Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications
© The Author(s) 2016
Received: 30 March 2016
Accepted: 3 August 2016
Published: 11 August 2016
Recombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome. Recently, heterologous and endogenous autonomously replicating sequences (ARS) were identified in P. pastoris by genome mining, opening the possibility of expanding the available toolbox to include efficient episomal plasmids. The aim of this technical report is to validate a 452-bp sequence (“panARS”) in context of P. pastoris expression vectors, and to compare their performance to classical integrative plasmids. Moreover, we aimed to test if such episomal vectors would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells.
A panARS-based episomal vector was evaluated using blue fluorescent protein (BFP) as a reporter gene. Normalized fluorescence from colonies carrying panARS-BFP outperformed the level of signal obtained from integrative controls by several-fold, whereas endogenous sequences, identified from the P. pastoris genome, were not as efficient in terms of protein production. At the single cell level, panARS-BFP clones showed lower interclonal variability but higher intraclonal variation compared to their integrative counterparts, supporting the idea that heterologous protein production could benefit from episomal plasmids. Finally, efficiency of 2-fragment and 3-fragment in vivo recombination was tested using varying lengths of overlapping regions and molar ratios between fragments. Upon optimization, minimal background was obtained for in vivo assembled vectors, suggesting this could be a quick and efficient method to generate of episomal plasmids of interest.
An expression vector based on the panARS sequence was shown to outperform its integrative counterparts in terms of protein productivity and interclonal variability, facilitating recombinant protein expression and screening. Using optimized fragment lengths and ratios, it was possible to perform reliable in vivo recombination of fragments in P. pastoris. Taken together, these results support the applicability of panARS episomal vectors for synthetic biology approaches.
The methylotrophic yeast Pichia pastoris is widely considered an industrial workhorse for recombinant protein production (RPP, [1–3]); insights into P. pastoris genomic arrangement [4, 5] and metabolism [6, 7] are starting to accumulate in the literature, as a testament to the interest in this host. Despite an increasing repertoire of molecular tools to enable efficient protein production, including newly identified natural promoters , engineered sequences [9, 10], and secretion signals , the vast majority of vectors available for RPP are based on genomic integration of expression cassettes in the P. pastoris genome. Although in general stable clones derived from genomic integration are preferred for RPP, disadvantages of their use are related to relatively low efficiency of transformation (recently at least mitigated by technical developments ), to a certain degree of heterogeneity in protein production due to non-specific integration and to genetic instability of multi-copy integration in presence of stress conditions . The classical solution to this problem—episomal expression vectors—is deemed to alleviate only the chromosomal instability of multi-copy integrative clones, since other reasons of heterogeneity, recently addressed analysing micro engraved P. pastoris secretive clones, are hypothesized to be related to stochastic post-translational events, especially relevant in secreted protein expression [14, 15]. Nonetheless, episomal expression systems present advantages such as simpler protocols and higher efficiencies of transformation; however, such tools are unavailable in most non-canonical protein production hosts, often due to lack of efficient replication origins that promote in vivo plasmid replication and maintenance. Recent high-throughput work has identified novel autonomously replicating sequences (ARSs) in different organisms, to bring to light novel regions conferring self-replicating properties and understand their features [16–18]. These elements may help expedite RPP efforts through addition of stable expression plasmids to the available molecular toolkit; moreover, another intriguing possibility is represented by the possibility of self-assembly recombinant DNA fragments in its nucleus, eliminating the cloning process for plasmid assembly ; such strategy is theoretically facilitated by self-replication of the assembled fragment in the recipient cells, and has been successfully applied to Saccharomyces cerevisiae [20, 21]. In vivo recombination in P. pastoris was first observed when a library of Rhizopus chinensis lipase mutants was assembled directly by the host and generated a linear expression cassette integrated at the targeted genomic locus. Overlapping ends as short as 50 nucleotides were reported to be sufficient to promote assembly at a relatively high efficiency .
In this technical report, we aimed to functionally characterize the use of panARS, a 452-nt element originally isolated from Kluyveromyces lactis and synthetically optimized, as well as two endogenous sequences derived from P. pastoris, using blue fluorescent protein (BFP) as a reporter protein. Moreover, we evaluated in vivo recombination of a panARS-based vector, to establish its technical feasibility for efficient gene assembly in P. pastoris.
Results and discussion
Evaluation of ARSs in Pichia pastoris GS115
Recently, endogenous autonomously replicating sequences from P. pastoris have been identified and described . In order to evaluate the general performance of two sequences in comparison with a wide-range ARS (panARS, ), sequences A76 and C937, previously described to possess respectively strong and moderately weak self-replicating activity, were tested in the same genetic context as panARS. To do so, blue fluorescent protein (BFP)-expressing plasmids, containing AOX1 promoter, zeocin resistance cassette and each of the three different ARS sequences, were constructed starting from the commercial vector pSEC-SUMO (see ‘‘Methods’’ section). BFP was selected as reporter gene, due to its fast maturation, high photostability and pH-stability [24–26].
Copy number determination for integrated or episomal BFP-expressing vectors
Copy number Zeo Set 1
Copy number BFP Set 1
Evaluation of potential positional effect of panARS
Specific fluorescence evaluation at the single cell level
In vivo recombination: proof-of-principle
In vivo recombination: cloning simulation
Summary of results for in vivo recombination (3-fragments assembly)
Overlapping ends (nts)
Coefficient of variation
Fluorescent colonies (%)b
1 + 2 + 3
1 + 2 + 3
1 + 2 + 3
1 + 2 + 3
1 + 2 + 3
1 + 2 + 3
In this technical report, a 452-nt sequence (panARS) was tested for its capacity to confer stable replicative maintenance to a commercial plasmid used for recombinant protein production in P. pastoris. Plasmids carrying the panARS sequence complement the existing toolbox for RPP in P. pastoris. It is hereby showed that episomal panARS plasmids outperformed the corresponding integrative plasmids in terms of protein production, efficiency of transformation, and macroscopic clonal homogeneity. Endogenous P. pastoris sequences were not nearly as efficient in BFP expression compared to panARS-based constructs. At the single cell level, discrete subpopulations of high- and low-producers were consistently detected in clones carrying episomal panARS vectors. In addition, panARS episomal plasmids could be successfully used for quick in vivo self-ligation cloning directly using fragments with overlapping DNA sequences. This method was validated for gene assembly with panARS-based vectors of varying overlapping lengths and molar ratios between fragments: the resulting minimal amount of background, and reported high reproducibility of clone behaviour will allow the application of panARS plasmids to in vivo recombination for synthetic biology applications, where complex, multigene cassettes might be more easily assembled in P. pastoris. Taken together, this report characterizes for the first time the use of panARS episomal plasmids to improve the P. pastoris molecular toolbox for synthetic biology.
Strains, plasmids, and materials
Pichia pastoris strain GS115 (Life Technologies, USA) and E. coli Top10F (Life Technologies, USA) were used in this study. Plasmid pSEC-SUMO (Lifesensors, USA) was used as a backbone vector to generate pARS vector for P. pastoris. Vector Gateway® TagBFP-AS-C (Evrogen, Russia) was used as the source of a Blue Fluorescent Protein (BFP) gene codon-optimized for S. cerevisiae. All primers and gBlocks, available as supplementary information, were synthesized by IDT (Singapore). All restriction enzymes, as well as Gibson cloning kits, were purchased from NEB (Singapore). DNA amplification was performed using Q5 DNA polymerase from NEB (Singapore), while colony PCR for screening was performed with Dream Taq polymerase (Promega, USA) following standard molecular biology protocols . DNA sequencing was performed by Axil Scientific Support (Singapore). All chemicals were purchased from Sigma (USA).
Cloning and transformation
Primers used in this study
qPCR-PpACT1-Fw (ACT1, Set 1)
qPCR-PpACT1-Rv (ACT1, Set 1)
qPCR-PpACT1_2_Fw (ACT1, Set 2)
qPCR-PpACT1_2_Rv (ACT1, Set 2)
qPCR_pEM7_1_Fv (Zeo, Set 1)
qPCR_ZEO_1_Rv (Zeo, Set 1)
qPCR_pEM7_2_Fv (Zeo, Set 2)
qPCR_ZEO_2_Rv (Zeo, Set 2)
qPCR_BFP_1_Fw (BFP, Set 1)
qPCR_BFP_1_Rv (BFP, Set 1)
qPCR_BFP_2_Fw (BFP, Set 2)
qPCR_BFP_2_Rv (BFP, Set 2)
Deep well plate cultivation and fluorescence measurement
Pichia pastoris cultures were grown in 96 Well Masterblock, 2 mL, V-bottom plates (Greiner, Germany), sealed with BREATHseal™ gas-permeable sealer (Greiner, Germany) in a variant of BMD1 media , where 1 % d-glucose was substituted with 1 % sorbitol (BMS media), and using 100 mg/L of zeocin as selection marker. A final concentration of 1 % methanol was added to the media to induce protein production. All cultivations were performed at least in duplicate. After 48 h cell suspensions were diluted 1:10 in PBS and evaluated for OD600 and fluorescence emission in a Tecan Infinite® 200 PRO series (Tecan, Austria), using Microplate PS, 96 Well, F-Bottom clear plates (Greiner, Germany) or Microplate PS, 96 Well, F-Bottom Black plates (Greiner, Germany), for OD600 or fluorescence respectively; the latter was determined using 402 nm and 452 nm as excitation and emission wavelength, respectively, and normalizing all values against OD600. Every reading of both OD600 and fluorescence was performed in triplicate (technical replicates).
Genomic DNA extraction
20 OD of yeast cultures were pelleted and washed in 500 µL distilled H2O. Pellet was re-suspended in breaking buffer (2 % (v/v) Triton X-100, 1 % (v/v) SDS, 100 mM NaCl, 10 mM Tris–Cl pH 8.0, 1 mM EDTA pH 8.0). 200 µL of glass beads and 200 µL of phenol/chloroform/isoamylalcohol were added. Samples were vortexed at highest speed for 5 min, supplemented with 200 µL of TE Buffer (10 mM Tris–Cl, 0.1 mM EDTA pH 8.0), vortexed briefly, and centrifuged at 13,000g for 5 min at room temperature. Samples were extracted two more times with phenol/chloroform/isoamylalcohol and one final time with chloroform. 1 mL of 100 % freeze-cold ethanol was added to the aqueous phase, mixing by inversion, and samples were incubated at −20 °C for 1 h. Samples were mixed several times by inversion, centrifuged at 13,000g for 3 min at 4 °C and supernatant aspirated. Pellet was air-dried; 400 µL of TE were then added to pellets and incubated at 65 °C for 10 min, followed by addition of 30 µL of RNase A and incubation for 1 h at 37 °C. 10 µL of 4 M ammonium acetate was added, samples were mixed by inversion, and quickly spun. 1 mL of 100 % ethanol (−20 °C) was added and mixed by inversion, followed by 13,000g centrifugation (3 min) at 4 °C. Pellet was air-dried, resuspended in 50 µL of TE buffer and incubated in 65 °C water bath for 10 min.
Picogreen DNA quantification
Lambda DNA (Life Technologies, USA) was serially diluted to produce DNA standards at the following concentrations (ng/µL): DNA Standards Set #1—50, 33.33, 16.66, 8.3, 4.16, 2.08, 1.042, 0.5208, 0; DNA Standards Set #2—75, 50, 25, 12.5, 6.25, 3.125, 1.5262, 0. Quant-iT™ PicoGreen® dsDNA reagent (Life Technologies, USA), diluted 200× in 1x Tris–EDTA (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0) was transferred to wells of a white 96-well plate (Greiner Bio-One, Germany), 195 μL per well (“DNA sample plate”).
Based on previous spectrophotometer readings (NanoDrop 2000c), genomic DNA samples were diluted 5x, 10x, 20x, or 40x, to fall within the range of the DNA standards made. 2 µL of each lambda DNA standard and diluted genomic DNA sample was transferred to wells of the DNA sample plate. Sample plate was scanned on a SpectraMax M5 microplate reader (Molecular Devices, USA) using the preconfigured Quant-It Picogreen protocol in the accompanying SoftMax Pro software, measuring fluorescence signal at 525 nm (excitation wavelength: 490 nm). Sample concentrations were calculated from RFU readings using the linear regression equation derived from the DNA standards. All samples were evaluated in triplicate.
Digital droplet PCR (ddPCR) copy number determination
Purified genomic DNA was digested with KpnI (NEB, USA) for 1 h at 37 °C. No heat inactivation was performed, following ddPCR manufacturer indications. An Evagreen ddPCR mastermix was assembled, mixing 10 µL of Eva Green 2x master solution (Biorad, Singapore), 2 µL of primer set (100 nM each, Table 3), 1 µL of genomic DNA sample (0.5 ng/µl, as reported in ) and mQ H2O up to 20 µL. Primers were designed following published indications , although different sets of primers were tested for optimal signal. Following droplet generation with a Droplet generator (Biorad, Singapore), samples were transferred to a 96-well plate and sealed. PCR was performed adjusting the ramp rate on a C1000 Touch Deep Well PCR system (Biorad, Singapore) to 2 °C/s, applying the following cycle: 95 °C, 5 min; 40x (95 °C, 30 s; annealing/extension 57–60 °C); 4 °C, 5 min; 90 °C, 5 min; 4 °C, infinite hold). Droplet detection was carried out in a QX200 Droplet Digital PCR system (Biorad, Singapore) and analysed using the software QuantaSoft v. 1.7.4.0917 (Biorad), following an absolute quantification protocol.
Copy number determination of in vivo recombined fragments
qPCR was conducted to accurately evaluate the copy number of individual fragments upon in vivo recombination. All qPCR reactions were performed in triplicates for each transformed clone and each diluted standard, using FastStart Essential DNA Green Master (Roche, USA) and LightCycler® 96 Instrument (Roche, USA). A 5 ng DNA sample of each transformed clone was used as template for qPCR reaction. Two primer sets, namely Zeo, Set 1 and BFP, Set 2, were designed to determine the copy number of the two transformed fragments. To determine PCR efficiency of the two primer sets, a 5-log dilution series of PARS-BFP plasmid, started from 10 ng, was used. Each diluted standard was mixed with 5 ng of untransformed parental genomic DNA, to compensate for any non-specific amplification arising from genomic DNA contamination in the plasmid preparation. During the qPCR reaction, the enzyme was activated at 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s, 57 °C for 10 s and 72 °C for 10 s.
Standard curve of each primer set amplified the 5-log diluted plasmid was plotted, and primer amplification efficiency was determined. The adjusted copy number ratio of the two transformed fragments in each transformed clone was calculated using formula: E Zeo Ct ZEO /E BFP Ct BFP , where EZeo and EBFP are primer efficiencies of Zeo and BFP primer set, respectively, while CtZEO and CtBFP are Ct values of a transformed clone amplified using ZEO and BPF primer set, respectively.
FACS analysis and hierarchial clustering
Samples were normalized to OD600 = 0.1 using a Tecan EVO 150 liquid-handling robot. After washing with PBS twice, samples were analysed in 96-well batches on a MACSQuant VYB instrument (Miltenyi), acquiring ~80,000 cells per clone. The Blue Florescence Protein signal profile was gated into 10 equally spaced areas (P1–P10) using FlowJo_vX.0.7 software. The blue fluorescence intensity for each gate was visualized in heat maps built using customized R scripts. Hierarchial clustering was generated based on the relative frequencies of fluorescence events in every gate, using MATLAB R2014a (version 22.214.171.1242, Mathworks, Natick, MA). An agglomerative ‘bottom-up’ clustering algorithm  was used where the fluorescence of each clone initiated as its own cluster, which then merged with clones possessing similar profiles.
Data analysis was performed with GraphPad Prism 6 (GraphPad Software, USA).
AC conceived the outline of the work, and performed all cloning, transformation, and cultivation tasks in collaboration with AG. AG and SWN performed copy number determination with ddPCR and qPCR, under the supervision of AHMT. IL and MJD provided support for sequence selection; GR, AT and GL performed DNA extraction, quantification and flow cytometry experiments (including analysis and data processing). LYY processed and evaluated the clustering data. All authors read and approved the final manuscript.
IL and MJD declare a possible competing interest (co-inventors in “Pan-yeast autonomously replicating sequence”, WO 2014131056 A1). AC, AG, LYY, AHMT, SWN, AT, GL and GR declare that they no competing interests.
Availability of data and materials
The datasets during and/or analysed during the current study are available from the corresponding author on reasonable request.
Consent for publication
This manuscript does not contain data from any individual person.
Ethics approval and consent to participate
This manuscript does not report on or involve the use of any animal or human data or tissue.
This study was supported by the Biomedical Research Council of the Singapore Agency for Science, Technology and Research. GR lab is supported by A-STAR Investigatorship award 1437a00119.
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