RESEARCH ARTICLE

Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae Jun Ishii1, Takashi Kondo1, Harumi Makino1, Akira Ogura1, Fumio Matsuda1,2 & Akihiko Kondo2,3 1

Organization of Advanced Science and Technology, Kobe University, Kobe, Japan; 2RIKEN Biomass Engineering Program, Yokohama, Japan; and 3Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan

Correspondence: Akihiko Kondo, Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan. Tel.: +81-78-803-6196; fax: +81-78-803-6196; e-mail: [email protected] Present address: Takashi Kondo, Division of Natural Environment and Information, Faculty of Environment and Information Sciences, Yokohama National University, Japan Fumio Matsuda, Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Japan Received 18 July 2013; revised 8 November 2013; accepted 15 January 2014. Final version published online 10 February 2014.

Abstract Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering.

DOI: 10.1111/1567-1364.12138

YEAST RESEARCH

Editor: Jens Nielsen Keywords genetic engineering; metabolic engineering; multiple gene expression; biofuel and biochemical production.

Introduction The yeast species Saccharomyces cerevisiae is a traditional microorganism in brewing industries (Donalies et al., 2008). During the last two decades, ethanol fermentation with yeast has been studied for use in biofuel production (Lynd et al., 2005; Hasunuma et al., 2013; Kato et al., 2013; Suga et al., 2013). Interest in the production of other fuels and chemicals by microbial fermentation processes has recently increased (Atsumi et al., 2008; Mainguet & Liao, 2010; Yim et al., 2011). Yeast is a promising host organism for biofuel and chemical production due to its potential for bulk-scale production of fermentative compounds, tolerance for low pH and robustness for autolysis that reduce the risk of contamination, and abilFEMS Yeast Res 14 (2014) 399–411

ity to support long-term repeated or continuous fermentation (Matsuda et al., 2011; Kondo et al., 2012, 2013; Buijs et al., 2013). Genetic engineering is an indispensable and continually evolving technique in the biotechnology field (Kittleson et al., 2012). In cooperation with advancements in gene manipulation techniques, it is becoming necessary to introduce multiple genes into cells for dramatic changes in the characteristics of the host organisms. In metabolic engineering, genetically engineered microbes have been generated to produce diverse fuels and chemicals (Atsumi et al., 2008; Mainguet & Liao, 2010; Yim et al., 2011). For example, isobutanol production in S. cerevisiae was engineered by introducing five genes including two genes for the Ehrlich pathway and three genes for the cytosolic ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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valine biosynthesis pathway (Matsuda et al., 2012). Moreover, Escherichia coli that produced isoprenoid-derived amorphadiene was engineered by introducing more than ten genes, including three genes for the mevalonate (MEV) pathway, five genes for synthesis of farnesyl pyrophosphate (FPP), two genes encoding formate dehydrogenase (fdh1) and amorpha-4,11-diene synthase (ADS), and the lacI gene (Ma et al., 2011). While the prokaryote E. coli can easily express many genes as operons, the eukaryotic yeast S. cerevisiae does not permit transcription of mRNAs in a polycistronic fashion (Kondo et al., 2013). Therefore, a set of promoters in front of each expression gene is needed to express multiple genes in S. cerevisiae. However, it is laborious and complicated to prepare the expression cassettes or plasmids for various genes, and the inability to easily express multiple genes in S. cerevisiae has slowed yeastbased progress in metabolic engineering despite the growing need for bulk chemical production. In the present study, we developed systematic and convenient genetic engineering tools for introducing multiple genes into S. cerevisiae with the future aim to facilitate highly advanced modifications of yeast metabolism. We constructed a series of vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three promoters into the pRS yeast shuttle vectors (Sikorski & Hieter, 1989; Ishii et al., 2009). Comparative expression capabilities of the constructed vectors were tested with green fluorescent protein, and the concurrent expression of three genes was checked with three different fluorescent proteins.

Materials and methods Yeast strain and media

Saccharomyces cerevisiae YPH499 (MATa ura3-52 lys2-801 ade2-101 trp1-D63 his3-D200 leu2-D1) (Sikorski & Hieter, 1989) was used as the host yeast strain. Synthetic dextrose (SD) media contained 6.7 g L 1 of yeast nitrogen base without amino acids (YNB) (BD-Diagnostic Systems, Sparks, MD) and 20 g L 1 of glucose. Amino acids and nucleotides (40 mg L 1 adenine for transformation or 150 g L 1 adenine for cultivation 20 mg L 1 histidine, 60 mg L 1 leucine, 20 mg L 1 lysine, 40 mg L 1 tryptophan or 20 mg L 1 uracil) were supplemented into each medium to provide the relevant auxotrophic components. Plasmid constructions

All plasmids used in this study are listed in Table 1. All primers used for plasmid construction are listed in Supporting information, Table S1. Flow diagrams for conª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

J. Ishii et al.

struction of two or three gene expression vectors (pAT and pATP vectors) are presented in Figs S1 and S2. A flow diagram for the construction of fluorescent protein expression pATP plasmids is presented in Fig. S3. The DNA fragment containing ADH1 promoter (PADH1) and ADH1 terminator (TADH1) was prepared by digesting pADH404 (Ishii et al., 2013) with XhoI and NotI. The DNA fragments corresponding to TDH3 promoter (PTDH3) and TDH3 terminator (TTDH3) were respectively amplified from YPH499 genomic DNA, digested with XhoI/NheI and NheI/NotI, and combined at NheI sites. The obtained fragments containing PADH1TADH1 and PTDH3-TTDH3 (containing NheI and EcoRI sites between each promoter and terminator) were inserted into the XhoI/NotI sites of pRS416 (American Type Culture Collection, Manassas, VA), resulting in pADH416 and pTDH416, respectively. The DNA fragment encoding enhanced green fluorescent protein, the EGFP gene, was prepared by digesting pGK416-EGFP (Ishii et al., 2009) with NheI and EcoRI, and then the fragment was inserted into the same sites of pADH416 and pTDH416 to create pADH416-EGFP and pTDH416-EGFP, respectively. The DNA fragments corresponding to PADH1, PTDH3 or PGK1 promoter (PPGK1) and TADH1, TTDH3 or PGK1 terminator (TPGK1) were amplified from pADH416, pTDH416 or pGK416 (Ishii et al., 2009) (NBRP ID: BYP7367) and digested with PmeI, MluI or XmaI, respectively. The DNA fragments corresponding to TEF1 promoter (PTEF1) and TEF1 terminator (TTEF1) were amplified from YPH499 genomic DNA and digested with XmaI. The digested fragments were respectively combined to generate the multiple cloning site (MCS) and then inserted into the blunt end EcoRV site of pBlueScript II KS(+) (Stratagene/Agilent Technologies, Santa Clara, CA), resulting in the pBlue-ADH1PT, pBlue-TDH3PT, pBluePGK1PT or pBlue-TEF1PT plasmid. The DNA fragments containing PADH1-MCS1-TADH1 and PTDH3-MCS2-TTDH3 were prepared by digesting pBlue-ADH1PT and pBlue-TDH3PT with XhoI/SacII and SacII/SacI, respectively. The obtained fragments were combined and then inserted into the XhoI and SacI sites of pRS405 + 2 lm yeast multi-copy vector (Ishii et al., 2009), creating pAT425. The DNA fragment containing PPGK1-MCS3-TPGK1 was prepared by digesting pBluePGK1PT with PacI/SacI and then inserted into the PacI and SacI sites of pAT425 vector, producing pATP425. The DNA fragment containing PADH1-MCS1-TADH1 and PTDH3-MCS2-TTDH3 was prepared by digesting pAT425 with XhoI/SacI and then inserted into the XhoI and SacI sites of pRS40x + 2 lm (x = 2, 4 and 6) yeast multi-copy vectors (Ishii et al., 2009), constructing pAT422, pAT424 and pAT426, respectively. The DNA fragment containing PADH1-MCS1-TADH1, PTDH3-MCS2FEMS Yeast Res 14 (2014) 399–411

401

Three gene expression vector sets for eukaryotic yeast

Table 1. Yeast strains and plasmids used in this study Plasmids

Specific features

Source

pRS402 pRS403 pRS404 pRS405 pRS406 pRS402 + 2 lm pRS403 + 2 lm pRS404 + 2 lm pRS405 + 2 lm pRS406 + 2 lm pRS416 pADH404 pADH416 pTDH416 pGK416 pADH416-EGFP pTDH416-EGFP pGK416-EGFP pBlueScript II KS(+) pBlue-ADH1PT pBlue-TDH3PT pBlue-PGK1PT pBlue-TEF1PT pTagBFP-H2B pmKate2-endo pTagGFP2-tubulin pBlue-B1 pBlue-R2 pBlue-G1 pBlue-G2 pBlue-G3 pAT402

Integration vector (w/o yeast origin), ADE2 marker Integration vector (w/o yeast origin), HIS3 marker Integration vector (w/o yeast origin), TRP1 marker Integration vector (w/o yeast origin), LEU2 marker Integration vector (w/o yeast origin), URA3 marker Multi-copy vector (2l origin), ADE2 marker Multi-copy vector (2l origin), HIS3 marker Multi-copy vector (2l origin), TRP1 marker Multi-copy vector (2l origin), LEU2 marker Multi-copy vector (2l origin), URA3 marker Single-copy vector (CEN/ARS origin), URA3 marker PADH1, MCS (NheI and EcoRI), and TADH1 in pRS404 PADH1, MCS (NheI and EcoRI), and TADH1 in pRS416 PTDH3, MCS (NheI and EcoRI), and TTDH3 in pRS416 PTDH3, MCS, and TTDH3 in pRS416 (NBRP ID: BYP7367) pADH416, expressing EGFP gene pTDH416, expressing EGFP gene pGK416, expressing EGFP gene Cloning vector SacII–PADH1–MCS1 (AvrII–PmeI–FseI) –TADH1– XhoI in pBlueScript II KS(+) SacII–PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3–PacI–SacI in pBlueScript II KS(+) PacI–PPGK1–MCS3 (XmaI–AscI)–TPGK1–SacI in pBlueScript II KS(+) PacI–PTEF1–MCS3 (XmaI–AscI)–TTEF1–SacI in pBlueScript II KS(+) H2B–TagBFP fusion gene in mammalian expression vector mKate2–RhoB GTPase fusion gene in mammalian expression vector TagGFP2–a-tubulin fusion gene in mammalian expression vector AvrII–TagBFP–FseI in pBlueScript II KS(+) SalI–mKate2–NotI in pBlueScript II KS(+) AvrII–TagGFP2–FseI in pBlueScript II KS(+) SalI–TagGFP2–NotI in pBlueScript II KS(+) XmaI–TagGFP2–AscI in pBlueScript II KS(+) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in pRS402 (NBRP ID: BYP7579) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in AvrII-disrupted pRS403 (NBRP ID: BYP7580) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in pRS404 (NBRP ID: BYP7581) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in pRS405 (NBRP ID: BYP7582) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in pRS406 (NBRP ID: BYP7583) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in pRS402+2lm (NBRP ID: BYP7584) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in AvrII-disrupted pRS403+2lm (NBRP ID: BYP7585) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in pRS404+2lm (NBRP ID: BYP7586) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in pRS405+2lm (NBRP ID: BYP7587) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1 and PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 in pRS406+2lm (NBRP ID: BYP7588) TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3 (XmaI–AscI)–TPGK1 in pRS402 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3 (XmaI–AscI)–TPGK1 in AvrII-disrupted pRS403

ATCC ATCC ATCC ATCC ATCC Ishii et al. (2009) Ishii et al. (2009) Ishii et al. (2009) Ishii et al. (2009) Ishii et al. (2009) ATCC Ishii et al. (2013) This study This study Ishii et al. (2009) This study This study Ishii et al. (2009) Stratagene This study This study This study This study Evrogen Evrogen Evrogen This study This study This study This study This study This study

pAT403 pAT404 pAT405 pAT406 pAT422 pAT423 pAT424 pAT425 pAT426 pATP402 pATP403

This study This study This study This study This study This study This study This study This study This study This study

[Correction added after online publication 25 February 2014: pAGK416 changed to pGK416].

FEMS Yeast Res 14 (2014) 399–411

ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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402

Table 1. Continued Plasmids

Specific features

pATP404

TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in pRS404 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in pRS405 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in pRS406 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in pRS402+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in pRS403+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in AvrII-disrupted pRS403+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in pRS404+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in pRS405+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TPGK1 in pRS406+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF1 in pRS402 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF1 in AvrII-disrupted pRS403 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF1 in pRS404 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF1 in pRS405 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF1 in pRS406 TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF in pRS402+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF in AvrII-disrupted pRS403+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF1 in pRS404+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF1 in pRS405+2lm TADH1–MCS1 (FseI–PmeI–AvrII) –PADH1, PTDH3–MCS2 (XmaI–AscI)–TTEF1 in pRS406+2lm pATP402, expressing TagGFP2 gene by PADH1 pATP404, expressing TagGFP2 gene by PADH1 pATP405, expressing TagGFP2 gene by PADH1 pATP406, expressing TagGFP2 gene by PADH1 pATP422, expressing TagGFP2 gene by PADH1 pATP423, expressing TagGFP2 gene by PADH1 pATP424, expressing TagGFP2 gene by PADH1 pATP425, expressing TagGFP2 gene by PADH1 pATP426, expressing TagGFP2 gene by PADH1 pATP402, expressing TagGFP2 gene by PTDH3 pATP404, expressing TagGFP2 gene by PTDH3 pATP405, expressing TagGFP2 gene by PTDH3 pATP406, expressing TagGFP2 gene by PTDH3 pATP422, expressing TagGFP2 gene by PTDH3 pATP423, expressing TagGFP2 gene by PTDH3 pATP424, expressing TagGFP2 gene by PTDH3 pATP425, expressing TagGFP2 gene by PTDH3 pATP426, expressing TagGFP2 gene by PTDH3

pATP405 pATP406 pATP422 pATP423temp pATP423 pATP424 pATP425 pATP426 pATT402 pATT403 pATT404 pATT405 pATT406 pATT422 pATT423 pATT424 pATT425 pATT426 pATP402-G1 pATP404-G1 pATP405-G1 pATP406-G1 pATP422-G1 pATP423-G1 pATP424-G1 pATP425-G1 pATP426-G1 pATP402-G2 pATP404-G2 pATP405-G2 pATP406-G2 pATP422-G2 pATP423-G2 pATP424-G2 pATP425-G2 pATP426-G2

ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

Source (SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PPGK1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PTEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PTEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PTEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PTEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PTEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PTEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PTEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and PTEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and P

TEF1–MCS3

This study

(SalI–MluI–NotI) –TTDH3 and P

TEF1–MCS3

This study This This This This This This This This This This This This This This This This This This

study study study study study study study study study study study study study study study study study study

FEMS Yeast Res 14 (2014) 399–411

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Three gene expression vector sets for eukaryotic yeast

Table 1. Continued Plasmids

Specific features

pATP402-G3 pATP404-G3 pATP405-G3 pATP406-G3 pATP422-G3 pATP423-G3 pATP424-G3 pATP425-G3 pATP426-G3 pATT402-G3 pATT403-G3 pATT404-G3 pATT405-G3 pATT406-G3 pATT422-G3 pATT423-G3 pATT424-G3 pATT425-G3 pATT426-G3 pATP402-B1 pATP422-B1 pATP402-B1R2 pATP422-B1R2 pATP402-B1R2G3 pATP422-B1R2G3

pATP402, pATP404, pATP405, pATP406, pATP422, pATP423, pATP424, pATP425, pATP426, pATT402, pATT403, pATT404, pATT405, pATT406, pATT422, pATT423, pATT424, pATT425, pATT426, pATP402, pATP422, pATP402, pATP422, pATP402, pATP422,

expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing expressing

Source TagGFP2 gene by PPGK1 TagGFP2 gene by PPGK1 TagGFP2 gene by PPGK1 TagGFP2 gene by PPGK1 TagGFP2 gene by PPGK1 TagGFP2 gene by PPGK1 TagGFP2 gene by PPGK1 TagGFP2 gene by PPGK1 TagGFP2 gene by PPGK1 TagGFP2 gene by PTEF1 TagGFP2 gene by PTEF1 TagGFP2 gene by PTEF1 TagGFP2 gene by PTEF1 TagGFP2 gene by PTEF1 TagGFP2 gene by P TEF1 TagGFP2 gene by P TEF1 TagGFP2 gene by P TEF1 TagGFP2 gene by P TEF1 TagGFP2 gene by P TEF1 TagBFP gene by PADH1 TagBFP gene by PADH1 TagBFP gene by PADH1 and mKate2 gene by PTDH3 TagBFP gene by PADH1 and mKate2 gene by PTDH3 TagBFP gene by PADH1, mKate2 gene by PTDH3 and TagGFP2 gene by PPGK1 TagBFP gene by PADH1, mKate2 gene by PTDH3 and TagGFP2 gene by PPGK1

TTDH3 and PPGK1-MCS3-TPGK1 was prepared by digesting pATP425 with XhoI/SacI and then inserted into the XhoI and SacI sites of pRS40x + 2 lm (x = 2, 3, 4 and 6) yeast multi-copy vectors, generating pATP422, pATP423temp, pATP424 and pATP426, respectively. The DNA fragment encoding a partial HIS3 gene was amplified from pATP423temp to insert a point mutation and disrupt the AvrII site in the HIS3 marker. In-Fusion cloning (Clontech Laboratories/Takara Bio, Shiga, Japan) into NheI-digested pATP423temp plasmid was performed to substitute the amplified fragment for the corresponding sequence. The reconstituted vector that successfully disrupted the AvrII site in the HIS3 marker was named pATP423. The DNA fragment containing PADH1-MCS1TADH1 and PTDH3-MCS2-TTDH3 was prepared by digesting pAT425 with XhoI/SacI and then inserted into the XhoI and SacI sites of pATP423 to replace the promoter regions, producing pAT423 vector with AvrII disruption in the HIS3 marker. The DNA fragments containing PADH1-MCS1-TADH1 and PTDH3-MCS2-TTDH3 and PADH1-MCS1-TADH1, PTDH3-MCS2-TTDH3 and PPGK1-MCS3-TPGK1 were respectively prepared by digesting pAT425 and pATP425 with XhoI/SacI and then inserted into the XhoI and SacI sites of pRS40x (x = 2, 4, 5 and 6) (American Type Culture Collection) yeast integration vectors, producing pAT402, FEMS Yeast Res 14 (2014) 399–411

This This This This This This This This This This This This This This This This This This This This This This This This This

study study study study study study study study study study study study study study study study study study study study study study study study study

pAT404, pAT405 and pAT406, and pATP402, pATP404, pATP405 and pATP406, respectively. The 2l yeast multi-copy origins were removed by digesting pAT423 and pATP423 with AatII, and then the linear fragments were recircularized at the same sites. The resultant vectors with AvrII disruption in the HIS3 marker were named pAT403 and pATP403, respectively. The DNA fragment containing PTEF1-MCS3-TTEF1 was prepared by digesting pBlue-TEF1PT with PacI/SacI and then inserted into the PacI and SacI sites of pAT42x and pAT40x vectors, producing pATT42x and pATT40x (x = 2–6). The DNA fragments encoding blue fluorescent protein TagBFP (mTagBFP) and far-red fluorescent protein mKate2 genes with AvrII/FseI and SalI/NotI sites at both ends were respectively amplified from pTagBFP-H2B (Evrogen, Moscow, Russia) and pmKate2-endo (Evrogen) and cloned into the blunt end EcoRV site of pBlueScript II KS(+), resulting in the pBlue-B1 and pBlue-R2 plasmids. The DNA fragments encoding green fluorescent protein TagGFP2 (mTagGFP) genes with AvrII/FseI, SalI/NotI and XmaI/AscI sites at both ends were respectively amplified from pTagGFP2-tubulin (Evrogen) and cloned into the blunt end EcoRV site of pBlueScript II KS(+), generating the pBlue-G1, pBlue-G2 and pBlue-G3 plasmids. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

J. Ishii et al.

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The DNA fragments encoding TagGFP2 genes were respectively prepared by digesting pBlue-G1, pBlue-G2 and pBlue-G3 with AvrII/FseI, SalI/NotI and XmaI/AscI and inserted into the AvrII/FseI, SalI/NotI and XmaI/AscI sites of pATP42x (x = 2–6) and pATP40x (x = 2 and 4– 6), creating pATP42x-G1, pATP42x-G2 and pATP42x-G3 (x = 2–6), and pATP40x-G1, pATP40x-G2 and pATP40xG3 (x = 2 and 4–6). The DNA fragment prepared by digesting pBlue-G3 with XmaI/AscI also inserted into the same sites of pATT42x and pATT40x (x = 2–6), creating pATT42x-G3 and pATT40x-G3 (x = 2–6). The DNA fragment encoding the TagBFP gene was prepared by digesting pBlue-B1 with AvrII and FseI and inserted into the same sites of pATP422 and pATP402, constructing pATP422-B1 and pATP402-B1. The DNA fragment encoding the mKate2 gene was prepared by digesting pBlue-R2 with SalI and NotI and inserted into the same sites of pATP422-B1 and pATP402-B1, producing pATP422-B1R2 and pATP402-B1R2. The DNA fragment encoding the TagGFP2 gene was prepared by digesting pBlue-G3 with XmaI and AscI and inserted into the same sites of pATP422-B1R2 and pATP402-B1R2, creating pATP422-B1R2G3 and pATP402-B1R2G3.

All transformants were grown in the appropriate SD selectable media overnight and were then inoculated into10 mL of fresh SD media to give an initial optical density of 0.1 at 600 nm. Yeast cells were grown at 30 °C on a rotary shaker set at 150 r.p.m. for 24 h (or up to 96 h). Cultured cells were harvested for use in the following experiments.

Yeast transformation and cultivation

Fluorescence microscope

Yeast transformation procedures were conducted with the lithium acetate method (Gietz et al., 1992) and cultivation conditions basically followed the previously described procedures (Ishii et al., 2009). The integration plasmids pATP40x-G1, pATP40x-G2, pATP40x-G3, pATT40x-G3 and pATP402-B1R2G3 were linearized with a single restriction site in the selectable markers (x = 2, EcoRV; x = 4, BspEI; x = 5, EcoRV and x = 6, EcoRV), and YPH499 was transformed with the linear DNA fragments. The integration pATT40x-G3 plasmids were linearized with a single restriction site in the selectable markers (x = 2, EcoRV; x = 3, NdeI; x = 4, BspEI; x = 5, EcoRV and x = 6, EcoRV), and W303-1A was transformed with the linear DNA fragments. Three colonies grown on the SD selectable media were picked up, and then single-colony isolations were performed for each. The isolated three single colonies were used for the next experiments as the transformants. All multi-copy plasmids pATP42x-G1, pATP42x-G2, pATP42x-G3 and pATP422-B1R2G3 (x = 2–6) transformed YPH499 without the restriction enzyme digestions. Multi-copy plasmids pATT42x-G3 (x = 2–6) transformed YPH499 and W303-1A without the restriction enzyme digestions. Three colonies grown on the SD selectable media were picked up and used for the next experiments as the transformants without single-colony isolations.

The harvested yeast cells (YPH499/pATP422-B1R2G3 and YPH499/pATP402-B1R2G3) were washed and resuspended in distilled water. The cell suspensions were observed by using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan). Fluorescence images were acquired with a DAPI-BP filter (ex. 360/40 nm, em. 460/ 50 nm), a GFP-BP filter (ex. 470/40 nm, em. 535/50 nm), and a Texas-Red filter (ex. 560/40 nm, em. 630/60 nm).

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Flow cytometry

Quantitative measurements of TagGFP2 expression levels in yeast cells (YPH499/pATP42x-G1, -G2, -G3 and YPH499/pATP40x-G1, -G2, -G3) (YPH499/pATT42x-G3, YPH499/pATT40x-G3, W303-1A/pATT42x-G3 and W3031A /pATT40x-G3) were performed by basically following previous procedures (Ishii et al., 2012a, b). In brief, the harvested cells were diluted into test tubes containing sheath solution and TagGFP2 fluorescence was measured by using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). The green fluorescence signal from 10 000 cells was excited with a blue laser and collected through a 530/30 nm band-pass (GFP) filter. The data were analyzed by using BD FACSDIVA software (BD Biosciences).

Results and discussion Construction of yeast two and three gene expression vector sets (pAT and pATP series)

To construct the yeast three gene expression vectors, we chose three prominent promoters derived from ADH1, TDH3 and PGK1 that are often used for protein overexpression in S. cerevisiae. To check the promoter activities, we first prepared CEN/ARS single-copy EGFP gene expression plasmids, whose transcription was controlled under the single PADH1, PTDH3 or PPGK1 promoter (pADH416-EGFP, pTDH416-EGFP or pGK416-EGFP) (Table 1), respectively. All transformants with each plasmid presented clearly higher green fluorescence intensities than those with the corresponding EGFP-lacking mock plasmid, resulting in the following intensity order; PTDH3 > PADH1 > PPGK1 (data not shown). FEMS Yeast Res 14 (2014) 399–411

Three gene expression vector sets for eukaryotic yeast

Second, we constructed a series of two gene expression vectors harboring a yeast multi-copy replication origin (2l) and 5 auxotrophic selectable markers with PADH1 and PTDH3 promoters (pAT42x; x = 2, ADE2; x = 3, HIS3; x = 4, TRP1; x = 5, LEU2; and x = 6, URA3) (deposited to National BioResource Project (NBRP) – Yeast; NBRP IDs are BYP7584–7588) (Fig. 1a, Table 1, Fig. S1 and Fig. S2). The PADH1 promoter was oriented opposite to the PTDH3 promoter. For convenience, multiple-cloning sites (MCS1 and MCS2) were added between respective promoters and terminators (MCS1, PADH1–AvrII–PmeI–FseI–TADH1; MCS2, PTDH3–SalI–MluI–NotI– TTDH3). The integration-type of two gene expression vector sets harboring auxotrophic selectable markers without yeast replication origins were also generated in a similar fashion (pAT40x; x = 2–6) (deposited to NBRP – Yeast; NBRP IDs are BYP7579–7583) (Fig. 1a, Table 1 and Fig. S2). In the HIS3 marker sequences, a point mutation was introduced to disrupt the AvrII site, because the MCS1 also contains an AvrII site (Fig. S2). Third, by inserting the PPGK1 and TPGK1 fragments with the multiple-cloning site (MCS3, PPGK1–XmaI–AscI– TPGK1) downstream of the TTDH3 terminator of pAT42x vectors, we fabricated a series of three gene expression vectors possessing three PADH1, PTDH3 and PPGK1 promoters in addition to a yeast multi-copy 2l origin and 5 auxotrophic selectable markers (pATP42x; x = 2–6) (Fig. 1b, Table 1, Fig. S1 and Fig. S2). Similarly, the integration-type of three gene expression vector sets without yeast replication origins were also produced based on pAT40x vectors (pATP40x; x = 2–6) (Fig. 1b, Table 1 and Fig. S2). Expression of single fluorescent protein with pATP42x vector sets

To evaluate the performance of the constructed three gene expression vectors, the TagGFP2 gene encoding green fluorescent protein was separately inserted into the respective PADH1, PTDH3 and PPGK1 promoters of each pATP42x and pATP40x vector, producing pATP42x-G1, -G2 and -G3 (x = 2–6), and pATP40x-G1, -G2 and -G3 (x = 2, 4–6). YPH499 was used as the host strain to test TagGFP2 expression, because it contains multiple auxotrophic alleles that permit the introduction of multiple plasmids with the corresponding auxotrophic selectable markers (ADE2, HIS3, TRP1, LEU2, URA3 and LYS2). Prior to the construction of TagGFP2 expression plasmids, we omitted the pATP403 integration vector with HIS3 marker due to the his3-D200 allele in YPH499. The his3-D200 allele, which contains a 1.0-kb deletion of the entire coding region of the HIS3 gene (Sikorski & Hieter, 1989), is extremely difficult to replace with the linearized FEMS Yeast Res 14 (2014) 399–411

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pRS403-based plasmid due to its low homology to the HIS3 marker sequence (Ishii et al., 2009). The LYS2 marker was primarily omitted from the vector constructs due to its extremely long marker sequences (over 5000 bp). To generate the transformants, all 2l multi-copy pATP42x-G1, -G2 and -G3 TagGFP2 expression plasmids (x = 2–6) and pATP42x control vectors without TagGFP2 (mock) were introduced directly into YPH499. For integration of pATP40x-G1, -G2 and -G3 TagGFP2 expression plasmids (x = 2 and 4–6) and pATP40x control vectors (mock), all integration plasmids were linearized and then introduced into YPH499. EcoRV restriction enzyme was used to prepare the linear fragment because the middle of all selectable markers contained a single EcoRV site (x = 2, ADE2; x = 4, TRP1; x = 5, LEU2; x = 6, URA3). The ade2-101 (ochre mutation), leu2-D1 (D0.6-kb) and ura3-52 (Ty1 insertion) alleles successfully provided the correct transformants with EcoRV-digested linear plasmids, while the trp1-D63 (D0.6-kb) allele could not provide the transformants (data not shown). The XbaI-digested pATP404-G1, -G2 and -G3 plasmids also never generated transformants. Whereas the use of a single BspEI site (Ishii et al., 2009) in pATP404-G1, -G2 and -G3 (replicated in SCS110, free from Dam methylation) allowed the appearance of a few colonies, only one or two transformants were produced (data not shown). The transformation efficiency to integrate the TRP1-based plasmid into YPH499 would probably depend on not only the digested form of the TRP1 marker but also the length of linear plasmid. Although the one or two transformants that were obtained correctly exhibited green fluorescence, we decided not to evaluate the pATP404-based transformants of YPH499 with trp1-D63 allele. These results suggest that different trp1-deficient alleles or improved TRP1 marker sequences are needed for the use of pATP404 vector or YPH499, respectively. After cultivation of the transformants in SD selectable media for 24 h, the green fluorescence intensities of yeast cells were measured by flow cytometry (Fig. 2). All pATP42x- and pATP40x-based transformants harboring TagGFP2 genes displayed higher green fluorescence intensities than the control mock strains (Fig. 2a and b). Although the relative fluorescence intensities were considerably different among three promoters, the order of fluorescence intensities was equivalent to the abovedescribed EGFP evaluations of the plasmids harboring only the single promoters (PTDH3 > PADH1 > PPGK1). Especially in the pATP42x-based transformants, the PTDH3 promoter displayed significantly higher fluorescence, while the PPGK1 promoter showed relatively lower fluorescence (Fig. 2a). This difference might be attributed to the substantial transcription bias generated by the multiple copy numbers (20–50 copies) of three expression ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Fig. 1. Two gene expression pAT vector sets (a) and three gene expression pATP vector sets (b). pAT vector series has two gene expression cassettes of PADH1–MCS1–TADH1 and PTDH3–MCS2–TTDH3. pATP vector series has three gene expression cassettes of PADH1–MCS1–TADH1, PTDH3– MCS2–TTDH3 and PPGK1–MCS3–TPGK1. Multi-copy–type autonomous replicating pATP42x vectors contain 2l replication origins. Integration-type pATP40x vectors never contain replication origins.

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Fig. 2. Green fluorescence intensities of YPH499 yeast cells transformed with TagGFP2 single expression plasmids. All transformants were grown in SD selectable media for 24 h. The mean values of the green fluorescence signal of 10 000 cells are displayed. (a) Transformants with pATP42x-based multi-copy plasmids. (b) Transformants with pATP40x-based integration plasmids. Mock cells were transformed with the control pATP42x and pATP40x vectors without TagGFP2 genes. Error bars represent the standard deviations of three independent transformants.

plasmids. The green fluorescence intensities of multi-copy pATP42x-based transformants were naturally higher than those of single-copy integration pATP40x-based transformants, even though the pATP402-G2-integrated transformants unexpectedly exhibited inexplicably high green fluorescence intensities (Fig. 2a and b). Subsequently, to check the dependence of promoter activities on the cultivation times, time courses of TagGFP2 expression in pATP425-based transformants were examined (Fig. 3). The green fluorescence of each transformant was maintained from 0 h up to 24 h or 48 h and then gradually diminished. The order of TagGFP2 expression levels was unchanged among PADH1, PTDH3 and PPGK1 promoters. Since the ADH1, TDH3 and PGK1 genes encoded the enzymes involved in glycolysis (alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase), the time-dependent expression characteristics would have been similar among the three promoters. From all these aspects, the search for more robust and fitted promoters, as alternatives especially to the PPGK1 promoter, will be important to make the three gene expression vectors more accessible. Expression of single fluorescent protein with pATT42x vector sets

As a candidate alternative promoter, we replaced the PPGK1 of pATP42x and pATP40x vectors by the PTEF1 FEMS Yeast Res 14 (2014) 399–411

promoters that are also often used for protein overexpression in S. cerevisiae, producing pATT42x and pATT40x vectors. To evaluate the performance of the TEF1 promoter, the TagGFP2 gene was inserted into the PTEF1 promoter of each pATT42x and pATT40x vector, producing the pATT42x-G3 and pATP40x-G3 (x = 2–6). In addition to YPH499, W303-1A was used as the host strains to test TagGFP2 expression. It has been shown that W303-1A hardly maintains 2l plasmids but permits the integrations of pRS-based vectors with all five selectable markers (x = 2–6; ADE2, HIS3, TRP1, LEU2 and URA3) (Ishii et al., 2009). To generate the transformants, all multi-copy pATT42x-G3 plasmids (x = 2–6) and pATT42x control vectors (mock) were introduced into YPH499 and W3031A. Integration of pATT40x-G3 (x = 2, 5 and 6) and pATT40x control vectors (mock) into YPH499 followed the above-described procedures. For integration into W303-1A, single EcoRV sites (x = 2 and 4–6) and a single NdeI site (x = 3) were used to obtain the linear fragments. All transformants were successfully generated, although the transformation efficiencies were a little different between pATT40x and pATT40x-G3, or YPH499 and W303-1A (Table S2). After cultivation of the transformants in SD selectable media for 24 h, the green fluorescence intensities of yeast cells were measured by flow cytometry (Figs S4 and S5). All YPH499 transformants harboring TagGFP2 genes ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Expression of three fluorescent proteins with pATP422 and pATP402 vectors

Fig. 3. Time courses of green fluorescence intensities of YPH499 yeast cells transformed with pATP425-based TagGFP2 single expression plasmids. All transformants were grown in SD selectable media for up to 96 h. The mean values of the green fluorescence signal of 10 000 cells are displayed. Mock cells were transformed with the control pATP425 vector without TagGFP2 gene. Error bars represent the standard deviations of three independent transformants.

displayed increased green fluorescence intensities than the control mock strains (Fig. S4a and b). However, the fluorescence intensities were comparably similar to the case with pATP4xx vectors. Most of W303-1A transformants also showed TagGFP2 fluorescence, although they showed relatively higher background fluorescence derived from the cell own. Prolonged cultivation time also never induced the TagGFP2 expression in these transformants (Figs S6 and S7). These results indicate that the systematic exploration for more robust and fitted promoters as alternatives to the PPGK1 promoter or the elucidation for hidden factors will be needed to design the more accessible three gene expression vectors.

Finally, to test the simultaneous expression of three genes, the plasmids for expressing three fluorescent proteins were constructed. TagBFP, mKate2 and TagGFP2 genes encoding the blue, far-red and green fluorescent proteins were respectively inserted downstream of the PADH1, PTDH3 and PPGK1 promoters of pATP422 and pATP402 vectors, creating pATP422-B1R2G3 and pATP402B1R2G3 (Fig. S3 and Table 1). Each plasmid was used for producing YPH499 transformants, and the blue, red and green fluorescence of cultured cells was observed by fluorescence microscopy (Fig. 4). Yeast cells transformed with pATP422-B1R2G3 or pATP402-B1R2G3 displayed blue, red and green fluorescence (Fig. 4a and b). pATP422-B1R2G3 yielded more vivid fluorescence than pATP402-B1R2G3 (Fig. 4a and b; blue, red and green), reflecting the results that the TagGFP2 intensities in multi-copy–type pATP42x-based transformants were higher than those in the integrationtype pATP40x-based transformants (Fig. 2). Almost all cells integrated with pATP402-B1R2G3 were fluorescent (Fig. 4b; overlay – bright/blue, bright/red and bright/ green), whereas not all transformants with pATP422B1R2G3 were fluorescent (Fig. 4a). The retention rates of plasmids were in agreement with the previous report (Ishii et al., 2009). Overlays of blue, red and/or green images (cyan, purple, yellow and white) were nearly overlapped with the patterns of the single-fluorescent cells (Fig. 4a and b), indicating that the three PADH1, PTDH3 and PPGK1 promoters simultaneously worked in yeast cells maintaining the pATP422-B1R2G3 and pATP402B1R2G3 plasmids.

Conclusion In the present study, we constructed a series of two and three gene expression vectors with 2l origins (pAT42x and pATP42x) and without yeast replication origins (pAT40x and pATP40x), respectively, for multi-copy autonomous replication and genomic integration in S. cerevisiae. The ADH1, TDH3 and PGK1 promoters on the single vectors permitted the simultaneous expression of three different genes, even though the choice of promoters may admittedly need improvement. Because yeast S. cerevisiae can generally harbor (or integrate) the

Fig. 4. Fluorescence images of YPH499 yeast cells transformed with TagBFP, mKate2 and TagGFP2 triple expression plasmids. Yeast cells transformed with multi-copy pATP422-B1R2G3 plasmid (a) and integration pATP402-B1R2G3 plasmid (b) were grown in SD selectable media for 24 h. Blue, red and green fluorescence images were acquired with a DAPI-BP filter (ex. 360/40 nm, em. 460/50 nm), a GFP-BP filter (ex. 470/ 40 nm, em. 535/50 nm), and a Texas-Red filter (ex. 560/40 nm, em. 630/60 nm), respectively. Exposure time; (a) bright, 1/20 s; blue, 3.5 s; red, 1/70 s; green, 1 s; and for (b) bright, 1/20 s; blue, 10 s; red, 1/10 s; green, 3 s.

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plasmids in a number corresponding to the types of available selectable markers, a series of the constructed vectors must be able to concurrently express a considerable number of different genes. In parallel with the present study, we have been progressing the construction of isobutanol-producing yeast, and at this time we have succeeded in the engineering of metabolic pathway by expressing nine genes using three distinct types of pATP vectors in S. cerevisiae (F. Matsuda, J. Ishii, T. Kondo, K. Ida, H. Tezuka & A. Kondo, submitted). Our tools are definitely helpful to the advanced creation of genetically engineered yeast strains in research fields, such as metabolic engineering.

Acknowledgements This work was supported by the Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe; iBioK) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and in part by the Industrial Technology Research Grant Program in 2011 of the New Energy and Industrial Technology Development Organization (NEDO), Japan, and the commission for Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Flow diagram for the construction of the multicopy two and three gene expression vectors. Fig. S2. Flow diagram for point mutation to disrupt the AvrII site in the HIS3 marker and for the construction of integration vectors for expression of two and three genes. Fig. S3. Flow diagram for the construction of fluorescent protein expression plasmids.

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Fig. S4. Green fluorescence intensities of YPH499 yeast cells transformed with the plasmids that replaced PGK1 promoters of pATP4xx-G3 by TEF1 promoters. Fig. S5. Green fluorescence intensities of W303-1A yeast cells transformed with the plasmids that replaced PGK1 promoters of pATP4xx-G3 by TEF1 promoters. Fig. S6. Time courses of green fluorescence intensities of YPH499 yeast cells transformed with the plasmids that replaced PGK1 promoters of pATP4x5-G3 by TEF1. Fig. S7. Time courses of green fluorescence intensities of W303-1A yeast cells transformed with the plasmids that replaced PGK1 promoters of pATP4x5-G3 by TEF1. Table S1. List of primers. Table S2. Transformation efficiencies with pATT4xx and pATT4xx-G3.

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