- Email: [email protected]
The PARS sequence increase the efficiency of stable Pichia pastoris transformation
The PARS sequence increase the efficiency of stable Pichia pastoris transformation Claus Krogh Madsen, Gilles Vismans, Henrik Brinch-Pedersen PII: DOI: Reference:
S0167-7012(16)30187-7 doi: 10.1016/j.mimet.2016.07.015 MIMET 4961
To appear in:
Journal of Microbiological Methods
Received date: Revised date: Accepted date:
26 April 2016 12 July 2016 16 July 2016
Please cite this article as: Madsen, Claus Krogh, Vismans, Gilles, Brinch-Pedersen, Henrik, The PARS sequence increase the efficiency of stable Pichia pastoris transformation, Journal of Microbiological Methods (2016), doi: 10.1016/j.mimet.2016.07.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Revised transformation
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Claus Krogh Madsen1*, Gilles Vismans2, Henrik Brinch-Pedersen1
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The PARS sequence increase the efficiency of stable Pichia pastoris
Department of Molecular Biology and Genetics, Section for Crop Genetics and Biotechnology,
Aarhus University. Forsogsvej, 1 4200 Slagelse, Denmark
Wageningen University, 6708PB, Wageningen, The Netherlands
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*Corresponing author: [email protected] (+45)87158104
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Abstract
The methylotrophic yeast Pichia pastoris is a popular host for recombinant expression of proteins.
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Plasmids containing the Pichia autonomously replicating sequence (PARS) transform P. pastoris
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with higher efficiency than linear DNA equipped with termini designed for homologous recombination. Moreover, PARS containing constructs provide higher protein yields. Unfortunately,
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these autonomous plasmids are inherently unstable and the preferred method of P. pastoris transformation is therefore stable integration in the genome by homologous recombination. In the present study we report that a novel combination of PARS and linearization of plasmids for P. pastoris transformation serves to significantly increase the transformation efficiency. Moreover, it is
genome.
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demonstrated that the constructs do not re-circularize but integrate stably into the P. pastoris
Keywords Pichia pastoris, PARS, Transformation efficiency, Homologous recombination.
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Introduction Autonomously replicating sequences (ARS) were first identified in baker’s yeast (Saccharomyses
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cerevisiae) as sequences from the yeast genome which enable plasmids to replicate autonomously (Struhl et al., 1979). Genomic ARS sequences are believed to serve as origins of DNA replication
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during mitosis (Huberman et al., 1988). A functionally similar sequence (PARS) have been identified in the popular recombinant expression host Pichia pastoris (Cregg et al., 1985). S.
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cerevisiae and P. pastoris are transformed with high efficiency by circular plasmids containing ARS or PARS respectively (Cregg, Barringer, Hessler and Madden, 1985, Struhl, Stinchcomb, Scherer
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and Davis, 1979). Unfortunately, autonomous P. pastoris plasmids have a high propensity to integrate in the genome if they contain segments with homology to the P. pastoris genome, e.g.
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endogenous promoter or terminator sequences. A plasmid with more than 500 bp homology will
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integrate within the first 100 generations after transformation (Higgins and Cregg, 1998). Such
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uncontrolled integration events are undesired because they convert the originally isolated clones with autonomous plasmids into a heterogeneous mixture of cells with various patterns of construct integration. Transgene expression is associated with a considerable metabolic burden (Görgens et
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al., 2001). It should therefore be expected that clones where only the selectable marker is expressed will have the competitive advantage and gradually become dominant thus compromising expression levels. There are few publications where the PARS sequence has been used in constructs for routine recombinant expression. Of the 42 expression studies listed by Macauley-Patrick et al. (2005) only one (Aoki et al. (2003)) used a vector with the PARS sequence. Steinle et al. (2010) used His4 complementation to stabilize autonomous plasmids for the expression of cyanophycin synthases. In addition, the plasmids harbored zeocin resistance which was used to evaluate the stability of the plasmids. While this method does not distinguish between integrated and autonomous plasmids, the
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ACCEPTED MANUSCRIPT authors did show that between 12 and 15% of cells in a flask culture would lose zeocin resistance in the absence of selection. His4 complementation retained zeocin resistance in 98% of the cells
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cultivated in the absence of zeocin. Hong et al. (2006) evaluated the potential of pGAPz-B with
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PARS inserted at the BamHI site as an autonomous plasmid vector. The stability of this vector was
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only 35% after 24 hours or 10 generations without selection. The authors achieved improved yields of β-Galactosidase compared to a stable transformant control when their autonomous plasmid clone
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was grown under zeocin selection. The continued presence of autonomous plasmids was confirmed by re-transformation of E. coli but the percentage of cells which retained autonomous plasmids was
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not determined. Lueking et al. (2000) reported shuttling 29 PARS plasmids from E. coli to P. pastoris and back but did not quantify their stability.
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The most widely used vectors are the pPIC and pGAP series which do not contain the PARS
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sequence (Macauley-Patrick, Fazenda, McNeil and Harvey, 2005). These are “ends in” vectors and
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constructs in this type of vector are linearized prior to P. pastoris transformation to promote homologous recombination between promoter regions on the plasmids and in the genome. Here we show that linearized plasmids containing the PARS sequence retain high transformation
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efficiency and integrate into the genome by recombination at the linearization site. This means that transformation efficiencies are significantly increased without compromising the benefits of stable chromosomal integration. The functionality of the resulting transformants was confirmed by expression of the chromogenic reporter protein TinselPurple.
Materials and methods Molecular cloning The PARS1 sequence was amplified from pPICHOLI (MoBiTec, Germany) using primers PARS1 Fus Fw/Rv (table 1). The PARS1 amplicon was inserted in pGAPzαB (ThermoFisher scientific, USA) at the BglI site. This was achieved by In-Fusion cloning (Clontec, USA) and resulted in
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ACCEPTED MANUSCRIPT pGAPzαB-PARS. The TinselPurple coding sequence was amplified from plasmid CPB-38-902 (DNA2.0, USA) with
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primers Int TP Fw and TP His XbaI Rv (table 1). The TinselPuple amplicon was inserted into
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pGAPzαB, linearized with BstBI and XbaI, by In-Fusion cloning (Clontec, USA) resulting in
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pGAPzαB-intTP (figure 1, left).
A fragment containing the TinselPurple coding sequence was subcloned from pGAPzαB-intTP to
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pGAPzαB-PARS after AvrII and BamHI digestion, gel purification of the relevant fragments followed by ligation with T4 DNA ligase. The final plasmid was named pGAPzαB-PARS-intTP
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(figure 1, right). Pichia transformation
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Competent cells were prepared by a modified condensed protocol (Lin-Cereghino et al., 2005).
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Briefly, 20mL of YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) in a 250 mL
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baffled flask was inoculated with P. pastoris KM71H cell mass transferred from a plate culture with a 3 mm inoculation loop. The culture was incubated at 27°C with 230 rpm shaking overnight. A further 80 mL of YPD was added after 18 hours where after the incubation was proceeded for four
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hours. At this point, a dilution series for OD600 measurement was prepared and the cells were harvested by centrifugation for 10 min at 1500×g. Typical OD600 values ranged from 6-10. The cells were washed three times with 50 mL of sterile demineralized water at the above mentioned centrifuge settings. The washed cells were re-suspended in 25 mL buffer SED (1M sorbitol, 50 mM Tris HCl, 25 mM EDTA, 10 mM DTT pH 7.5) and incubated at ambient temperature for 18 min. The SED treated cells were spun down at 1500×g at 4°C for 5 min and washed twice with 25 mL ice cold 1M sorbitol at the same centrifuge settings. Finally, the cells were re-suspended in ice cold 1 M sorbitol to an OD600 of 100 based on the initial OD measurement.
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ACCEPTED MANUSCRIPT The cells were kept on ice and used for transformation immediately. Preparation of linearized DNA
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Plasmid midi preps were prepared using the Nucleobond Xtra kit (Macherey-Nagel) according to
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the manufactures instructions. Restriction digests with AvrII and BspHI were carried out over night
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at 37°C in 1×cutsmart buffer, with 0.1 u of enzyme per μg plasmid at a plasmid concentration of 0.5 μg/μL (all reagents from New England Biolabs, USA). The linearized plasmids were agarose gel
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purified and the concentration was measured on a Nanodrop spectrophotometer. Transformation
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The desired DNA amount was mixed with 400 μL of competent cells in a 0.2 cm Gene Pulser cuvette (BIO-RAD) and incubated on ice for 30 min before electroporation. The pulse settings were
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1.8 kV, 200Ω and 25μF. The cells were transferred to a 1.5 mL centrifuge tube with the help on 1
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mL 1M sorbitol and incubated at 28°C o/n before plating on YPD agar with 100 μg/mL zeocin.
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Plates were incubated at 28°C for two days at which point the colonies were counted. Colonies selected for DNA purification were propagated in liquid YPD and P. pastoris total DNA was
PCR
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purified according to (Lõoke et al., 2011).
Control PCR reactions were performed with Failsafe polymerase (Epicentre, USA) and primer pairs pUC-GAP Fw+pUC-GAP Rv2, GAPflank Fw+TPint Rv and pUC-GAP Fw+TPint R (table1). E. coli transformation E. coli strain “Stellar” (Clontech, USA) was transformed by heat chock according to the manufacturer’s instructions for the cloning DNA cloning procedures. ElectroMAX Stbl4 (Invitrogen, USA) was transformed with 2.5 μL P. pastoris total DNA by electroporation according to the manufacturer’s instructions in re-transformation experiments.
Results and discussion
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ACCEPTED MANUSCRIPT Transformation experiment 1 The expression constructs pGAPzαB-intTP and pGAPzαB-PARS-intTP are identical apart from the
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160 bp PARS1 sequence which make up 4.3% of the pGAPzαB-PARS-intTP plasmid. The two
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plasmids were linearized with AvrII and BspHI and gel purified. Competent P. pastoris cells from
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the same batch were transformed with 0.5 µg linear plasmid in triplicate. One and five hundred µL of the transformations were plated and the colonies were counted after two days. The counts were
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converted to colony forming units (cfu) per µg DNA per transformation (figure 2). Transformations with pGAPzαB-PARS-intTP produced more zeocin resistant colonies regardless of the enzyme used
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for linearization. When the vector was linearized with AvrII before transformation, the mean number of colonies was significantly higher with the PARS version of the construct (1180.7±366.6)
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compared to version without PARS (138±75.6) (T-test, N=3, p=0.009). An even bigger difference of
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65.3±9.3 versus 2401.1±572.2 (T-test, N=3, p=0.019) was seen with BspHI used for linearization.
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After a futher three days incubation, the ablity of the clones to express the TinselPurple protein was evalued by visual inspection for pink to purple color. Transformant colonies with the PARS sequence were more frequently expressing the transgene as illustrated in figure 3.
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Transformation experiment 2
Fresh DNA midipreps were prepared and linearized with BspHI to exclude batch to batch variation of DNA quality as an explanation for the observed differences in transformation efficiency. P. pastoris transformation was performed using three different DNA amounts (0.1 ,0.5 and 0.75µg) each in triplicate. Again, linear DNA with the PARS sequence showed the highest transformation efficiency at all DNA amounts (Figure 4A). There is high variation in the number of colonies between repetitions of the same treatment and the trend is therefore only statistically significant for the highest DNA amount 23±0 cfu/transformation vs. 535±206 cfu/transformation ( t-test, N=3, p=0.045). Increasing DNA amount improved both total colony count and specific transformation
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ACCEPTED MANUSCRIPT efficiencies (figure 4A+B). Genomic integration of linear DNA with PARS
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The observation that linear DNA with the PARS sequence retains the high transformation efficiency
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known from circular plasmids with PARS raises the question; do the linearized plasmids
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recircularize to form autonomous plasmids or do they integrate in the P. pastoris genome? To examine this, DNA was isolated from 10 randomly selected colonies of each treatment in
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transformation experiment 1 and one colony transformed with circular pGAPzαB-PARS-intTP as a control. Thus 41 colonies in total.
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The isolated DNA (2.5 µL/250-750 ng) was used to transform E. coli Stbl4. A control reaction was performed with 10 pg pUC19 supplied by ThermoFisher scientific, USA, and intended for quality
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control of competent cells. Of 500 µL transformation reaction we plated 100 µL and for pUC19, 1
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µL. None of the reactions with DNA from the experimental clones produced any colonies. The 1 µL
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pUC19 control produced 21 colonies and the control transformed with circular pGAPzαB-PARSintTP produced two colonies. We were able to recover and confirm the identity of the construct from the control colonies by restriction digest. This result suggests that the linearized plasmids do
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not simply re-circularize once inside P. pastoris cells. Position and mechanism of genomic integration The primers GAPflank Fw and TPint Rv were used for control PCRs to examine if the linearized plasmids integrate in the GAP promotor by homologous recombination at the left border. GAPflank Fw primes anneals in the P. pastoris genome upstream of the GAP promotor segment included in the constructs. TPint Rv primes anneals in the TinselPurple coding sequence. All 20 pGAPzαBintTP (i.e. non-PARS) clones produced the expected amplicon of 600 bp whereas for pGAPzαBPARS-intTP it was only the case for 12 out of 20 (figure 5). Nevertheless, this illustrates that linear DNA with PARS and termini capable of homologous recombination, mostly integrates at the target
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ACCEPTED MANUSCRIPT site in the P. pastoris genome. Integration at the right border was investigated using primers pUCGAP Fw and pUC-GAP Rv2 which primes at pUC orgin of the constructs and in the endogenous P.
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pastoris GAP coding sequence, respectively. Nineteen pGAPzαB-intTP clones produced the
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expected amplicon of 1363 bp which proves normal integration by homologous recombination.
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Eleven pGAPzαB-PARS-intTP clones produced an amplicon of the same size which is shorter than the expected 1520 bp. This suggests that the PARS containing constructs integrate by homologous
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recombination at the right border in approximately half of the clones. The foreign DNA is however slightly truncated so that no size difference is observed between PARS versus non PARS clones.
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The primer combination pUC-GAP Fw and Int TP Rv produce amplicons of 846 bp and 1006 bp from circular pGAPzαB-intTP and pGAPzαB-PARS-intTP plasmids, respectively. The same
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amplicons are produced from genomic DNA containing tandem integrated constructs. All 20
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pGAPzαB-intTP transformants produced the expected amplicon with this primer set. Eighteen of 20
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pGAPzαB-PARS-intTP transformants also produced the expected amplicon but all 20 produced an amplicon of similar size to the amplicon produced by the pGAPzαB-intTP transformants (figure 7). Sequencing of the bands originating from pGAPzαB-PARS-intTP transformants confirmed that the
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higher band was as expected whereas the PARS sequence had been deleted from the lower band. This shows that both constructs integrate predominantly in tandem at a zeocin concentration of 100 μg/mL but the PARS containing constructs integrated partially by a novel mechanism which results in the deletion of the PARS sequence. The clones transformed with the PARS containing construct generally produced fainter expected bands and more unspecific bands in the two PCR reactions which span the integration borders (figures 5 and 6) but not in the reaction that spans tandem head to tail inserts (figure 7). These clones are may have higher transgene copy numbers since this is likely to correlate with the increase in transformation efficiency. If this is the case, the higher copy number may in turn be inhibitory to
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ACCEPTED MANUSCRIPT the cross border PCR reactions because the construct specific primer would have multiple dead end priming sites. One on each construct copy which does not flank the border.
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Proposed mechanism
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As demonstrated, the linearized plasmids with the PARS sequence showed increased transformation
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efficiency compared to a sister construct without PARS. The genomic integration was almost exclusively by homologous recombination at both borders for the non-PARS construct pGAPzαB-
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intTP. In contrast, only approximately half of the PCR reactions across the expected integration borders responded in the pGAPzαB-PARS-intTP clones. Three of 20 PARS containing clones did
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not respond at either integration border. Furthermore, the right border of pGAPzαB-PARS-intTP copies frequently showed a deletion of the same size as the PARS sequence. Sequencing of a
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truncated amplicon that spans tandem integrated copies confirmed that this deletion is of the PARS
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sequence.
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We propose that these observations may be explained by partial replication of the constructs before genomic integration. The replication would start at the PARS sequence and proceed to the end of linearized molecule thus generating a single stranded and truncated partial replicon (figure 8). Being
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single stranded and numerous, the partial replicons are very likely to integrate in the genome. The lack of right border homology in these replicons may explain why the PARS clones to some extent integrate in an illegitimate fashion as the PCR results suggest. While this mechanism explains the deletion of the PARS sequence, it may not be the only factor influencing the transformation efficiency. Another possibility is that DNA with the PARS sequence is more efficiently imported to the nucleus. Although it is not known how foreign DNA passes the nuclear envelope some clues may be offered by the study of the cell cycle. The MCM 1-2 proteins are imported from the cytoplasm to the nucleus at the end of mitosis in Saccharomyces. Once inside the nucleus these replication regulators become associated with the ARS sequences (Yan et al., 1993). Thus, it is
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ACCEPTED MANUSCRIPT possible that foreign DNA containing the PARS sequence is co-imported with DNA binding proteins that shuttle between the cytoplasm and nucleus. The exact mechanism behind our
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observations require further investigation but it is clear that our discovery can be used to greatly
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enhance transformation efficiency of P. pastoris.
References
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ACCEPTED MANUSCRIPT Aoki, H., Nazmul Ahsan, M., Watabe, S., 2003. Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp. Protein Expression and Purification. 31, 213-221.
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Cregg, J.M., Barringer, K.J., Hessler, A.Y., Madden, K.R., 1985. Pichia pastoris as a host system for transformations. Molecular and Cellular Biology. 5, 3376-3385.
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Görgens, J.F., van Zyl, W.H., Knoetze, J.H., Hahn-Hägerdal, B., 2001. The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium. Biotechnology and bioengineering. 73, 238-245. Higgins, D.R., Cregg, J.M., 1998. Pichia protocols, Springer.
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Hong, I.P., Anderson, S., Choi, S.G., 2006. Evaluation of a new episomal vector based on the GAP promoter for structural genomics in Pichia pastoris. Journal of microbiology and biotechnology. 16, 1362-1368.
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Huberman, J.A., Zhu, J., Davis, L.R., Newlon, C.S., 1988. Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae. Nucleic Acids Research. 16, 6373-6384.
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Lin-Cereghino, J., Wong, W.W., Giang, W., Luong, L.T., Vu, J., Johnson, S.D., Lin-Cereghino, G.P., 2005. Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques. 38, 44.
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Lõoke, M., Kristjuhan, K., Kristjuhan, A., 2011. Extraction of genomic DNA from yeasts for PCRbased applications. Biotechniques. 50, 325. Lueking, A., Holz, C., Gotthold, C., Lehrach, H., Cahill, D., 2000. A system for dual protein expression in Pichia pastoris and Escherichia coli. Protein Expression and Purification. 20, 372-378.
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Macauley-Patrick, S., Fazenda, M.L., McNeil, B., Harvey, L.M., 2005. Heterologous protein production using the Pichia pastoris expression system. Yeast. 22, 249-270. Steinle, A., Witthoff, S., Krause, J.P., Steinb++chel, A., 2010. Establishment of Cyanophycin Biosynthesis in Pichia pastoris and Optimization by Use of Engineered Cyanophycin Synthetases. Applied and Environmental Microbiology. 76, 1062-1070. Struhl, K., Stinchcomb, D.T., Scherer, S., Davis, R.W., 1979. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proceedings of the National Academy of Sciences. 76, 1035-1039. Yan, H., Merchant, A.M., Tye, B.K., 1993. Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. Genes & Development. 7, 2149-2160.
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ACCEPTED MANUSCRIPT Legends Figure 1. The two constructs used for P. pastoris transformation
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Figure 2. Transformation efficiencies of the two constructs linearized with AvrII and BspHI. Error
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bars represent the standard deviation. Letters above the columns indicate statistical significance of
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the difference between the results.
Figure 3. Percentage of colonies with a pink to purple color indicative of transgene expression.
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Error bars represent the standard deviation. Letters above the columns indicate statistical significance of the difference between the results.
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Figure 4. Colony counts (A) and transformation efficiencies (B) of transformations with three different DNA amounts of the two constructs. Error bars represent the standard deviation. Letters
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above the columns indicate statistical significance of the difference between the results obtained
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with the two respective constructs at the given DNA amount.
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Figure 5. Agarose gel of 20 PCR amplicons across the left border (primers GAPflank Fw and TPint Rv). The DNA ladder is “GeneRuler 1Kb” from ThermoFisher scientific and “Wt” is a negative control reaction with DNA from untransformed P. pastoris.
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Figure 6. Agarose gel of PCR amplicons across the right border (primers pUC-GAP Fw and pUCGAP Rv2). The DNA ladder is “GeneRuler 1Kb” from ThermoFisher scientific and “Wt” is a negative control reaction with DNA from untransformed P. pastoris. Figure 7. Agarose gel of PCR amplicons across the right border between tandem integrated constructs (primers pUC-GAP Fw and Int TP Rv). The DNA ladder is “GeneRuler 1Kb” from ThermoFisher scientific. +PARS, “Wt” and –PARS are control reactions with pGAPzαB-PARSintTP, DNA from untransformed P. pastoris and pGAPzαB-intTP respectivly.
Figure 8. Start and stop sites of replication of linear pGAPzαB-PARS-intTP as defined by the
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ACCEPTED MANUSCRIPT location of the PARS sequence and the restriction site used for linearization (here AvrII).
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Table 1. PCR primers used for cloning and control PCR reactions.
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Primer sequence
PARS1 Fus Fw
5'GCATGAGATCAGATCCAATTAATATTTACTTATTTTGGT3'
PARS1 Fus Rv
5'TCTACAAAAAAGATCGATAAGCTGGGGGAACATT3'
Int TP Fw
5'ATTGAACAACTATTTAAAAATGGCATCTTTAGTCAAAAAGG3'
TP His XbaI Rv
5'GATGAGTTTTTGTTCTAGTTAATGATGATGATGATGATGGTCGAC
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Primer name
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GAATTCTGG3'
5’GCAGAGCGAGGTATGTAGGC3’
pUC-GAP Rv2
5’CCGTCAACGGTCTTTTGAGT3’
GAPflank Fw
5’CGATCAATGAAATCCATCAAGA3’
TPint Rv
5’CGGTACCTTCCATCGTCATT3’
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pUC-GAP Fw
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Table 1
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Highlights
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Linear plasmids with the PARS sequence transform P. pastoris with high efficiency. The linear plasmids integrate in the P. pastoris genome. They are not episomal. A novel mechanism for genomic integration is suggested. Constructs which are inherently more efficient for transformation can be designed.
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S0167-7012(16)30187-7 doi: 10.1016/j.mimet.2016.07.015 MIMET 4961
To appear in:
Journal of Microbiological Methods
Received date: Revised date: Accepted date:
26 April 2016 12 July 2016 16 July 2016
Please cite this article as: Madsen, Claus Krogh, Vismans, Gilles, Brinch-Pedersen, Henrik, The PARS sequence increase the efficiency of stable Pichia pastoris transformation, Journal of Microbiological Methods (2016), doi: 10.1016/j.mimet.2016.07.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Revised transformation
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Claus Krogh Madsen1*, Gilles Vismans2, Henrik Brinch-Pedersen1
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The PARS sequence increase the efficiency of stable Pichia pastoris
Department of Molecular Biology and Genetics, Section for Crop Genetics and Biotechnology,
Aarhus University. Forsogsvej, 1 4200 Slagelse, Denmark
Wageningen University, 6708PB, Wageningen, The Netherlands
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*Corresponing author: [email protected] (+45)87158104
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Abstract
The methylotrophic yeast Pichia pastoris is a popular host for recombinant expression of proteins.
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Plasmids containing the Pichia autonomously replicating sequence (PARS) transform P. pastoris
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with higher efficiency than linear DNA equipped with termini designed for homologous recombination. Moreover, PARS containing constructs provide higher protein yields. Unfortunately,
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these autonomous plasmids are inherently unstable and the preferred method of P. pastoris transformation is therefore stable integration in the genome by homologous recombination. In the present study we report that a novel combination of PARS and linearization of plasmids for P. pastoris transformation serves to significantly increase the transformation efficiency. Moreover, it is
genome.
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demonstrated that the constructs do not re-circularize but integrate stably into the P. pastoris
Keywords Pichia pastoris, PARS, Transformation efficiency, Homologous recombination.
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Introduction Autonomously replicating sequences (ARS) were first identified in baker’s yeast (Saccharomyses
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cerevisiae) as sequences from the yeast genome which enable plasmids to replicate autonomously (Struhl et al., 1979). Genomic ARS sequences are believed to serve as origins of DNA replication
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during mitosis (Huberman et al., 1988). A functionally similar sequence (PARS) have been identified in the popular recombinant expression host Pichia pastoris (Cregg et al., 1985). S.
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cerevisiae and P. pastoris are transformed with high efficiency by circular plasmids containing ARS or PARS respectively (Cregg, Barringer, Hessler and Madden, 1985, Struhl, Stinchcomb, Scherer
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and Davis, 1979). Unfortunately, autonomous P. pastoris plasmids have a high propensity to integrate in the genome if they contain segments with homology to the P. pastoris genome, e.g.
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endogenous promoter or terminator sequences. A plasmid with more than 500 bp homology will
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integrate within the first 100 generations after transformation (Higgins and Cregg, 1998). Such
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uncontrolled integration events are undesired because they convert the originally isolated clones with autonomous plasmids into a heterogeneous mixture of cells with various patterns of construct integration. Transgene expression is associated with a considerable metabolic burden (Görgens et
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al., 2001). It should therefore be expected that clones where only the selectable marker is expressed will have the competitive advantage and gradually become dominant thus compromising expression levels. There are few publications where the PARS sequence has been used in constructs for routine recombinant expression. Of the 42 expression studies listed by Macauley-Patrick et al. (2005) only one (Aoki et al. (2003)) used a vector with the PARS sequence. Steinle et al. (2010) used His4 complementation to stabilize autonomous plasmids for the expression of cyanophycin synthases. In addition, the plasmids harbored zeocin resistance which was used to evaluate the stability of the plasmids. While this method does not distinguish between integrated and autonomous plasmids, the
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ACCEPTED MANUSCRIPT authors did show that between 12 and 15% of cells in a flask culture would lose zeocin resistance in the absence of selection. His4 complementation retained zeocin resistance in 98% of the cells
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cultivated in the absence of zeocin. Hong et al. (2006) evaluated the potential of pGAPz-B with
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PARS inserted at the BamHI site as an autonomous plasmid vector. The stability of this vector was
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only 35% after 24 hours or 10 generations without selection. The authors achieved improved yields of β-Galactosidase compared to a stable transformant control when their autonomous plasmid clone
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was grown under zeocin selection. The continued presence of autonomous plasmids was confirmed by re-transformation of E. coli but the percentage of cells which retained autonomous plasmids was
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not determined. Lueking et al. (2000) reported shuttling 29 PARS plasmids from E. coli to P. pastoris and back but did not quantify their stability.
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The most widely used vectors are the pPIC and pGAP series which do not contain the PARS
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sequence (Macauley-Patrick, Fazenda, McNeil and Harvey, 2005). These are “ends in” vectors and
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constructs in this type of vector are linearized prior to P. pastoris transformation to promote homologous recombination between promoter regions on the plasmids and in the genome. Here we show that linearized plasmids containing the PARS sequence retain high transformation
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efficiency and integrate into the genome by recombination at the linearization site. This means that transformation efficiencies are significantly increased without compromising the benefits of stable chromosomal integration. The functionality of the resulting transformants was confirmed by expression of the chromogenic reporter protein TinselPurple.
Materials and methods Molecular cloning The PARS1 sequence was amplified from pPICHOLI (MoBiTec, Germany) using primers PARS1 Fus Fw/Rv (table 1). The PARS1 amplicon was inserted in pGAPzαB (ThermoFisher scientific, USA) at the BglI site. This was achieved by In-Fusion cloning (Clontec, USA) and resulted in
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ACCEPTED MANUSCRIPT pGAPzαB-PARS. The TinselPurple coding sequence was amplified from plasmid CPB-38-902 (DNA2.0, USA) with
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primers Int TP Fw and TP His XbaI Rv (table 1). The TinselPuple amplicon was inserted into
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pGAPzαB, linearized with BstBI and XbaI, by In-Fusion cloning (Clontec, USA) resulting in
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pGAPzαB-intTP (figure 1, left).
A fragment containing the TinselPurple coding sequence was subcloned from pGAPzαB-intTP to
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pGAPzαB-PARS after AvrII and BamHI digestion, gel purification of the relevant fragments followed by ligation with T4 DNA ligase. The final plasmid was named pGAPzαB-PARS-intTP
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(figure 1, right). Pichia transformation
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Competent cells were prepared by a modified condensed protocol (Lin-Cereghino et al., 2005).
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Briefly, 20mL of YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) in a 250 mL
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baffled flask was inoculated with P. pastoris KM71H cell mass transferred from a plate culture with a 3 mm inoculation loop. The culture was incubated at 27°C with 230 rpm shaking overnight. A further 80 mL of YPD was added after 18 hours where after the incubation was proceeded for four
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hours. At this point, a dilution series for OD600 measurement was prepared and the cells were harvested by centrifugation for 10 min at 1500×g. Typical OD600 values ranged from 6-10. The cells were washed three times with 50 mL of sterile demineralized water at the above mentioned centrifuge settings. The washed cells were re-suspended in 25 mL buffer SED (1M sorbitol, 50 mM Tris HCl, 25 mM EDTA, 10 mM DTT pH 7.5) and incubated at ambient temperature for 18 min. The SED treated cells were spun down at 1500×g at 4°C for 5 min and washed twice with 25 mL ice cold 1M sorbitol at the same centrifuge settings. Finally, the cells were re-suspended in ice cold 1 M sorbitol to an OD600 of 100 based on the initial OD measurement.
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Plasmid midi preps were prepared using the Nucleobond Xtra kit (Macherey-Nagel) according to
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the manufactures instructions. Restriction digests with AvrII and BspHI were carried out over night
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at 37°C in 1×cutsmart buffer, with 0.1 u of enzyme per μg plasmid at a plasmid concentration of 0.5 μg/μL (all reagents from New England Biolabs, USA). The linearized plasmids were agarose gel
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purified and the concentration was measured on a Nanodrop spectrophotometer. Transformation
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The desired DNA amount was mixed with 400 μL of competent cells in a 0.2 cm Gene Pulser cuvette (BIO-RAD) and incubated on ice for 30 min before electroporation. The pulse settings were
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1.8 kV, 200Ω and 25μF. The cells were transferred to a 1.5 mL centrifuge tube with the help on 1
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mL 1M sorbitol and incubated at 28°C o/n before plating on YPD agar with 100 μg/mL zeocin.
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Plates were incubated at 28°C for two days at which point the colonies were counted. Colonies selected for DNA purification were propagated in liquid YPD and P. pastoris total DNA was
PCR
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purified according to (Lõoke et al., 2011).
Control PCR reactions were performed with Failsafe polymerase (Epicentre, USA) and primer pairs pUC-GAP Fw+pUC-GAP Rv2, GAPflank Fw+TPint Rv and pUC-GAP Fw+TPint R (table1). E. coli transformation E. coli strain “Stellar” (Clontech, USA) was transformed by heat chock according to the manufacturer’s instructions for the cloning DNA cloning procedures. ElectroMAX Stbl4 (Invitrogen, USA) was transformed with 2.5 μL P. pastoris total DNA by electroporation according to the manufacturer’s instructions in re-transformation experiments.
Results and discussion
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ACCEPTED MANUSCRIPT Transformation experiment 1 The expression constructs pGAPzαB-intTP and pGAPzαB-PARS-intTP are identical apart from the
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160 bp PARS1 sequence which make up 4.3% of the pGAPzαB-PARS-intTP plasmid. The two
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plasmids were linearized with AvrII and BspHI and gel purified. Competent P. pastoris cells from
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the same batch were transformed with 0.5 µg linear plasmid in triplicate. One and five hundred µL of the transformations were plated and the colonies were counted after two days. The counts were
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converted to colony forming units (cfu) per µg DNA per transformation (figure 2). Transformations with pGAPzαB-PARS-intTP produced more zeocin resistant colonies regardless of the enzyme used
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for linearization. When the vector was linearized with AvrII before transformation, the mean number of colonies was significantly higher with the PARS version of the construct (1180.7±366.6)
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compared to version without PARS (138±75.6) (T-test, N=3, p=0.009). An even bigger difference of
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65.3±9.3 versus 2401.1±572.2 (T-test, N=3, p=0.019) was seen with BspHI used for linearization.
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After a futher three days incubation, the ablity of the clones to express the TinselPurple protein was evalued by visual inspection for pink to purple color. Transformant colonies with the PARS sequence were more frequently expressing the transgene as illustrated in figure 3.
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Transformation experiment 2
Fresh DNA midipreps were prepared and linearized with BspHI to exclude batch to batch variation of DNA quality as an explanation for the observed differences in transformation efficiency. P. pastoris transformation was performed using three different DNA amounts (0.1 ,0.5 and 0.75µg) each in triplicate. Again, linear DNA with the PARS sequence showed the highest transformation efficiency at all DNA amounts (Figure 4A). There is high variation in the number of colonies between repetitions of the same treatment and the trend is therefore only statistically significant for the highest DNA amount 23±0 cfu/transformation vs. 535±206 cfu/transformation ( t-test, N=3, p=0.045). Increasing DNA amount improved both total colony count and specific transformation
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ACCEPTED MANUSCRIPT efficiencies (figure 4A+B). Genomic integration of linear DNA with PARS
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The observation that linear DNA with the PARS sequence retains the high transformation efficiency
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known from circular plasmids with PARS raises the question; do the linearized plasmids
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recircularize to form autonomous plasmids or do they integrate in the P. pastoris genome? To examine this, DNA was isolated from 10 randomly selected colonies of each treatment in
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transformation experiment 1 and one colony transformed with circular pGAPzαB-PARS-intTP as a control. Thus 41 colonies in total.
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The isolated DNA (2.5 µL/250-750 ng) was used to transform E. coli Stbl4. A control reaction was performed with 10 pg pUC19 supplied by ThermoFisher scientific, USA, and intended for quality
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control of competent cells. Of 500 µL transformation reaction we plated 100 µL and for pUC19, 1
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µL. None of the reactions with DNA from the experimental clones produced any colonies. The 1 µL
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pUC19 control produced 21 colonies and the control transformed with circular pGAPzαB-PARSintTP produced two colonies. We were able to recover and confirm the identity of the construct from the control colonies by restriction digest. This result suggests that the linearized plasmids do
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not simply re-circularize once inside P. pastoris cells. Position and mechanism of genomic integration The primers GAPflank Fw and TPint Rv were used for control PCRs to examine if the linearized plasmids integrate in the GAP promotor by homologous recombination at the left border. GAPflank Fw primes anneals in the P. pastoris genome upstream of the GAP promotor segment included in the constructs. TPint Rv primes anneals in the TinselPurple coding sequence. All 20 pGAPzαBintTP (i.e. non-PARS) clones produced the expected amplicon of 600 bp whereas for pGAPzαBPARS-intTP it was only the case for 12 out of 20 (figure 5). Nevertheless, this illustrates that linear DNA with PARS and termini capable of homologous recombination, mostly integrates at the target
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ACCEPTED MANUSCRIPT site in the P. pastoris genome. Integration at the right border was investigated using primers pUCGAP Fw and pUC-GAP Rv2 which primes at pUC orgin of the constructs and in the endogenous P.
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pastoris GAP coding sequence, respectively. Nineteen pGAPzαB-intTP clones produced the
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expected amplicon of 1363 bp which proves normal integration by homologous recombination.
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Eleven pGAPzαB-PARS-intTP clones produced an amplicon of the same size which is shorter than the expected 1520 bp. This suggests that the PARS containing constructs integrate by homologous
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recombination at the right border in approximately half of the clones. The foreign DNA is however slightly truncated so that no size difference is observed between PARS versus non PARS clones.
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The primer combination pUC-GAP Fw and Int TP Rv produce amplicons of 846 bp and 1006 bp from circular pGAPzαB-intTP and pGAPzαB-PARS-intTP plasmids, respectively. The same
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amplicons are produced from genomic DNA containing tandem integrated constructs. All 20
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pGAPzαB-intTP transformants produced the expected amplicon with this primer set. Eighteen of 20
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pGAPzαB-PARS-intTP transformants also produced the expected amplicon but all 20 produced an amplicon of similar size to the amplicon produced by the pGAPzαB-intTP transformants (figure 7). Sequencing of the bands originating from pGAPzαB-PARS-intTP transformants confirmed that the
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higher band was as expected whereas the PARS sequence had been deleted from the lower band. This shows that both constructs integrate predominantly in tandem at a zeocin concentration of 100 μg/mL but the PARS containing constructs integrated partially by a novel mechanism which results in the deletion of the PARS sequence. The clones transformed with the PARS containing construct generally produced fainter expected bands and more unspecific bands in the two PCR reactions which span the integration borders (figures 5 and 6) but not in the reaction that spans tandem head to tail inserts (figure 7). These clones are may have higher transgene copy numbers since this is likely to correlate with the increase in transformation efficiency. If this is the case, the higher copy number may in turn be inhibitory to
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ACCEPTED MANUSCRIPT the cross border PCR reactions because the construct specific primer would have multiple dead end priming sites. One on each construct copy which does not flank the border.
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Proposed mechanism
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As demonstrated, the linearized plasmids with the PARS sequence showed increased transformation
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efficiency compared to a sister construct without PARS. The genomic integration was almost exclusively by homologous recombination at both borders for the non-PARS construct pGAPzαB-
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intTP. In contrast, only approximately half of the PCR reactions across the expected integration borders responded in the pGAPzαB-PARS-intTP clones. Three of 20 PARS containing clones did
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not respond at either integration border. Furthermore, the right border of pGAPzαB-PARS-intTP copies frequently showed a deletion of the same size as the PARS sequence. Sequencing of a
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truncated amplicon that spans tandem integrated copies confirmed that this deletion is of the PARS
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sequence.
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We propose that these observations may be explained by partial replication of the constructs before genomic integration. The replication would start at the PARS sequence and proceed to the end of linearized molecule thus generating a single stranded and truncated partial replicon (figure 8). Being
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single stranded and numerous, the partial replicons are very likely to integrate in the genome. The lack of right border homology in these replicons may explain why the PARS clones to some extent integrate in an illegitimate fashion as the PCR results suggest. While this mechanism explains the deletion of the PARS sequence, it may not be the only factor influencing the transformation efficiency. Another possibility is that DNA with the PARS sequence is more efficiently imported to the nucleus. Although it is not known how foreign DNA passes the nuclear envelope some clues may be offered by the study of the cell cycle. The MCM 1-2 proteins are imported from the cytoplasm to the nucleus at the end of mitosis in Saccharomyces. Once inside the nucleus these replication regulators become associated with the ARS sequences (Yan et al., 1993). Thus, it is
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ACCEPTED MANUSCRIPT possible that foreign DNA containing the PARS sequence is co-imported with DNA binding proteins that shuttle between the cytoplasm and nucleus. The exact mechanism behind our
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observations require further investigation but it is clear that our discovery can be used to greatly
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enhance transformation efficiency of P. pastoris.
References
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ACCEPTED MANUSCRIPT Aoki, H., Nazmul Ahsan, M., Watabe, S., 2003. Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp. Protein Expression and Purification. 31, 213-221.
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Cregg, J.M., Barringer, K.J., Hessler, A.Y., Madden, K.R., 1985. Pichia pastoris as a host system for transformations. Molecular and Cellular Biology. 5, 3376-3385.
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Görgens, J.F., van Zyl, W.H., Knoetze, J.H., Hahn-Hägerdal, B., 2001. The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium. Biotechnology and bioengineering. 73, 238-245. Higgins, D.R., Cregg, J.M., 1998. Pichia protocols, Springer.
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Hong, I.P., Anderson, S., Choi, S.G., 2006. Evaluation of a new episomal vector based on the GAP promoter for structural genomics in Pichia pastoris. Journal of microbiology and biotechnology. 16, 1362-1368.
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Huberman, J.A., Zhu, J., Davis, L.R., Newlon, C.S., 1988. Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae. Nucleic Acids Research. 16, 6373-6384.
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Lin-Cereghino, J., Wong, W.W., Giang, W., Luong, L.T., Vu, J., Johnson, S.D., Lin-Cereghino, G.P., 2005. Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques. 38, 44.
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Lõoke, M., Kristjuhan, K., Kristjuhan, A., 2011. Extraction of genomic DNA from yeasts for PCRbased applications. Biotechniques. 50, 325. Lueking, A., Holz, C., Gotthold, C., Lehrach, H., Cahill, D., 2000. A system for dual protein expression in Pichia pastoris and Escherichia coli. Protein Expression and Purification. 20, 372-378.
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Macauley-Patrick, S., Fazenda, M.L., McNeil, B., Harvey, L.M., 2005. Heterologous protein production using the Pichia pastoris expression system. Yeast. 22, 249-270. Steinle, A., Witthoff, S., Krause, J.P., Steinb++chel, A., 2010. Establishment of Cyanophycin Biosynthesis in Pichia pastoris and Optimization by Use of Engineered Cyanophycin Synthetases. Applied and Environmental Microbiology. 76, 1062-1070. Struhl, K., Stinchcomb, D.T., Scherer, S., Davis, R.W., 1979. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proceedings of the National Academy of Sciences. 76, 1035-1039. Yan, H., Merchant, A.M., Tye, B.K., 1993. Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. Genes & Development. 7, 2149-2160.
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ACCEPTED MANUSCRIPT Legends Figure 1. The two constructs used for P. pastoris transformation
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Figure 2. Transformation efficiencies of the two constructs linearized with AvrII and BspHI. Error
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bars represent the standard deviation. Letters above the columns indicate statistical significance of
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the difference between the results.
Figure 3. Percentage of colonies with a pink to purple color indicative of transgene expression.
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Error bars represent the standard deviation. Letters above the columns indicate statistical significance of the difference between the results.
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Figure 4. Colony counts (A) and transformation efficiencies (B) of transformations with three different DNA amounts of the two constructs. Error bars represent the standard deviation. Letters
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above the columns indicate statistical significance of the difference between the results obtained
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with the two respective constructs at the given DNA amount.
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Figure 5. Agarose gel of 20 PCR amplicons across the left border (primers GAPflank Fw and TPint Rv). The DNA ladder is “GeneRuler 1Kb” from ThermoFisher scientific and “Wt” is a negative control reaction with DNA from untransformed P. pastoris.
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Figure 6. Agarose gel of PCR amplicons across the right border (primers pUC-GAP Fw and pUCGAP Rv2). The DNA ladder is “GeneRuler 1Kb” from ThermoFisher scientific and “Wt” is a negative control reaction with DNA from untransformed P. pastoris. Figure 7. Agarose gel of PCR amplicons across the right border between tandem integrated constructs (primers pUC-GAP Fw and Int TP Rv). The DNA ladder is “GeneRuler 1Kb” from ThermoFisher scientific. +PARS, “Wt” and –PARS are control reactions with pGAPzαB-PARSintTP, DNA from untransformed P. pastoris and pGAPzαB-intTP respectivly.
Figure 8. Start and stop sites of replication of linear pGAPzαB-PARS-intTP as defined by the
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ACCEPTED MANUSCRIPT location of the PARS sequence and the restriction site used for linearization (here AvrII).
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Table 1. PCR primers used for cloning and control PCR reactions.
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Primer sequence
PARS1 Fus Fw
5'GCATGAGATCAGATCCAATTAATATTTACTTATTTTGGT3'
PARS1 Fus Rv
5'TCTACAAAAAAGATCGATAAGCTGGGGGAACATT3'
Int TP Fw
5'ATTGAACAACTATTTAAAAATGGCATCTTTAGTCAAAAAGG3'
TP His XbaI Rv
5'GATGAGTTTTTGTTCTAGTTAATGATGATGATGATGATGGTCGAC
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Primer name
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GAATTCTGG3'
5’GCAGAGCGAGGTATGTAGGC3’
pUC-GAP Rv2
5’CCGTCAACGGTCTTTTGAGT3’
GAPflank Fw
5’CGATCAATGAAATCCATCAAGA3’
TPint Rv
5’CGGTACCTTCCATCGTCATT3’
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pUC-GAP Fw
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Table 1
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Highlights
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Linear plasmids with the PARS sequence transform P. pastoris with high efficiency. The linear plasmids integrate in the P. pastoris genome. They are not episomal. A novel mechanism for genomic integration is suggested. Constructs which are inherently more efficient for transformation can be designed.
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