluted with 6 mL of fresh SD medium in an L-shape tube to an OD660 of ∼0.1. The diluted cells were cultured at 30 ◦ C with the use of a rocking incubator (TN-1506, ADVANTEC, Tokyo, Japan) for 5–6 h until an OD660 of ∼0.6 was reached, the fluorescence of the cells were measured with a flow cytometer, as described below. 2.4. Medium exchange experiment The genome-integrated W303-1a strains containing the five terminator regions were pre-cultured and cultured in an L-shape tube containing SD medium at 30 ◦ C as described above until an OD660 of ∼0.6 was reached. The yeast cells were then transferred into the appropriate media (YPD, SD with 100 mM NaCl or 1 M sorbitol, or SC medium containing 2% acetate, 2% ethanol, 2% galactose, 10% glucose, or 2% glycerol, respectively). Following an additional incubation at 30 ◦ C for 6 h with shaking on the rocking incubator for acclimation, the GFP fluorescence of the cells was measured with a flow cytometer, as described below. 2.5. Growth-phase dependency test

2.2. Construction of genome-integrated strains All genome integration gene cassettes were constructed as described in the previous study (Fig. 1) (Yamanishi et al., 2011). In separate constructs, each terminator region was inserted between the KpnI and BamHI sites located downstream of a reporter gene, which encoded either codon-optimized green fluorescent protein (opGFP) (Yamanishi and Matsuyama, 2012) or monomeric Kusabira Orange 2 (mKO2) fluorescent protein (Amalgaam, Tokyo, Japan). The reporter gene opGFP was expressed under the control of TDH3pro or ADH1pro, and the mKO2 reporter gene was expressed under the control of TDH3pro. The terminator regions used in this study were: PGK1t (+4 to +429, where “+1” is defined as the nucleotide immediately downstream of the stop codon), RPL41Bt (+1 to +455), RPL15At (−3 to +426), DIT1t (+1 to +434), RPL3t

After the overnight cultures were diluted, as described above, they were incubated in SD medium for 24 h with shaking on the rocking incubator. During the incubation period, sampling was performed at 6 h, 12 h, and 24 h, and the samples were used immediately for flow cytometry (FCM) and for quantification the concentrations of ethanol and glucose using enzyme-coupled electrodes (Bio Flow 5i, Oji Scientific Instruments, Amagasaki, Japan). 2.6. Flow cytometry Measurements were performed as described previously (Matsuyama et al., 2011). After the cell cultures were diluted 10–40 fold with physiological saline, the fluorescence intensity (FI) of approximately 10,000 cells in each sample was measured by

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using a flow cytometer (Cell Lab Quanta SC MPL, Beckman-Coulter, Brea, CA) equipped with a filter set (570/15) for mKO2 or a filter set (510/10) for GFP and a 488 nm laser. The histogram of the ratio of FI/side-scatter intensity (SS) in each cell was fitted to a log-normal distribution calculated by using IgorPro software (ver. 6.1, Wavemetrics, Lake Oswego, OR), as described in our previous study (Yamanishi et al., 2013). Because the cell-to-cell variability within each integrated strain harboring a terminator region was negligible (data not shown), the peak value of the fitted log-normal distribution was used as the 3 -UTR activity for each strain.

Relative 3'-UTR intensity (a.u.)

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2.7. Quantitative PCR analysis The level of GFP mRNA expression in each yeast strain was measured as described in our previous paper (Yamanishi et al., 2013). Briefly, total RNA was isolated from the same cultures as those used for FCM by using a High Pure RNA Isolation Kit (Roche, Basel, Switzerland). cDNA templates were synthesized from 1 ␮g of each total RNA by using a High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad CA). To quantify target cDNAs, realtime PCR analysis with SYBR Green I was performed with the use of a SYBR Green PCR Master Mix in an ABI PRISM 7000 Sequence Detection System (both from Life Technologies). TUB1 expression was used as the internal standard. 2.8. Secretory production of cellulase The secretory expression vector was constructed by inserting TDH3pro, the secretion signal sequence of the glucoamylase gene from Rhizopus oryzae, the TrEG2 gene, and the respective terminator regions into SmaI-digested CEN/ARS-based plasmid pAUR112 (GenBank Accession No., AB012283; Takara Bio, Shiga, Japan) by using the In-Fusion Advantage PCR Cloning Kit. The sequences for TDH3pro and the secretion signal were obtained from pRS436GAP (DDBJ accession no., 304862; (Ohto et al., 2009)), and the TrEG2 gene was artificially synthesized by Genscript (Piscataway, NJ). The terminators inserted into the secretory expression vectors were the top five–ranked terminators and PGK1t described above, MFA2t and ICY2t (Yamanishi et al., 2013), CYC1t (Yamanishi et al., 2011), and ADH1t from pESC-His (Agilent Technology, Santa Clara, CA). The plasmids were introduced into the protease-deficient yeast strain BJ5465 by using a Frozen-EZ Yeast Transformation II Kit and plated on SD-URA agar. Colonies were picked into 500 ␮l SD-Ura medium supplemented with 1% casamino acids in a 96 deep well plate and grown at 30 ◦ C with shaking at 1800 rpm for 24 h in a specialized shaker for deep well plates (MBR-022UP, Taitec, Aichi, Japan). A 100 ␮l aliquot of each culture was inoculated into 500 ␮l SD-URA supplemented with 1% casamino acids and grown at 30 ◦ C with shaking at 1800 rpm for 24 h. Cultures were centrifuged, and 5 ␮l aliquots of the supernatants were used for measuring cellulase activity with phosphoric acid-swollen cellulose (PASC) as the substrate and reducing sugar as the product assayed, as described in our previous paper (Ito et al., 2013), except that the cellulase reaction was at 50 ◦ C for 1.5 h. All the measurements were performed in sextuplicate. To avoid the problem of instable secretory expression, twelve colonies each of PGK1t, DIT1t, RPL41Bt, ICY2t strain and a strain harboring pAUR112 (negative control) were cultured as described above. After the final culture step, each culture was mixed with other cultures of the same strain and centrifuged to obtain a supernatant with an average level of secreted cellulase. Each supernatant was concentrated 50 times by using an ultrafiltration device (Vivaspin 500-30K, GE Healthcare), and then 20 ␮l aliquots were used for SDS-PAGE. Following CBB staining, the gel was scanned by

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Fig. 2. Effects of promoter and reporter gene exchange on 3 -UTR activities. Relative outputs were calculated as the ratio between the 3 -UTR activity (the peak value of log-normal fit of the FI/SS values measured by FCM) of the strain containing the indicated terminator region and that of the respective PGK1t strain. The terminator strains expressed either the GFP gene or the mKO2 gene under the TDH3pro or ADH1pro, as indicated. Data for GFP under the TDH3pro (left group) are provided as a control. The terminator strains examined were RPL41Bt (open bars), RPL15At (hatched bars), DIT1t (black bars), RPL3t (light gray bars), and IDP1t (gray bars). Values are the mean of three independent experiments, and error bars represent standard deviation.

using an image analyzer (F-9000, Fuji-Film, Japan) and the image was analyzed by using Multi gauge software (Fuji-Film, Japan). 2.9. Statistical tests Student’s t-tests with Bonferroni correction were used to determine the significance of the differences between groups of values. P values less than 0.05 were considered to be statistically significant. 3. Results 3.1. Effects of promoter and reporter gene swaps on 3 -UTR activity To determine whether inclusion of the strong terminator regions identified in our previous study (Yamanishi et al., 2013) leads to more effective transgene expression in general, we examined the 3 -UTR activities of the top five terminator regions, RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t relative to that of PGK1t in transgenic yeasts harboring a strong promoter, ADH1pro instead of TDH3pro (Fig. 2, middle), or the mKO2 reporter gene instead of the GFP reporter gene (Fig. 2, right). All genome integration cassettes were introduced into the yeast strains at the PDC6 locus, the deletion of which does not change the phenotype or pyruvate decarboxylase activity (Hohmann, 1991). Although genome-integrated strains harboring the GFP gene under ADH1pro or the mKO2 gene under TDH3pro exhibited comparable relative 3 -UTR activity values compared with those exhibited by the equivalent strains harboring the GFP gene under TDH3pro, the five terminator regions conferred about twice the relative 3 -UTR activity as PGK1t (all P < 0.05, Fig. 2). These results indicate that the presence of any of the five terminator regions was sufficient to substantially increase the protein yield of a transgene even in the presence of the strong promoter, and this effect was not dependent on the nature of the promoter or transgene sequence.

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Fig. 3. Effects of carbon sources on 3 -UTR activity. The FI and SS values of terminator strains with the GFP gene under the TDH3pro were measured by FCM and used to calculate the relative 3 -UTR activity (see Section 2). The PGK1t strain was used as a standard (relative 3 -UTR activity = 1). The investigated media were SC with 2% acetate, 2% ethanol, 2% galactose, 2% glycerol, or 10% glucose, and YPD, respectively. The terminator strains examined were RPL41Bt (open bars), RPL15At (hatched bars), DIT1t (black bars), RPL3t (light gray bars), and IDP1t (gray bars). Values are the mean of three independent experiments, and error bars represent standard deviation.

3.2. Effects of carbon source and stress on 3 -UTR activity Promoter activity is substantially dependent on the growth conditions (Sun et al., 2012), especially carbon source and stress factors. Therefore, we investigated whether 3 -UTR activity is affected by various carbon sources. In the previous analysis (Yamanishi et al., 2013), the yeast cells were grown in medium containing 2% w/v glucose. Here, we examined four carbon sources (acetate, ethanol, galactose, and glycerol) at the concentration used for glucose in the previous study, as well as a higher concentration of 10% w/v glucose (Fig. 3). Of the five strains, the DIT1t strain exhibited the strongest 3 -UTR activity in all conditions tested except acetate-containing medium (P < 0.05 in the four conditions). In the presence of glycerol, the DIT1t strain showed almost 3 times the 3 -UTR activity of the PGK1t strain. When the strains were incubated in nutrient-rich YPD medium, similar relative activities were observed in the DIT1t, RPL41Bt, and RPL3t strains (P < 0.05). Salt stress (100 mM NaCl) and osmotic stress (1 M sorbitol) had a slight but significant effect on 3 -UTR activity (Fig. 4). So even under stress conditions, the activity of the five terminator regions was more than twice that of the PGK1t strain. These results indicate that the five terminator regions would be useful in any growth conditions. 3.3. Comparison of 3 -UTR activity between five S. cerevisiae strains during log and stationary growth phases Among various wild-type S. cerevisiae strains, considerable numbers of genes are differentially expressed at the protein level (Rogowska-Wrzesinska et al., 2001). These differences among strains might be regulated via the 3 -UTR of the respective mRNAs. In our previous study, we used the haploid strain W303-1a for our comprehensive analysis (Yamanishi et al., 2013). Here, we compared the activity of the five terminator regions in four haploid laboratory strains, W303-1a, A451, BY4741, and YPH499, and one diploid strain, TDO2 (Fig. 5 and Fig. S1), which were arbitrarily selected from among the stocks available in-house. All genome integration cassettes were introduced into the above host strains at the PDC6 locus. Additionally, to determine the effect of growth phase, yeast cells (OD660 = 0.1) were inoculated into fresh SD medium (0 h time point), and the relative 3 -UTR activities of the cells were measured at 6 h, 12 h, and 24 h of incubation. For all

2

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100 mM NaCl

1M Sorbitol

Fig. 4. Effects of salt and osmotic stress on 3 -UTR activity. The FI and SS values of terminator strains with the GFP gene under the TDH3pro were measured by FCM and used to calculate the relative 3 -UTR activity (see Section 2). The PGK1t strain was used as a standard (relative 3 -UTR activity = 1). The investigated media were SD with 100 mM NaCl or 1 M sorbitol. The terminator strains examined were RPL41Bt (open bars), RPL15At (hatched bars), DIT1t (black bars), RPL3t (light gray bars), and IDP1t (gray bars). Values are the mean of three independent experiments, and error bars represent standard deviation.

strains and growth-phase conditions, the five terminator strains showed significantly higher 3 -UTR activity than that of the PGK1t strain (i.e., relative 3 -UTR activities were all >1, all P < 0.05). The relative 3 -UTR activities of the RPL41Bt, RPL15At, RPL3t, and IDP1t strains slightly decreased as the yeasts progressed through the growth phases (Fig. 5). In contrast, the relative 3 -UTR activity of the DIT1t strain increased with time in culture (Fig. 5). This result indicates that DIT1t acts by a different mechanism to the others. 3.4. Application for secretory expression of cellulase To explore the potential application of the strong terminator regions for metabolic engineering, we assessed their ability to increase secretory production of cellulase from protease-deficient yeast strain BJ5465, which is generally suitable for production of secreted products (Ito et al., 2013; Morawski et al., 2000). Here, we chose to use not only the top five terminator regions and the standard terminator, PGK1t, but also weaker terminators, MFA2t and ICY2t, and commonly used terminators, CYC1t and ADH1t, for comparison. The cellulase activity in an aliquot of the yeast-free supernatant of the yeast culture containing secreted TrEG2 was higher for the top five terminator strains than for the commonly used strains (PGK1t, CYC1t, and ADH1t strains, Fig. 6A). The DIT1t strain showed significantly higher cellulase activity than the other strains (Fig. 6A and Table S3, P < 0.05). Fig. 6B shows the estimated TrEG2 production level in various strains plotted against the GFP FI data from our previous genome-integration analysis (Yamanishi et al., 2013). Although each of the GFP strains harboring a top five terminator showed almost the same GFP FI, the level of cellulase secretory production in the DIT1t cellulase strain (2.2 times that in the PGK1t cellulase strain), was higher than that of the other 4 strains (each slightly less than 1.5 times that in the PGK1t cellulase strain). The average level of secretory production was also confirmed by measuring the density of each corresponding band after SDS PAGE (Fig. 6C). The secretory production levels of the DIT1t, RPL41Bt, and ICY2t strains relative to the PGK1t strain were 2.0, 1.6, and 0.33, respectively. This production level, which was measured directly, corresponded well with the estimated production level. Because the DIT1t cellulase strain (Fig. 6) and the DIT1t GFP strain (Fig. 5) were both cultured until stationary phase (i.e., for 24 h), the results suggest that the DIT1t cellulase strain was more

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Relative 3'-UTR activity (a.u.)

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Fig. 5. Effect of host strain on the 3 -UTR activity in various growth phases. Each of the wild-type S. cerevisiae strains (W303-1a, A451, BY4741, YPH499, and TDO2) was separately transfected with constructs harboring the top five terminators. The FI and SS values of terminator strains with the GFP gene under the TDH3pro were measured by FCM and used to calculate the relative 3 -UTR activity (see Section 2). The PGK1t strain was used as a standard (relative 3 -UTR activity = 1). Sampling times were 6 h, 12 h, and 24 h. The terminator strains examined were DIT1 (closed circles), RPL41Bt (open circles), RPL15At (open triangles), RPL3t (open squares), and IDP1t (closed squares). Values are the mean of three independent experiments.

efficient than the other cellulase strains for secretory production under these experimental conditions. 3.5. Quantitative RT-PCR analysis of the top 5 terminator strains The molecular mechanism by which strong terminator regions increase transgene expression remains to be elucidated. The strong terminator regions addressed in this study effectively increased protein yield, regardless of the strong promoter used (TDH3pro or ADH1pro) in transgenic yeasts harboring low-copy numbers of transgenes. To investigate whether the regulation of the terminator regions was at the post-transcriptional or translational level, RT-PCR analysis was performed for the top five terminator GFP

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strains (Fig. 7). The relative GFP mRNA levels of the DIT1t and RPL3t strains were higher than those of the other three top terminator strains, which was a similar trend to that observed for GFP protein production, suggesting that attenuation of mRNA decay caused the increase in GFP production. In contrast, the IDP1t and RPL15At strains showed nearly the same GPF mRNA level as the PGK1t strain, in spite of the GFP protein production level of those strains being twice that of the PGK1t strain (Fig. 2), suggesting that these strains could be controlled by post-transcriptional or translational regulation. 4. Discussion In S. cerevisiae, little attention has been paid to the selection of terminator regions in efforts to increase transgene expression. Here, we investigated whether the five strong terminator regions RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t, identified in our previous study (Yamanishi et al., 2013), could be a useful alternative to the commonly used terminator regions PGK1t, ADH1t, and CYC1t. Two model fluorescent proteins and a cellulase were used as a demonstration for showing the higher 3 -UTR activities of the top five terminator regions. In particular, the cellulase experiment was performed under the condition of a secretory production. Our results indicate that all five terminator regions were promising candidates, with the activity of DIT1t being the highest under almost all conditions in transgenic yeast with a single genome-integrated transgene and a single copy plasmid.

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Fig. 6. TrEG2 secretory production. (A) The cellulase activity of secreted TrEG2 from each strain containing the indicated terminator region is shown. The PGK1t strain was used as a standard. (B) Comparison between TrEG2 secretory production and GFP production level for each terminator strain. The TrEG2 secretory production level was calculated from the cellulase activity (A) and its compensation experiment (Figure S2). Data for GFP FI were taken from our previous study (Yamanishi et al., 2013). (C) SDS-PAGE of secreted TrEG2 from some terminator strains. Strains with no cellulase gene (negative control, lane 1), and strains harboring PGK1t (lane 2), DIT1t (lane 3), RPL41Bt (lane 4), and ICY2t (lane 5) are indicated. The arrow indicates the band derived from secreted TrEG2. The size of the secreted TrEG2 (∼50 kDa) was a little higher than that estimated (42 kDa) because of the surface glycosylation required for secretory production of cellulase.

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Fig. 7. mRNA levels of the reporter GFP gene under the control of TDH3pro. GFP mRNA concentrations in the top 5 strains were analyzed by conducting quantitative RT-PCR. Relative GFP mRNA concentrations were then calculated as the ratio between the concentration in the strain containing the indicated terminator and that in the PGK1t strain. Data are means ± SD (n = 3).

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The Dit1 protein is a sporulation-specific enzyme that is involved in the production of a soluble LL-dityrosine-containing precursor of the spore wall that is required for spore wall maturation (Briza et al., 1990, 1994). Expression of the DIT1 gene is restricted to sporulating cells at the time of prospore enclosure, and during the vegetative stage it is negatively regulated more than 1000-fold at the transcriptional level by a negative regulatory element (NRE) in the promoter (Friesen et al., 1997). In this study, even during the vegetative stage, the strains harboring DIT1t showed strong fluorescence (Fig. 5). DIT1 gene is mainly regulated at the transcriptional level (Friesen et al., 1997), but could also be regulated at the post-transcriptional and/or translational levels. Our working hypothesis is that such regulations could be regulated by the interaction between cis RNA elements in the 3 -UTR of DIT1t and trans-acting factors. Once the DIT1 gene is transcribed, if the trans-acting factor were constantly present in the cell, Dit1p would efficiently accumulate to the quantities required to produce a mature spore cell wall. This “ready-to-produce” state might be essential for rapid response to severe environments in which nutrient deficiency triggers sporulation. The identification of the cis RNA element and the trans-acting factor is currently under investigation. The selection of terminator should complement, not compete, with other approaches for improving protein yield in yeast cells, such as strong or inducible promoters, single or multiple copy number plasmid vectors, and so forth (Da Silva and Srikrishnan, 2012; Kim et al., 2012). It offers the means to extend the dynamic range of transgene expression in the desired manner. For example, promoters regulate the transgenes at the transcriptional level, whereas terminators modulate the protein production of the transgenes at the post-transcriptional or translational levels. Thus, a promoter and terminator could control the protein yield of a transgene synergistically. In our previous work, use of the GAL1 promoter and the RPL41B terminator together increased the level of a transgene protein production to twice that obtained with the same promoter and the PGK1 terminator (Yamanishi et al., 2013). Transgenic yeasts harboring GAL1pro and RPL41Bt or GAL1pro and PGK1t showed a normal phenotype after galactose induction. Thus, like promoters, terminators can be combined with many other genetic approaches to modulate transgene expression. In conclusion, we identified five terminator regions that are more effective for increasing transgene expression in S. cerevisiae strains under various growth conditions than the traditional terminator region, PGK1t. Among the terminator regions in the cerevisiae genome, DIT1t was the most versatile. Even though more verification studies are needed, we put forward the use of these terminator regions as an alternative approach to enhance the expression of target genes, and propose that DIT1t could be applied to the development of useful metabolically engineered yeasts. Acknowledgments We thank Professor Rinji Akada for the supply of several plasmids. We also thank Mami Kanemitsu, Takako Nakamura, and Eriko Ohno for technical assistance. This work was financially supported in part by a grant from the New Energy and Industrial Technology Development Organization (NEDO), Japan (C.I.), and JSPS Grant-inAid for Exploratory Research Number 25660071 (T.M.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec. 2013.09.024.

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