Journal of Microbiological Methods 120 (2016) 44–49

Contents lists available at ScienceDirect

Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

Development of a system for multicopy gene integration in Saccharomyces cerevisiae Marta V. Semkiv a, Kostyantyn V. Dmytruk a, Andriy A. Sibirny a,b,⁎ a b

Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601, Poland

a r t i c l e

i n f o

Article history: Received 1 July 2015 Received in revised form 19 October 2015 Accepted 28 October 2015 Available online 31 October 2015 Keywords: Multicopy integration Saccharomyces cerevisiae Geneticin resistance Alkaline phosphatase

a b s t r a c t In this study we describe construction and evaluation of a vector for multicopy integration in yeast Saccharomyces cerevisiae. In this vector a modified selective marker and a reporter gene PHO8 (encoding alkaline phosphatase) were flanked with delta sequences of the Ty1 transposon. Modified by error-prone PCR version of selection marker kanMX4 was obtained from Escherichia coli clone with impaired geneticin (G418) resistance. The attenuation of kanMX4 gene provides an opportunity to select for explicitly multicopy integration of the module in S. cerevisiae using moderate (200 mg L−1) antibiotic concentrations. The developed system provided integration of 3–10 copies of the module in the genome of S. cerevisiae. High copy integration events were confirmed by qRT-PCR, Southern hybridization and reporter enzyme activity measurements. © 2015 Published by Elsevier B.V.

1. Introduction Baker's yeast Saccharomyces cerevisiae is a key component of traditional biotechnological processes such as baking, brewing of beer, wine and strong alcoholic beverages. Nowadays, it is also used as a producer of the fuel ethanol and a wide range of other important compounds, e.g. glycerol (Wang et al., 2001), isobutanol (Generoso et al., 2015), malic acid (Zelle et al., 2008), dihydroxyacetone (Nguyen and Nevoigt, 2009), farnesene (Tippmann et al., 2015) and even morphine (Fossati et al., 2015). Also, S. cerevisiae is a suitable host for heterologous protein production, in particular, for complex mammalian proteins (Romanos et al., 1992). Genetic engineering is widely used to change the profile of produced metabolites or improve the robustness of S. cerevisiae (Ostergaard et al., 2000). Construction of stable and efficient recombinant strains requires suitable vectors. For stable enhancement of a certain protein production, the vectors providing both integration of the expression cassette into the genome and its maintenance in several copies per cell are particularly desirable. Initially the integration of exogenous DNA sequences into S. cerevisiae nuclear DNA was achieved through homologous recombination of yeast integrating plasmid (YIp) vectors that targeted a single-copy gene (e.g. HIS3, URA3 or TRP1) resulting in one-copy-number vector integration (Romanos et al., 1992). Alternative episomal multi-copy vectors are mitotically unstable and could be lost after prolonged cultivation under non-selective conditions (Murray and Szostak, 1983). Also, replicative ⁎ Corresponding author at: Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine. E-mail address: [email protected] (A.A. Sibirny).

http://dx.doi.org/10.1016/j.mimet.2015.10.023 0167-7012/© 2015 Published by Elsevier B.V.

vectors are less suitable for simultaneous stable coexpression of several proteins (Jensen et al., 2014). Subsequently, more efficient integrative vectors with enhanced targeting of specific DNA sequences were developed; for example, a set of vectors based on integration of the desired genes into the rDNA locus (Lopes et al., 1989). The S. cerevisiae rDNA locus is a 1–2 Mb-long sequence containing about 200 copies of tandemly repeated 9.1 kb fragment. A detailed analysis of DNA integration into the rDNA revealed that despite the high number of possible integration targets, foreign DNA integrates only in a few sites and after that get amplified by selective pressure created by specially designed markers (Lopes et al., 1991). Also the insert size was found to be limited to 9.1 kb (Lopes et al., 1996). Another widely spread variant of multicopy integrative vectors are plasmids bearing δ sequences (Lee and Da Silva, 1997; Oliveira et al., 2007). The δ sequences are LTR (long terminal repeats) of the yeast retrotransposon Ty1, that are essential for the transposon insertion into genomic DNA (Eichinger and Boeke, 1990). They exist in more than 400 copies in the yeast genome, both associated with Ty1 transposon or as independent elements (Dujon, 1996). Integrative vectors may include only one δ sequence; however, constructs with two δ sequences flanking the expression cassette and lacking bacterial sequences (which are excised from the vector prior to yeast transformation) are much more efficient (Lee and Da Silva, 1997). Despite the abundance of δ sequences in the yeast genome, such vectors provide insertion of desired sequences only into one or two sites of genomic DNA (“hot spots”) in one copy or in tandem array. Systems for selection of multicopy integrants using appropriate selection markers appear to be superior. An example of such marker is leu2-d gene, which bears a truncated promoter region resulting in impaired expression (Erhart and Hollenberg, 1983). Only several copies

M.V. Semkiv et al. / Journal of Microbiological Methods 120 (2016) 44–49

of such gene can support growth without leucine, therefore, its selection gives rise to very high plasmid copy numbers. The main drawback of such a system is the necessity of leu2 mutation in the recipient strain. Industrial S. cerevisiae strains are predominantly prototrophic and often polyploid, requiring dominant selection markers for their transformation. The bacterial neo gene derived from the transposon Tn5 (Beck et al., 1982) is widely used as a heterologous dominant selection marker conferring resistance to geneticin (G418) in S. cerevisiae. A S. cerevisiae-adapted version of this gene (kanMX4) combines neo gene ORF with native yeast TEF1 promoter (Taxis and Knop, 2006). Combining this selection marker with the advantages of δ sequences, Wang et al. (1996) obtained stable high-copy S. cerevisiae integrants. The transformation efficiency and integration frequency of such vector decreased with the inclusion of an additional expression cassette, apparently due to increased vector size. The percentage of multicopy integrants among all obtained G418-resistant strains may be enhanced by increasing geneticin concentration in the medium, which constitutes another advantage of this vector (Parekh et al., 1996). The use of kanMX4-based vectors also enables the increase in integrated gene copy number through post-transformational vector amplification (Aw and Polizzi, 2013). Still today, there is a strong demand for yeast strains with increased expression of target genes from stable multicopy integration events. Moreover, such strains should be easy to distinguish from low-copy integrants. Direct (reverse transcriptase PCR) or indirect (Westernblot, enzymatic activity measurement) methods may be used to estimate the expression of the inserted gene; however, they are quite laborious. Enzymatic activity can be roughly evaluated on plates using color-generating substrates. For example, Oliveira et al., (2007) selected recombinant strains with high β-galactosidase production levels on X-gal selective plates by the deep blue color of the colonies. Hribar et al., (2008) suggested a vector for simultaneous expression of a desired gene along with a reporter gene (in this case gene encoding β-lactamase) for selection of strains with high reporter enzyme activity, and thus, a high-copy number integration. In this article we address the question whether the attenuation of kanMX4 selectable marker (mimicking leu2-d gene) could lead to preferential selection of transformants with multicopy vector integration using relatively low geneticin concentrations. We have introduced reporter gene PHO8 to the δ-based multi-copy integration module to simplify the selection and verification of S. cerevisiae multicopy integrants. 2. Material and methods 2.1. Strains, media and growth conditions The Escherichia coli DH5α strain (Φ80dlacZΔM15, recA1, endA1, + gyrA96, thi-1, hsdR17(r − K , m K ), supE44, relA1, deoR, Δ(lacZYAargF)U169) was used as a host for propagation of plasmids and for selection of the appropriate modified form of kanMX4 gene. Strain DH5α was grown at 37 °C in LB medium. Transformed E. coli cells were maintained in medium containing 100 mg L− 1 of ampicillin or from 2 to 50 mg L−1 of geneticin. IPTG was used together with the chromogenic substrate X-gal (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer specifications. The S. cerevisiae strain BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0) was used for the verification of the efficacy of multicopy integration. For selection of yeast transformants on YPD, 200 mg L−1 of geneticin was added. When required, histidine (20 mg L−1), leucine (60 mg L−1), lysine (20 mg L−1), or uracil (20 mg L−1) was added. S. cerevisiae cells were propagated at 30 °C in rich YPD (10 g L− 1 yeast extract, 10 g L−1 peptone and 20 g L−1 glucose) or mineral YNB (6.7 g L−1 yeast nitrogen base without amino acids, DIFCO, 5 g L− 1 ammonium sulfate, 20 g L− 1 glucose) media. Solid media contained 20 mg L−1 agar.

45

2.2. DNA manipulations Plasmid DNA from E. coli was isolated using the Wizard® Plus SV Minipreps DNA Purification System (Promega). Genomic DNA from S. cerevisiae was isolated using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Total RNA from S. cerevisiae was isolated using EURX universal RNA purification kit. Taq and High Fidelity polymerase mix, T4 DNA ligase, T4 DNA polymerase and restriction enzymes were used according to the supplier's recommendations (Thermo Scientific). E. coli transformation was performed by electroporation (Dower et al., 1988). S. cerevisiae transformation was performed by Li-acetate/PEG method (Kawai et al., 2010). 2.3. Plasmids construction Gene kanMX4 was amplified with primers Ko446 (CCG GGA TCC TCT AGA GTG ATG ACG GTG AAA ACC TCT G), Ko447 (CCG GGA TCC TCT AGA CTT ACG CAT CTG TGC GGT ATT TC) from plasmid pRS303K (Taxis and Knop, 2006) using either standard PCR protocol and High Fidelity polymerase mix or error-prone PCR protocol and Taq polymerase mix. The last protocol differed from the standard by using 7 mM MgCl2 and 0.5 mM MnCl2, as well as unbalanced deoxynucleotides (0.2 mM dGTP, 0.2 mM dATP, 1 mM dTTP, 1 mM dCTP). Obtained fragments were digested with XbaI and cloned into the corresponding site of the plasmid pUC57. The plasmid harboring wild-type form of kanMX4 gene was named pKanMX. Among E. coli transformants bearing the plasmid with the modified form of kanMX4 gene one was selected as described in Section 3. The plasmid isolated from this transformant was named pKanMXmod. To introduce a mutation leading to D120G amino acid substitution, two parts of the kanMX4 gene were amplified from plasmid pRS303K using primers SM47 (TGC TCT AGA ATT AAG GCG CGC CAG ATC TGT TTA GC)/SM48 (CGC AGG AAC ACT GCC AGC GCA CCA ACA ATA TTT TCA CCT GAA TC) and SM49 (GAT TCA GGT GAA AAT ATT GTT GGT GCG CTG GCA GTG TTC CTG CG)/SM50 (TGC TCT AGA GTT TTC GAC ACT GGA TGG CGG CG) (the substituted nucleotide is shown in bold underlined font). Then parts were fused into one gene via overlap PCR using primers SM47 and SM50, digested with XbaI and cloned into XbaI-linearized plasmid pUC57. The resulting plasmid was named pKanMXm1. To introduce a mutation leading to E267D amino acid substitution, similarly, two parts of kanMX4 gene were amplified from plasmid pRS303K using primers SM47/SM51 (GTC AGT ACT GAT TAG AAA AAA TCA TCG AGC ATC AAA TGA AAC) and SM52 (GTT TCA TTT GAT GCT CGA TGA TTT TTT CTA ATC AGT ACT GAC)/SM50, fused by overlap PCR and cloned into XbaI site of the plasmid pUC57. Constructed plasmid was designated as pKanMXm2. The same strategy and primers SM47/SM51 and SM52/SM50 were used to combine both mutations. For this, plasmid pKanMXm1 was used as a template. The obtained plasmid was named pKanMXm1m2. All plasmids were verified by sequencing. S. cerevisiae YJRWdelta12 sequence was divided into 5′- and 3′-part. 154 bp 5′-part and 180 bp 3′-part were amplified from genomic DNA of S. cerevisiae strain BY4742 using pairs of primers SM16 (CCG GAA TTC GAC GGG CAG TCT GTT GGA ATA GAA ATC AAC TAT C)/SM17 (CAT CAT TTT ATA TGT TTA TAT TCA TCT AGA CCC GGG GTC GAC TTG ATC CTA TTA CAT TAT CAA TCC) and SM18 (GGA TTG ATA ATG TAA TAG GAT CAA GTC GAC CCC GGG TCT AGA TGA ATA TAA ACA TAT AAA ATG ATG)/SM19 (CCC AAG CTT GAC GGG CAG TCT GAG AAA TAT GTG AAT GTT GAG), respectively. Then both fragments containing delta sequences were fused via overlap PCR using primers SM16 and SM19, digested with EcoRI and HindIII and cloned into EcoRI/HindIIIlinearized plasmid pUC57. The resulting plasmid was named pDel. To create the expression module, promoter of ADH1 gene (807 bp) encoding alcohol dehydrogenase and terminator of CYC1 gene (269 bp) encoding cytochrome c, were amplified from genomic DNA of BY4742 by pairs of primers Ко419 (CGC GTC GAC TTA ATT AAA GTC

46

M.V. Semkiv et al. / Journal of Microbiological Methods 120 (2016) 44–49

CAA TGC TAG)/Ко420 (GAT ATC GAC AAA GGA AAA GGG GCG GCC GC GGA TCC CTC GAG TGT ATA TGA GAT AGT TGA TTG) and Ко453 (CAA TCA ACT ATC TCA TAT ACA CTC GAG GGA TCC GCG GCC GC CCC TTT TCC TTT GTC GAT ATC)/Ко454 (CCC CCC GGG GCA AAT TAA AGC CTT CGA GC), respectively, and subsequently fused by overlap PCR using primers Ко419/Ко454. The amplified expression module was SalI/ XmaI cloned into the corresponding sites of the plasmid pDel resulting in the plasmid pDel-ADH-CYC. A 1701 bp DNA fragment bearing the ORF of PHO8 gene coding for unspecific alkaline phosphatase (Kaneko et al., 1982) was amplified from genomic DNA of the BY4742 strain using primers Ko508 (CGC GGA TCC ATG ATG ACT CAC ACA TTA CCA AGC) and Ko509 (TTT GCG GCC GC TCA GTT GGT CAA CTC ATG GTA GTA TTC). This fragment was digested with BamHI and NotI and subcloned into BamHI/NotI-digested plasmid pDel-ADH-CYC. This plasmid was designated as pDel-PHO. A 1470 bp DNA fragment corresponding to either wild-type or modified form of the selection marker kanMX4 was cut out from the plasmid pKanMX or pKanMXmod, with restriction endonucleases SacI and SmaI, blunt-ended and cloned into XbaI-digested and blunted plasmid pDel-PHO yielding the plasmids pDel-PHO-kanMX or pDel-PHO-kanMXmod (Fig. 1). 2.4. Selection of S. cerevisiae transformants Approximately 1 μg of the vector DNA containing wild-type or modified versions of the kanMX4 gene was digested with AhdI. AhdIfragments containing expression cassettes and selectable marker flanked by δ-sequences, were eluted from an agarose gel and used for transformation of S. cerevisiae strain BY4742. The transformation was performed by Li-Ac procedure developed by Ito et al. (1983) and modified by Schiestl and Gietz (1989). The amounts of DNA added to competent cells were estimated by the UV light absorbance using NanoDrop2000c (Thermo Scientific). After the heat shock, yeast cells were incubated for 2 h in non-selective YPD medium prior to plating on selective medium. The transformants were selected on solid YPD medium supplemented with 200 mg L−1 of geneticin. The transformants were stabilized by cultivation in liquid non-selective medium for one day (approximately 8–10 generations) and consecutive growth on non-selective solid YPD medium for approximately 30–40 generations with subsequent shift to selective medium with G418. The persistence of the vector in the genomic DNA was verified by diagnostic PCR. 2.5. Alkaline phosphatase assay The specific activity of alkaline phosphatase was assayed in cell-free extracts using the chromogenic substrate p-nitrophenyl phosphate. One cuvette contained 50 mM Tris–HCl buffer pH 8.5, 10 mM MgCl2, 10 mM p-nitrophenylphosphate and cell-free extract containing 0.05 or 0.025 mg of total cellular protein. The cellular protein amount was determined by Lowry assay. The total volume was 1 mL. Kinetic parameters of alkaline phosphatase were determined from the appearance of a yellow-colored product. The amount of product, p-nitrophenol, was determined by reading the absorbance at 410 nm and using a molar extinction coefficient of 18,000 M− 1 cm− 1. The assay was repeated three times and the measurement reported is the average of these determinations. The numbers reported use one unit of alkaline phosphatase activity as the amount of enzyme that liberates 1 μmol of p-nitrophenol per min under the assay conditions. Alkaline phosphatase activity of yeast strains was visualized on plates. Spots with identical quantities of cells of different strains were loaded on the surface of solid YNB medium; the cells were allowed to grow for 2 days and then the plates were overlaid with the reaction mixture containing 0.7% agarose, 50 mM Tris–HCl buffer pH 8.5, 10 mM MgCl2, 3 mg mL− 1 hexadecyltrimethylammonium bromide and 0.5 mg mL−1 of 5-bromo-4-chloro-3-indolyl phosphate disodium salt or 1 mg mL−1 of p-nitrophenylphosphate as substrates. Alkaline

phosphatase activity was estimated by the intensity of the yellow color of released p-nitrophenol or blue color of 5-bromo-4-chloro-3indol. 2.6. Quantitative Reverse Transcriptase-PCR (qRT-PCR) Quantitative PCR was carried out using the Applied Biosystems 7500 Fast Real-Time PCR System and the SG OneStep qRT-PCR kit plus ROX Solution (EURx Ltd) according to the manufacturer's instructions. Both cDNA synthesis and PCR were performed in a single tube using genespecific primers and total RNA. In brief, 100 ng of total RNA was used in a total reaction volume of 25 μl with 1 μM of each primer. The following primer pairs were used: SM89 (CTA TCC AAG ACA AAC TGA ATG AC)/SM90 (GTG TGT TTG GTG TCC CTA ATC) for the 3′ fragment of S. cerevisiae PHO8 gene and Ko348 (ATG AAG TGT GAT GTC GAT GTC)/ Ko349 (TTT GAG ATC CAC ATT TGT TGG AA) for the 3′ fragment of S. cerevisiae ORF of the ACT1 gene. The cycling parameters were 30 min at 50 °C and then 3 min at 95 °C at preparation step, followed by 40 cycles of 15 s at 95 °C, 30 s at 60 °C and 35 s at 72 °C. The fold change of each amplicon in each sample relative to the control sample was measured in triplicates, normalized to the internal control gene ACT1 and calculated according to the comparative Ct (ΔΔCt) method. 2.7. Dot-blot hybridization For quantitative Southern dot-blot, preparations of serial dilutions of yeast genomic DNA were denatured in 0.4 M NaOH and spotted 5 μL per dot onto dry nylon membrane (Hybond N+, Amersham Pharmacia Biotech). The amounts of DNAs were estimated by the UV light absorbance at NanoDrop2000c (Thermo Scientific). The labeling of the probe DNA and hybridization was performed using non-radioactive ECL direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech) according to the manufacturer's manual. 3. Results and discussion To develop a multi-copy integration module for S. cerevisiae we combined δ sequences for site-specific integration with a defective dominant selection marker, allowing growth under selective conditions only for S. cerevisiae transformants with high copy number of the insertion vector. Gene kanMX4 renders S. cerevisiae and E. coli resistant to geneticin. First, we set out to use an error-prone PCR to obtain mutated kanMX4 alleles, which would provide resistance to geneticin only after insertion to the genome in several copies. E. coli was used for selection of the desired mutant kanMX4 alleles. Growth tests were performed to reveal the minimal toxic geneticin concentration for E. coli cells. This revealed that E. coli wild-type strain is unable to grow in the medium containing 2 mg L−1 of geneticin. E. coli cells bearing modified kanMX4 gene were obtained as described in the subsection 2.3 of the section Material and methods and plated on the selective medium containing 100 mg L−1 of ampicillin and 2 mg L−1 of geneticin. Further colonies were replica-plated onto medium containing 50 mg L− 1 of geneticin. Several transformants unable to growth on higher antibiotic concentrations were picked up and analyzed. A single transformant that failed to survive on 10 mg L−1 of geneticin was chosen (Fig. 2, 1). Plasmid pKanMXmod harboring mutated kanMX4 gene was isolated from the selected E. coli strain. Sequencing of the mutated kanMX4 allele revealed four nucleotide substitutions: A(− 268)T in the promoter region; A(+ 359)G, T(+438)C, G(+ 801)T in the ORF (the numbers in parentheses show the position of the replaced nucleotide in kanMX4 gene, with “A” of the ORF ATG (start codon) being at position +1). Two of them lead to amino acid substitutions in neomycin phosphotransferase: aspartic acid in position 120 to glycine (D(120)G) and glutamic acid in position 267 to aspartic acid (E(267)D).

M.V. Semkiv et al. / Journal of Microbiological Methods 120 (2016) 44–49

47

Fig. 1. Linear scheme of plasmids pDel-PHO-kanMX and pDel-PHO-kanMXmod containing the multi-copy integration module with modified or wild-type selection marker.

To evaluate the impact of the mutations located in the ORF, three forms of kanMX4 gene were constructed. The first and second forms contained single D(120)G and E(267)D mutations, respectively, while the third form contained both substitutions. In order to compare the impact of each substitution, E. coli recombinant strains bearing different modified forms of kanMX4 gene were grown on media with different geneticin concentrations. We found that single identified mutations did not affect geneticin resistance of E. coli cells, whereas two substitutions, present simultaneously, significantly increased geneticin sensitivity (Fig. 2, 2). Therefore, the cumulative effect of both introduced missense mutations is important for the reduction in marker efficiency. We have constructed a multicopy integration module for S. cerevisiae pDel-PHO-kanMXmod containing modified kanMX4 gene with four nucleotide substitutions (A(−268)T; A(+359)G, T(+438)C, G(+801)T) (for details see Section 2). The ORF of the gene PHO8, encoding alkaline phosphatase, was inserted between ADH1 promoter and CYC1 terminator to evaluate the efficacy of multicopy integration. The same plasmid containing wild-type kanMX4 gene was constructed and used in our study. Both plasmids were transformed into S. cerevisiae BY4742 strain and plated on solid YPD medium containing 200 mg L−1 of geneticin. The vector with the wild-type selectable marker gave high transformation frequency (~ 103 transformants per μg of DNA), whereas the vector with the mutated selectable marker provided quite low transformation frequency (several clones per μg of DNA). To estimate the copy numbers of integrative modules, six and two transformants bearing pDel-PHO-kanMXmod and pDel-PHO-kanMX plasmids, respectively, were randomly chosen. These transformants were cultivated in liquid non-selective YPD medium for 8–10 generations and subsequently plated onto solid YPD medium; approximately 20 colonies of each strain were analyzed by diagnostic PCR. Each of the analyzed colonies retained the integration module. Copy numbers

Fig. 2. 1. Growth of E.coli cells containing plasmid pKanMX with wild-type kanMX4 gene (A) or pKanMXmod with kanMX4 mutated by error-prone PCR (B) on LB medium supplemented with 2 mg L−1 or 10 mg L−1 of geneticin. 2. Growth of E.coli cells containing plasmid pKanMXm1 with a mutation in kanMX4 gene causing D(120)G substitution (C); plasmid pKanMXm2 with E(267)D substitution (D) or plasmid pKanMXm1m2 with both mutations (E) on LB medium supplemented with 2 mg L−1 or 10 mg L−1 of geneticin. The three spots represent serial 10-fold dilutions of the cell suspension.

of vectors, integrated into the S. cerevisiae genome were analyzed by dot-blot hybridization. Samples of known quantities of genomic DNA were hybridized with labeled PHO8 gene. Two transformants strains with the wild-type marker revealed one or two additional copy of PHO8 gene integrated into the genome. Recombinant strains harboring the modified marker contained from 3 up to 10 copies of the reporter gene integrated into the genome (Fig. 3). In our previous work (Semkiv et al., 2014) we showed that constructed δ sequences-based vector provided tandem multi-copy integration in up to three sites of yeast genomic DNA in the so called “head-to-tail” conformation, which is in good agreement with other published results (Lee and Da Silva, 1997). To confirm the obtained result, the amount of synthesized mRNA of PHO8 gene in analyzed strains was estimated by quantitative RT-PCR (qRT-PCR). The relative expression levels of PHO8 transcript in recombinant strains with modified selection marker were up to 50- or 7-fold higher than that in the wild-type strain BY4742 or in strains with the intact form of kanMX4 gene, respectively (Fig. 4). The specific activity of the reporter enzyme alkaline phosphatase was also assayed. Strains containing a module with modified kanMX4 gene possessed strongly increased specific alkaline phosphatase activity compared to the parental and recombinant strains with intact kanMX4 gene (Fig. 5). BY-mut strains (multicopy integrants) revealed around 5–7-fold increase in Pho8 activity relative to BY-kanMX strains. Increased specific activity of alkaline phosphatase was additionally visualized by a plate patches assay. Strains with increased Pho8 activity stained more intensively in blue or yellow depending on chromogenic substrates (Fig. 5 lower panel). Plate patches or colony assay can be used for screening and selection of strains with maximal copy number of target plasmid, similarly, the assay of β-lactamase activity was used for selection by Hribar et al. (2008). Thus, the developed module provides one order of magnitude higher integration rate of the insertion cassette with the reporter PHO8 gene, as confirmed at DNA, RNA and protein levels. The frequency of integration events obviously depends on multiple factors: the efficacy of the transformation procedure; the length of the integration module (Lee and Da Silva, 1997); the severity of metabolic burden caused by the expression system in the host cells (Wang et al., 1996), etc. We used a developed integration module for overexpression of the PHO8 gene (Semkiv et al., 2014) or the GPD1 gene encoding glycerol-3-phosphate

Fig. 3. Estimation of integrated module copy number by dot-blot hybridization with labeled PHO8 gene. BY4742 – wild type strain; BY-mut and BY-kanMX – strains bearing module with modified and wild-type kanMX4 gene, respectively.

48

M.V. Semkiv et al. / Journal of Microbiological Methods 120 (2016) 44–49

when fast selection of multicopy integrants is required and one- or two-copy integrants should be avoided. We have demonstrated that the integrants are stable in nonselective rich media for 50 generations. Analysis of the reasons which provided efficiency of the modified selection marker revealed cumulative effect of several mutations inside of both promoter and ORF regions of kanMX4 gene. The developed δ-based multi-copy integration module harboring modified kanMX4 gene could be a powerful tool for exploring the effects of gene dosage on stable expression of target proteins in yeast. Since the activity of Pho8 acts as an indicator of copy number events of the integrated vector, expression of the gene of interest can be estimated directly and simply by assaying of alkaline phosphatase activity.

Acknowledgment Fig. 4. Relative expression level of PHO8 gene in recombinant strains bearing module with modified kanMX4 gene (BY-mut) or wild-type kanMX4 gene (BY-kanMX).

dehydrogenase (unpublished data). The developed system has similarities with other δ sequences-based modules, for example Parekh et al. (1996) obtained 1–30 copies of integrated module per cell after a single transformation using an improved δ integration vector with antibiotic selection provided by the weakly expressed (in yeast) NEO kanamycin resistance gene of transposon Tn903. NEO gene allowed copy number to be tuned by varying G418 resistance. Unfortunately we were unable to reach the highest integration frequency reported in this article. But neither did we observe single or two-copy integrants among randomly chosen transformants obtained with integration module bearing modified kanMX4 gene. The lowest number of integrations isolated is three, therefore three copies of the modified (i.e. less effective) kanMX4 gene are sufficient to overcome the 200 mg/L geneticin, but higher-copy integration events were also frequent. These observations indicate the applicability of the constructed module, especially in the conditions

The authors declare no conflict of interest. This work was supported in part by the Archer Daniels Midland Company (20.03.2013) (Decatur, IL) and a Polish grant from the National Scientific Center (NCN) DEC2012/05/B/NZ1/01657 awarded to A. Sibirny. We are grateful to Anastasiya Sybirna (Gurdon Institute, Cambridge, UK) and Petro Starokadomskyy (University of Texas Southwestern Medical Center, US) for the critical reading of the manuscript.

References Aw, R., Polizzi, K.M., 2013. Can too many copies spoil the broth? Microb. Cell Factories 12, 128. Beck, E., Ludwig, G., Auerswald, E., Reiss, B., Schaller, H., 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19, 327–336. Dower, W., Miller, J., Ragsdale, C., 1988. High efficiency transformation of E.coli by high voltage electroporation. Nucleic Acids Res. 16 (13), 6127–6145. Dujon, B., 1996. The yeast genome project: what did we learn? Trends Genet. 12, 263–270. Eichinger, D.J., Boeke, J.D., 1990. A specific terminal structure is required for Ty1 transposition. Genes Dev. 4, 324–330.

Fig. 5. Specific alkaline phosphatase activity measured in cell-free extracts or visualized on plates for strains bearing a module with modified kanMX4 gene (BY-mut) or unmodified kanMX4 gene (BY-kanMX). Substrates used for enzyme activity visualization: X-phosphate — 5-Bromo-4-chloro-3-indolyl phosphate disodium salt; p-NPP — para-Nitrophenylphosphate.

M.V. Semkiv et al. / Journal of Microbiological Methods 120 (2016) 44–49 Erhart, E., Hollenberg, C.P., 1983. The presence of a defective LEU2 gene in 2 pm DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number. J. Bacteriol. 156, 625–635. Fossati, E., Narcross, L., Ekins, A., Falgueyret, J.-P., Martin, V.J.J., 2015. Synthesis of morphinan alkaloids in Saccharomyces cerevisiae. PLoS One 10. Generoso, W.C., Schadeweg, V., Oreb, M., Boles, E., 2015. Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers. Curr. Opin. Biotechnol. 33, 1–7. Ito, H., Fukuda, Y., Murata, K., Kimura, A., 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168. Jensen, N.B., Strucko, T., Kildegaard, K.R., David, F., Maury, J., Mortensen, U.H., Forster, J., Nielsen, J., Borodina, I., 2014. EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res. 14, 238–248. Hribar, G., Smilović, V., Zupan, A.L., Gaberc-Porekar, V., 2008. Β-lactamase reporter system for selecting high-producing yeast clones. Biotechniques 44, 477–484. Kaneko, Y., Akio, T.-E., Oshima, Y., 1982. Identification of the genetic locus for the structural gene and a new regulatory gene for the synthesis of repressible alkaline phosphatase in Saccharomyces cerevisiae. Mol. Cell. Biol. 2 (2), 127–137. Kawai, S., Hashimoto, W., Murata, K., 2010. Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism. Bioeng. Bugs 1, 395–403. Lee, F.W.F., Da Silva, N., 1997. Improved efficiency and stability of multiple cloned gene insertions at the δ sequences of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 48, 339–345. Lopes T.S., Klootwijk J., Veenstra A.E., Aar P.C. van der, Heerikhuizen H. van, Raue H.A., Planta R.J., 1989. High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene 79, 199–206. Lopes, T.S., Hakkaart, G.J., Koerts, B.L., Raue, H.A., Planta, R.J., 1991. Mechanism of highcopy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae. Gene 105, 83–90. Lopes, T.S., de Wijs, I.J., Steenhauer, S.I., Verbakel, J., Planta, R.J., 1996. Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae. Yeast 12 467–477. Murray, W., Szostak, J.W., 1983. Pedigree analysis of plasmid segregation in yeast. Cell 34, 961–970.

49

Nguyen, H.T., Nevoigt, E., 2009. Engineering of Saccharomyces cerevisiae for the production of dihydroxyacetone (DHA) from sugars: a proof of concept. Metab. Eng. 11 (6), 335–346. Oliveira, C., Teixeira, J., Lima, N., Da Silva, N., Domingues, L., 2007. Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger betagalactosidase production. J. Biosci. Bioeng. 103, 318–324. Ostergaard, S., Olsson, L., Nielsen, J., 2000. Metabolic engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 64, 34–50. Parekh, R.N., Shaw, M.R., Wittrup, K.D., 1996. An integration vector for tunable, high copy, stable integration into the dispersed Ty δ sites of Saccharomyces cerevisiae. Biotechnol. Prog. 12, 16–21. Romanos, M., Scorer, C., Clare, J.J., 1992. Foreign gene expression in yeast: a review. Yeast 8, 423–488. Schiestl, R.H., Gietz, R.D., 1989. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16, 339–346. Semkiv, M.V., Dmytruk, K.V., Abbas, C., Sibirny, A., 2014. Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase. BMC Biotechnol. 14, 42. Taxis, C., Knop, M., 2006. System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. Biotechniques 40, 73–78. Tippmann, S., Scalcinati, G., Siewers, V., Nielsen, J., 2015. Production of farnesene and santalene by Saccharomyces cerevisiae using fed-batch cultivations with RQcontrolled feed. Biotechnol. Bioeng. 9999, 1–10. Wang, X., Wang, Z., Da Silva, N.A., 1996. G418 selection and stability of cloned genes integrated at the chromosomal d sequences of Saccharomyces cerevisiae. Biotechnol. Bioeng. 49, 45–51. Wang, Z.-X., Zhuge, J., Fang, H., Prior, B.A., 2001. Glycerol production by microbial fermentation: a review. Biotech. Adv. 19, 201–223. Zelle, R.M., de Hulster, E., van Winden, W.A., de Waard, P., Dijkema, C., Winkler, A.A., Geertman, J.-M.A., van Dijken, J.P., Pronk, J.T., van Maris, A.J.A., 2008. Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export. Appl. Environ. Microbiol. 74 (9), 2766–2777.