ORIGINAL RESEARCH published: 27 June 2019 doi: 10.3389/fmicb.2019.01472 Biotechnological Production of the Cell Penetrating Antifungal PAF102 Peptide in Pichia pastoris Crina Popa 1† , Xiaoqing Shi 1† , Tarik Ruiz 1 , Pau Ferrer 2 and María Coca 1* 1 Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Barcelona, Spain, 2 Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de Barcelona, Barcelona, Spain Antimicrobial peptides (AMPs) have potent and durable antimicrobial activity to a wide range of fungi and bacteria. The growing problem of drug-resistant pathogenic microorganisms, together with the lack of new effective compounds, has stimulated interest in developing AMPs as anti-infective molecules. PAF102 is an AMP that was rationally designed for improved antifungal properties. This cell penetrating peptide Edited by: has potent and specific activity against major fungal pathogens. Cecropin A is a László Galgóczy, natural AMP with strong and fast lytic activity against bacterial and fungal pathogens, Hungarian Academy of Sciences, Hungary including multidrug resistant pathogens. Both peptides, PAF102 and Cecropin A, are Reviewed by: alternative antibiotic compounds. However, their exploitation requires fast, cost-efficient Thalles Barbosa Grangeiro, production systems. Here, we developed an innovative system to produce AMPs in Universidade Federal do Ceará, Brazil Pablo E. Hernández, Pichia pastoris using the oleosin fusion technology. Oleosins are plant-specific proteins Complutense University of Madrid, with a structural role in lipid droplet formation and stabilization, which are used as Spain carriers for recombinant proteins to lipid droplets in plant-based production systems. *Correspondence: This study reports the efficient production of PAF102 in P. pastoris when fused to the María Coca maria.coca@cragenomica.es rice plant Oleosin 18, whereas no accumulation of Cecropin A was detected. The Ole18- † These authors have contributed PAF102 fusion protein targets the lipid droplets of the heterologous system where it equally to this work accumulates to high levels. Interestingly, the production of this fusion protein induces the formation of lipid droplets in yeast cells, which can be additionally enhanced by Specialty section: This article was submitted to the coexpression of a diacylglycerol transferase gene that allows a three-fold increase Fungi and Their Interactions, in the production of the fusion protein. Using this high producer strain, PAF102 a section of the journal Frontiers in Microbiology reaches commercially relevant yields of up to 180 mg/l of yeast culture. Moreover, the Received: 29 April 2019 accumulation of PAF102 in the yeast lipid droplets facilitates its downstream extraction Accepted: 13 June 2019 and recovery by flotation on density gradients, with the recovered PAF102 being Published: 27 June 2019 biologically active against pathogenic fungi. Our results demonstrate that plant oleosin Citation: fusion technology can be transferred to the well-established P. pastoris cell factory Popa C, Shi X, Ruiz T, Ferrer P and Coca M (2019) Biotechnological to produce the PAF102 antifungal peptide, and potentially other AMPs, for multiple Production of the Cell Penetrating applications in crop protection, food preservation and animal and human therapies. Antifungal PAF102 Peptide in Pichia pastoris. Front. Microbiol. 10:1472. Keywords: antimicrobial peptides, antifungal peptides, Pichia pastoris, plant oleosin, cecropin A, PAF peptides, doi: 10.3389/fmicb.2019.01472 lipid droplets Frontiers in Microbiology | www.frontiersin.org 1 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts INTRODUCTION for short peptides and high value applications. Their peptidic nature enables production through biotechnological systems, Antimicrobial peptides (AMPs) are natural compounds with but, depending on the nature of the AMPs, their production in antimicrobial activity toward a wide range of fungi and conventional microbial systems is frequently not very efficient bacteria. They are a diverse group of peptides with no due to toxicity toward host cells or proteolysis of the products consensus sequence associated to their biological activity but (Valore and Ganz, 1997; Bryksa et al., 2006; Jin et al., 2006; sharing common features: most of them are cationic, relatively Wang et al., 2018). Recently, our group has demonstrated that hydrophobic and amphipathic molecules (Zasloff, 2002). Being rice seeds are good biofactories for linear AMPs that, otherwise, amphipathic facilitates their selective interaction with negatively are difficult to produce in biological systems (Bundó et al., 2014, charged microbial membranes, so are capable of functioning 2019; Montesinos et al., 2016, 2017). We found that, interestingly, as membrane-disruption peptides or cell-penetrating peptides PAF102 and CecA could be efficiently produced in rice seeds as interfering with key intracellular processes (Zasloff, 2002; oleosin fusion proteins targeted to oil bodies (OBs) (Montesinos Nicolas, 2009). In general, AMPs seem to act on low-affinity et al., 2016; Bundó et al., 2019). OBs, also known as lipid droplets targets, so with little propensity to develop resistance in microbial (LDs), are specialized structures composed mainly of a core of targets (Zasloff, 2002; Hancock and Sahl, 2006; Peschel and neutral lipids (triacylglycerols and steryl esters) surrounded by Sahl, 2006; Sierra et al., 2017; Yu et al., 2018; Ghosh et al., a monolayer of phospholipids containing a number of proteins 2019). The growing problem of resistance to conventional that differ considerably between species (Chapman et al., 2012). antibiotics together with the lack of new effective molecules Oleosins are plant specific proteins with a structural role in has stimulated interest in developing AMPs as an alternative OB formation and stabilization (Tzen, 2012). They have been (Hancock and Sahl, 2006; Zhang and Gallo, 2016; Ghosh et al., used as a carrier of recombinant proteins to OBs, facilitating 2019). In addition, AMPs have different mechanisms of action their subsequent purification from plant material (van Rooijen to other antimicrobials, reinforcing their enormous potential as and Moloney, 1995). Consequently, plant OBs have emerged a complement to conventional antibiotics. They have multiple as a target for biotechnological production of recombinant applications in medical therapies, food preservation and crop proteins (Parmenter et al., 1995; Nykiforuk et al., 2006, 2011; protection (Marcos et al., 2008; López-García et al., 2012; Boothe et al., 2010). Montesinos et al., 2012; Kang et al., 2017). The purpose of the present study was to investigate whether Although research on AMP development has been highly oleosin fusion technology could be transferred to the well- active recently, few molecules have entered the market. Major established production system based on Pichia pastoris to challenges to be solved before natural AMPs can be fully exploited efficiently produce AMPs. P. pastoris has been developed as relate to their target specificity and stability, as well as production an excellent host for the production of biopharmaceuticals costs. The advances in peptide synthesis have enabled the de and industrial enzymes. The growth of yeast is fast, with very novo synthesis and rational design of AMPs with improved high cell densities producing grams of recombinant protein properties (Marcos et al., 2008), with the hexapeptide PAF26 per liter of culture (Ahmad et al., 2014). Here, PAF102 was (RKKWFW) identified through a combinatorial approach as a efficiently produced in P. pastoris when fused to the rice plant novel peptide with specific antifungal activity and cell penetrating Oleosin 18, whereas no accumulation of CecA was detected. properties (López-García et al., 2002), and PAF102 rationally The plant oleosin was correctly targeted to the LDs in the designed as a modified concatemer of PAF26 of 29 amino acid heterologous system and carries the PAF102 peptide, leading residues with high potency and specificity toward fungal cells, to high accumulation of the fusion protein in these vesicles. low toxicity to mammalian cells, high stability to proteolysis Moreover, the recombinant LDs were easily isolated by flotation and for optimal production in plants (López-García et al., on density gradients for PAF102 purification. We also show that 2015). Other strategies are based on the modification of natural the recovered PAF102 was biologically active against pathogenic AMPs, such as the cecropin::melittin hybrids that are designed fungi. Our results demonstrate that P. pastoris allows fast and to decrease the toxicity of the high hemolytic melittin peptide efficient production of AMPs through oleosin fusion technology. (Cavallarin et al., 1998; Badosa et al., 2007, 2013). However, some naturally occurring peptides, such as the 37 residues peptide cecropin A (CecA) isolated from insects, have potent lytic activity MATERIALS AND METHODS against major bacterial and fungal pathogens without additional modifications (Steiner et al., 1981; Cavallarin et al., 1998; Moreno Plasmid Constructs, Yeast Strains and et al., 2003). More importantly, CecA has no lytic activity against Growth Conditions mammalian erythrocytes or lymphocytes (Steiner et al., 1981; A construct was prepared to produce the fusion protein of Suttmann et al., 2008). These properties make this natural peptide Oleosin 18 to the Green Fluorescent Protein (Ole18-GFP) in a good candidate to be developed as an antimicrobial in multiple P. pastoris (Figure 1). The open reading frame of the rice Ole18 fields of applications. (LOC_Os03g49190) without the stop codon was amplified by The other major limitation for most AMPs is their production PCR from the plant vector pC::pOle18:Ole18-CecA (Montesinos costs. The amount of AMPs produced in living organisms is very et al., 2016) using the primers indicated in Supplementary low, and their extraction and purification requires complex and Table 1 (EcoRI_Ole18fwd and Ole18rev) and the Phusion high- costly procedures. Chemical synthesis is only economically viable fidelity DNA polymerase (Thermo Scientific, Spain). The GFP Frontiers in Microbiology | www.frontiersin.org 2 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts FIGURE 1 | Ole18-GFP is targeted to P. pastoris LDs (A) Gene construct used to produce the Ole18-GFP fusion protein in P. pastoris cells. Expression of the fusion gene encoding the rice Ole18 protein tagged with the Green Fluorescent Protein (Ole18-GFP) was under the control of the constitutive promoter GAP1 (promGAP1) and the AOX1 terminator (tAOX1). Arrows indicate the position of primers used for preparing the construct (B) Confocal laser microscopy images of cells accumulating the Ole18-GFP protein visualized under fluorescence for GFP, Nile red, and the merged GFP and Nile red image. The corresponding bright field image (BF) is also shown. Scale bar = 2 µm. open reading frame was amplified from the pYGWY yeast vector Dgat1_rev (Supplementary Table 1). The amplified fragment containing the GFP tag (Popa et al., 2016) using the GFPfwd was inserted into the pGAPZA plasmid (Invitrogen, Thermo and XbaI_GFPrev primers (Supplementary Table 1). Amplified Scientific, Spain) using the Gibson assembly reaction. This DNA fragments were assembled by the Gibson reaction using plasmid allows gene expression under the control of the GAP the NEbuilder HiFi DNA assembly master mix (New England promoter and confers ZeocinTM resistance. All the constructs Biolabs, United States). The assembled Ole18-GFP fragment was were verified by DNA sequencing. then digested with the EcoRI and XbaI restriction enzymes, Gene constructs were transferred to the wild-type P. pastoris and cloned into these restriction sites of the integrative yeast strain X-33 by electroporation. Recombinant colonies were vector pGAPHA for expression, driven by the Glyceraldehyde- isolated using the appropriate antibiotic selection at 100 µg/ml 3-Phosphate Dehydrogenase (GAP) constitutive promoter. This of ZeocinTM or 300 µg/ml of hygromycyn B, and then confirmed plasmid derives from pGAPZA (Invitrogen, Thermo Scientific, by PCR. Two to three independent colonies were analyzed by Spain), in which the ZeocinTM resistance marker was replaced by transformation event with similar results in the different assays. the hygromycin resistance one (Adelantado, 2016). Yeast cells were grown in YPD medium (1% yeast extract, 2% Two more constructs were prepared to produce the Ole18- peptone, and 2% glucose) at 30◦ C. Cell growth was monitored by CecA and Ole18-PAF102 fusion proteins in P. pastoris (Figure 2). measuring the optical density at 600 nm (OD600 ) of the culture The Ole18-CecA open reading frame was amplified by PCR at different time points, or by plating serial 1/10 dilutions of an from the plant vector pC::pOle18:Ole18-CecA (Montesinos et al., exponential growing culture on YPD agar plates. 2016) using the EcoRI_Ole18fwd and XhoI_CecArev primers, shown in Supplementary Table 1. The Ole18-PAF102 was Lipid Droplets Fractionation amplified from the pC::pOle18:Ole18-PAF102 (Bundó et al., Lipid droplets (LDs), along with their bound proteins, were 2019) using the same forward primer and the XhoI_PAF102rev isolated by density gradient centrifugation provided by Ficoll as the reverse primer (Supplementary Table 1). The fusion (Sigma-Aldrich, Spain), as previously described, with some genes were introduced into the pGAPHA plasmid as EcoRI- modifications (Mannik et al., 2014). Basically, the method was XhoI fragments behind the GAP promoter. As a control, we also adapted to lyse the cells by sonication on ice. Cells grown in prepared a construct for the single Ole18 protein (Figure 2). YPD medium to late logarithmic phase were resuspended in For that, the Ole18 open reading frame was amplified with Ficoll buffer I (12% Ficoll, 10 mM Tris–Hcl, 200 µM EDTA) EcoRI_Ole18fwd and XhoI_Ole18rev primers (Supplementary at a concentration of 2 ml/g of cells. Cells were then lysed Table 1), and transferred to the pGAPHA plasmid as an EcoRI- through seven consecutive cycles of sonication of 20 s each. XhoI fragment. After clarifying the lysate from the cell debris by low speed An additional construct was prepared for the production of centrifugation, the supernatant was overlaid with the same Diacylglycerol O-AcylTransferase 1 (Dgat1) from Arabidopsis volume of Ficoll buffer I and centrifuged at high speed (100,000 g) thaliana (At2g19450) in P. pastoris. In this case the open for 1 h in a SW40 rotor. LDs were recovered in the floating white reading frame was amplified from the G16373 clone provided layer, and then, were overlaid with a volume of Ficoll buffer II (8% by the Arabidopsis Biological Resource Center (ABRC, Ohio Ficoll, 10 mM Tris–HCl, 200 µM EDTA). After a second round State University, United States) using the primers Dgat1_fwd and of flotation-centrifugation under the same conditions, LDs were Frontiers in Microbiology | www.frontiersin.org 3 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts obtained at high purity in the floating fat pad. The purity and integrity of recovered LDs were confirmed by selective staining with Nile red (1 ng/ml, Sigma-Aldrich, Spain) and fluorescence microscopy visualization. Immunoblot Analysis of LD-Associated Proteins and PAF102 Purification LD-associated proteins were solubilized in loading buffer, separated by SDS-PAGE (15% acrylamide, Laemmli, 1970), transferred to nitrocellulose membranes and immunodetected with different antibodies: anti-Ole18 (1:2000 dilution, Montesinos et al., 2016), anti-CecA (1:500 dilution, Coca et al., 2006), anti-PAF102 (1:1000 dilution, Bundó et al., 2019), and anti-Erg6 (1:500 dilution, Paltauf et al., 1994). PAF102 accumulation was estimated on immunoblot by quantification of signal intensities of Ole18-PAF102 to known amounts of synthetic PAF102, using the Quantity Tools Image LabTM Software (Version 5.2.1) included in the ChemiDocTM Touch Imaging System (Bio-Rad, United States). OBs containing Ole18-CecA or Ole18-PAF102 proteins were isolated from pOle18:Ole18-CecA and pOle18:Ole18-PAF102 rice seeds as previously described (Montesinos et al., 2016; Bundó et al., 2019). OB fractions obtained from ten seeds and resuspended in 100 µl buffer (0.2 M sucrose, 10 mM phosphate buffer pH 7.6) were separated into four aliquots (25 µl each). Protease inhibitor cocktail (Sigma-Aldrich, Spain) was added to two of these, and then 25 µg of total protein extracts from the empty vector P. pastoris strain was added to all them. One with and one without the inhibitor were immediately frozen and the other two incubated at room temperature overnight. Proteins were then analyzed by immunoblot using anti-Ole18 specific antibodies. PAF102 was recovered from the LDs containing the Ole18- PAF102 fusion protein by digestion with Tobacco Etch Virus (TEV) protease (Invitrogen, Thermo Fisher Scientific, Spain, 1:100), as the Ole18 protein was linked to the PAF102 peptide through a TEV protease recognition site (ENLYFQ/G). Proteolytic digestion was overnight at 30◦ C in TEV protease buffer. The estimated efficiency of the TEV protease was based on the disappearance of the Ole18-PAF102 signal by immunoblot analysis. The released PAF102 was immunodetected using anti- PAF102 antibodies by western-blot, after acetone precipitation, separation in Tricine-SDS-PAGE (18% acrylamide, Schägger and FIGURE 2 | Ole18-PAF102 accumulates in P. pastoris LDs. (A) Gene von Jagow, 1987) and transfer to nitrocellulose membranes constructs used to produce the rice Ole18 and the Ole18-CecA and Ole18-PAF102 fusion proteins in P. pastoris cells. Ole18 protein and the CecA (Whatman Potran 0.2 µm). R and PAF102 antimicrobial peptides are linked through the TEV protease recognition site (PRS). Arrows indicate the position of primers used for preparing the constructs. (B) Amino acid sequence of the Ole18-CecA and Confocal and Fluorescence Microscopy Ole-PAF102 fusion proteins with Ole18 protein (red) linked through PRS (gray) Cells of P. pastoris carrying the different gene constructs to CecA or PAF102 peptides (black). Arrows indicate TEV protease cleavage were visualized under confocal fluorescent microscopy with a site. (C) Immunoblot analysis of equivalent total protein extracts (lysates) and LDs from P. pastoris cell cultures transformed with empty vector (EV) or the Leica TCS SP5 microscope. For that, cells grown overnight in indicated gene constructs, using anti-PAF102, anti-Ole18 or anti-Erg6 liquid medium to late logarithmic phase (1 ml of cultures) antibodies as indicated. Ponceau staining of the membrane is shown as were concentrated by centrifugation for 2 min at 600 g, protein loading control (25 µg of total protein). Red arrows indicate Ole18 washed in water, resuspended in phosphate-buffered saline (PBS) protein bands and black the Ole18-PAF102 fusion protein bands. Molecular weight markers are indicated in kDa on the left. containing 1 ng/ml of Nile red and directly visualized under a coverslip. GFP signal was detected at 500–550 nm through Frontiers in Microbiology | www.frontiersin.org 4 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts excitation with an Argon ion laser emitting a 488 nm and Nile cells. Hence, the rice Ole18 protein was efficiently produced red signal at 570–670 nm using DPSS laser emitting at 561 nm. and correctly targeted to LDs in this heterologous system, as Cell viability was evaluated by fluorescein diacetate (FDA, previously reported for other plant oleosin proteins produced in Sigma-Aldrich, Spain) staining (Jones and Senft, 1985). Cell Saccharomyces cerevisiae yeast cells (Ting et al., 1997; Beaudoin cultures grown at logarithmic phase (OD600 = 1) were et al., 2000; Parthibane et al., 2012; Jacquier et al., 2013; Vindigni concentrated, washed and resuspended in PBS containing FDA et al., 2013). Our results also suggest that Ole18 can be used as a (10 µg/ml) and stained for 20 min. After washing, the cells were carrier protein to LDs in Pichia cells. visualized under fluorescent microscopy using an Axiophot Zeiss upright wide field fluorescence microscope. The size of the intracellular LDs was estimated by image Ole18-PAF102 Accumulates in P. pastoris analysis using ImageJ software. The Z-projection fluorescent LDs images were converted to binary images by applying a threshold To investigate whether the oleosin fusion technology can be that highlights all the structures to be measured. The area of LDs used to produce AMPs in P. pastoris, we prepared two new was determined using the particle analysis tool and taking into gene constructs (Figure 2A). They were designed to produce the account only those droplets with spherical geometry. The 100 CecA and PAF102 antimicrobial peptides as fusion to the rice LDs measured per line were clustered in different size ranges and Ole18 protein (Figure 2B). Proteins were linked through a TEV the percentage of LDs in each group was calculated. To determine protease recognition site for the subsequent release of the AMPs the amount of LDs in the different cell lines, LDs were counted in from the fusion protein. We also prepared an additional construct three different replicas of the projection images of 30 cells from to produce the single Ole18 protein as a control (Figure 2A). at least three different experiments. Transformed colonies were obtained for each construct, and integration of the transgenes was confirmed by PCR. Total Antifungal Activity Assays protein extracts from the recombinant cultures grown in YPD Growth inhibition assays of Fusarium proliferatum were in 96- media to the late logarithmic phase were analyzed by SDS-PAGE well flat-bottom microtiter plates as previously described, with and immunoblotting (Figure 2C, right panels). Using specific minor changes (López-García et al., 2015). Basically, 50 µl of antibodies to the rice Ole18, we detected a band of the expected fungal conidia (5 × 104 conidia/ml), in 1/10 diluted potato size (around 18 kDa) in the lysates obtained from Ole18 strain dextrose broth (PDB) containing 0.02% (w/v) chloramphenicol, cultures (Figure 2C, red arrows). A higher band was detected in were mixed in each well with 50 µl of LD fractions. The the lysates from Ole18-PAF102 cultures (Figure 2C, black arrows) Ficoll buffer II of LD fractions was exchanged with sterile with an apparent size around 23 kDa, the expected size for the deionized H2 O containing 0.02% Tween-20 using ZebaTM Spin Ole18-PAF102 fusion protein (the theoretical molecular weight is Desalting columns (Thermo Fisher Scientific, Spain). Samples 22.17 kDa). This band was also immunodetected using the anti- were prepared in triplicate. Plates were incubated with agitation PAF102 specific antibodies. Our results indicate that the Ole18- for 72 h at 28◦ C. Fungal growth was monitored every 24 h by PAF102 fusion protein was produced and stably accumulated in measuring the OD600 using a Spectramax M3 reader (Molecular P. pastoris cells. Devices, United States), and mean values and standard deviation In contrast, no immunoreactive band was detected in the (SD) were calculated. Experiments were repeated twice with protein extracts from the Ole18-CecA cultures, using anti-Ole18 similar results. (Figure 2C) or anti-CecA antibodies (data not shown). The theoretical molecular weight of Ole18-CecA fusion proteins is 22.32 kDa. Up to five independent Ole18-CecA transformed RESULTS colonies confirmed for transgene integration were tested, with similar results. This data suggests that the Ole18-CecA Ole18-GFP Fusion Protein Is Targeted to fusion protein did not accumulate at detectable levels in P. pastoris LDs the yeast cells. A gene construct was prepared to study the subcellular We then isolated the LDs from the different recombinant localization of the rice Ole18 protein in P. pastoris (Figure 1A). yeast strains by gradient density centrifugation. LD fractions This construct was designed to express a Ole18-GFP fusion gene were stained with Nile red and visualized under fluorescence and under the control of the constitutive promoter GAP1 in the bright field microscopy. Samples had a high content of intact, integrative yeast vector pGAPHA. Cell colonies carrying the gene high purity LDs. After solubilizing in SDS loading buffer, the construct were obtained by genetic transformation. Their growth LD proteins were separated by SDS-PAGE and immunodetected rate in rich medium was similar to cells transformed with the using specific anti-Ole18 antibodies (Figure 2C, right upper empty vector (EV). Confocal fluorescence microscopy revealed panel). We clearly detected the presence of Ole18 (red arrow) the GFP signal surrounding spherical intracellular vesicles with and Ole18-PAF102 (black arrow) proteins in the LD fractions, but diameters of 0.1–1 µm (Figure 1B). These vesicles co-localized not the Ole18-CecA protein (Figure 2C, right upper panel). The with those labeled with Nile red, a fluorescent stain for neutral isolated fractions were additionally confirmed as LD fractions lipids used as an LD marker (Greenspan et al., 1985). These by immunodetection of the Erg6 protein (Figure 2C, right results indicate that the Ole18-GFP fusion protein is produced lower panel), an enzyme of the ergosterol biosynthetic pathway and accumulates mostly in the periphery of LDs of P. pastoris known to be associated with the LDs in P. pastoris cells Frontiers in Microbiology | www.frontiersin.org 5 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts (Ivashov et al., 2013). These results show that Ole18-PAF102 accumulates in P. pastoris LDs. Ole18-CecA Is Susceptible to P. pastoris Proteases Given that Ole18-CecA was not detected in total or LD protein extracts from the recombinant yeast strain, we hypothesized a potential toxicity problem related to the CecA antimicrobial activity, and this would lead to growth defects. However, when the growth of Ole18-CecA cells was monitored, we found they grew similarly to the strains transformed with the EV, the Ole18, and the Ole18-PAF102 gene constructs (Figure 3A). Additionally, the morphological appearance and viability of Ole18-CecA cells was similar to EV transformed cells when visualized under fluorescence microscopy after fluorescein diacetate viable cell staining (Figure 3B). We found no evidence of toxicity associated to the Ole18-CecA gene in our experiments. Another possible scenario could be that Ole18-CecA does not accumulate due to instability in the yeast cells. To test the stability, we obtained the fusion protein from promOle18:Ole18- CecA rice seeds previously generated in our group (Montesinos et al., 2016). These rice seeds accumulate large amounts of Ole18-CecA protein in OBs (40 µg per gram of rice seeds). Isolated rice OBs containing Ole18-CecA protein were incubated in vitro with P. pastoris protein extracts from EV strain, and then analyzed by immunoblotting (Figure 3C). There was a clear reduction in the amount of Ole18-CecA after incubation with Pichia protein extracts unless protease inhibitors were added. As a control, we also analyzed the Ole18-PAF102 fusion protein produced in rice seeds (Bundó et al., 2019). Interestingly, Ole18- PAF102 remained stable after overnight incubation with the Pichia protein extracts. These results suggest that Ole18-CecA FIGURE 3 | Ole18-CecA is susceptible to P. pastoris proteases. (A) Growth of does not stably accumulate in P. pastoris cells because it is recombinant strains carrying empty vector (EV), Olel8, Olel8-PAF102, susceptible to the yeast proteases, whereas Ole18-PAF102 is more Olel8-CecA. Aliquots of 5 µl of ten serial dilutions of log phase cultures resistant to yeast proteases (Figure 3C) and accumulates to high (OD600 = 1) were spotted on YPD agar media and allowed to grow for 2 days levels in the yeast system (Figure 2C). In contrast, accumulation at 30◦ C. (B) Images of fluorescence and bright field microscopy of EV and of Ole18-CecA is higher than Ole18-PAF102 in rice (Figure 3C), Olel8-CecA cells after fluorescein diacetate (FDA) viability staining. (C) Immunoblot of OB fractions isolated from rice seeds producing probably associated to a higher susceptibility of Ole18-PAF102 Ole18-CecA∗ or Ole18-PAF102∗ fusion proteins, in the presence of total to rice proteases. Therefore, P. pastoris seems to be an efficient protein extracts (25 µg) from EV yeast cells, supplemented without (–) or with system for the production of Ole18-PAF102, and hereinafter we (+) protease inhibitors, incubated (I) or not (N) overnight at room temperature. focus on this antifungal peptide. Ole18-PAF102 Production Induces induction of LD proliferation mediated by other plant oleosins Proliferation of LDs in P. pastoris in the yeast S. cerevisae (Ting et al., 1997; Jacquier et al., 2013; To further characterize the production of Ole18-PAF102 in Jamme et al., 2013; Vindigni et al., 2013). We also observed an the yeast cells, we closely inspected the recombinant cells increase in the size of the recombinant LDs, more pronounced in under confocal microscopy (Figure 4A). We observed that their those of Ole18-PAF102 (Figure 4C). Our results suggest that the morphological appearance was similar to EV and Ole18 cells, antifungal PAF102 peptide fused to Ole18 determines an increase but they contained a higher number of LDs, with the cell-to-cell in LDs in P. pastoris cells. LD number and size highly variable. As the number of LDs was difficult to quantify at the cell level, we used a large population (30 cells), detecting a statistically significant increase in the cells A. thaliana Dgat1 Expression Induces LD accumulating Ole18-PAF102 (Figure 4B). Although there was Formation in Ole18-PAF102 Yeast Cells also a tendency for a higher number of LDs in Ole18 cells, no With the purpose to increase the amount of LDs and presumably significant differences were quantified in comparison to EV cells. the accumulation of Ole18-PAF102 in the yeast cells, we These results differ from reports in the literature showing an expressed the Dgat1 gene from A. thaliana in P. pastoris cells. Frontiers in Microbiology | www.frontiersin.org 6 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts FIGURE 4 | Ole18-PAF102 production induces proliferation of LDs in P. pastoris cells (A) Confocal microscopy images of EV, Ole18, and Ole18-PAF102 recombinant cells at late log growth phase stained with Nile red. Bright field (BF) images, and fluorescence and BF merged images are also shown. Images correspond to single focal sections. Bars = 2 µm. (B) Total number of LDs per 30 cells of the indicated strains. LDs were counted in two replicates per assays and five independent assays (n = 10). Asterisk denotes statistically significant differences (Tukey test, p < 0.005) (C) Distribution of range sizes (area in µm2 ) from 50 LDs from cells of indicated strains. Dgat enzymes catalyze the acyl-CoA-dependent acylation of sn- visualized under confocal microscopy. Nile red staining of 1,2-diacylglycerol to produce triacylglycerol (TAG) (Liu et al., LDs revealed accumulation of cellular LDs mediated by the 2012). This is the rate-limiting activity for TAG biosynthesis and expression of the Dgat1 gene (Figure 5D). We found a statistically a target for engineering increase in oil content in microorganisms significant increase of around three times in the number of LDs (Liu et al., 2012). The expression of the Arabidopsis Dgat1 of double transformant cells (Figure 5E), which not only had in S. cerevisiae has been reported to enhance the content in more LDs, but were also larger in size (Figure 5F). Consistent TAG and LD formation (Bouvier-Navé et al., 2000; Aymé et al., with our previous observations, there was high variability in 2014). We prepared a new construct containing the coding the number and size of LDs in the cell population. Monitoring sequence of the AtDgat1 in the integrative pGAPZA vector, which cell growth through optical density (OD600 ), we also found carries another selection marker to allow double transformant that cells expressing Dgat1 were slower reaching high densities selection (Figure 5A). To easily monitor the LD content, Dgat1 (Figure 5G). This slow growth was not only measured in the transformation was first examined in the Ole18-GFP strain. Ole18-PAF102 strain but also in EV cells, and both EV and Visualization of cells under confocal microscopy revealed an Ole18-PAF102 single transformants had similar growth rates. increase in the content of LDs in the recovered transformant This indicates that the delay may be determined by Dgat1 gene cells (Figure 5B). Although there was a large variability among expression. Altogether, our results show that the LD content cells, most of Ole18-GFP possessed one or two LDs whereas is further enhanced in Ole18-PAF102 strain by expression of the double transformant Ole18-GFP/Dgat1 had three or four the Arabidopsis Dgat1 gene, albeit requiring a longer period LDs per cell. The number of LDs was significantly higher in of culture growth. those cells expressing the Dgat1 gene (Figure 5C), indicating that expression of the Arabidopsis Dgat1 gene induces LD formation in P. pastoris, as reported for S. cerevisiae (Bouvier-Navé et al., Biologically Active PAF102 Is Recovered 2000; Aymé et al., 2014). From Yeast Cells We then introduced the Dgat1 gene construct in the Knowing that double transformant Ole18-PAF102/Dgat1 cells Ole18-PAF102 strain. Double transformants were recovered and contain a high number of LDs, and that Ole18-PAF102 is Frontiers in Microbiology | www.frontiersin.org 7 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts FIGURE 5 | A. thaliana Dgat1 expression induces LD formation in Ole18-PAF102 yeast cells (A) Gene construct introduced into the pGAPZA vector for producing A. thaliana Diacylglycerol o-acyltransferase (Dgat1) enzyme (At2g19450) in P. pastoris. (B) Confocal microscopy images of Ole18-GFP cells with or without the Dgat1 construct, as indicated. Bright field (BF) and fluorescence merged images are shown. (C) Total number of LDs per 30 cells of the indicated strains. LDs were counted in three replicates. Asterisk denotes statistically significant differences (Tukey test, p < 0.005) (D) Confocal microscopy images of Nile red stained Ole18-PAF102 cells with or without the Dgat1 construct as indicated. Bright field (BF), and fluorescence and BF merged images are also shown. Images correspond to single focal sections. (E) Total number of LDs per 30 cells of the indicated strains. LDs were counted in three replicates. Asterisk denotes statistically significant differences (Tukey test, p < 0.005) (F) Distribution of range sizes (area in µm2 ) from 50 LDs from strain cells indicated. (G) Growth of indicated strain cultures. Graph represents the mean OD600 ± SD value of triplicate cultures measured every 2 h. Bars = 2 µm. Frontiers in Microbiology | www.frontiersin.org 8 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts accumulated in these LDs, we evaluated the amount of PAF102 Given that Ole18 LDs and TEV protease did not have any peptide produced by this strain. We isolated the LDs by density inhibitory activity (Figure 6E), TEV protease could be assisting gradient centrifugation and analyzed their Ole18-PAF102 content PAF102 activity. Our results demonstrate that when PAF102 is by immunodetection. As shown in Figure 6A, Ole18-PAF102 produced in P. pastoris using the oleosin fusion technology, accumulation was higher in the Dgat1 expressing strain and it is biologically active and equivalent to that produced by correctly targeted to LDs. We estimated that the fusion protein chemical synthesis. accumulates at 67 ± 2 mg/l in the Ole18-PAF102 strain, and reaches up to 180 ± 5 mg/l in the Ole18-PAF102/Dgat1 strain. This represents a near three-fold increase in the fusion protein DISCUSSION accumulation levels, congruently with the three-fold increase in cellular LD content (Figure 5E). This study reports that P. pastoris is an excellent cell factory Using this high producer Ole18-PAF102/Dgat1 strain, we for the fast and efficient biotechnological production of the assessed the recovery of the single PAF102 peptide from the antifungal PAF102 peptide, and probably other PAF peptides. The LDs containing the Ole18-PAF102 fusion protein. TEV protease process developed here is an innovative system based on the plant was used to digest the recombinant LD fraction since the oleosin fusion technology that we successfully transfer to the Ole18 and PAF102 polypeptides were linked through the TEV heterologous yeast system. It has so far been difficult to produce protease recognition site (PRS). In immunoblot analysis of LD rationally designed PAF antifungal peptides in conventional fractions after proteolytic digestion (Figure 6B), we observed microbial expression systems, probably due to toxicity toward that the fusion protein detected by anti-PAF102 antibodies was host cells. We recently demonstrated that these bioactive peptides almost absent (upper panel), and a protein slightly larger than could be produced in plant biofactories as oleosin fusion Ole18 appeared, recognized by anti-Ole18 antibodies (lower proteins (Bundó et al., 2019). The fusion of PAF102 to the rice panel). After TEV protease digestion, most of the PRS remained Ole18 protein reduces its toxicity to the host cells allowing its attached to the Ole18 protein. This gives the Ole18 released accumulation to large amounts. We show here that the Ole18- from the fusion protein by TEV protease digestion a theoretical PAF102 fusion protein is also produced in yeast cells and targeted molecular weight of 18.25 kDa, whereas the single Ole18 is to LDs where large amounts accumulate. By coexpressing the only 17.15 kDa. The bands detected in western-blot analysis Arabidopsis Dgat1 gene, P. pastoris cells produced up to 180 mg/l are consistent with this difference in size and suggest that of PAF102 peptide in only 4 days. This yield was obtained from the fusion protein was processed by the TEV protease. Based shake-flash cultures, and typically recombinant protein yield can on the disappearance of the fusion protein in different assays, be increased in fermenter cultures. This demonstrates that our the TEV protease efficiency was calculated at 86% ± 10.5 on new system is a considerable improvement on the previous rice- average. This data indicates a high efficiency of proteolytic seed based system in terms of production times and yields of processing of the fusion protein Ole18-PAF102 on intact LDs. commercial relevance. We then investigated the presence of the PAF102 single peptide Nevertheless, the natural CecA peptide is not produced in the protease digested fractions. We immunodetected a at detectable levels in this P. pastoris system, although it polypeptide in the Ole18PAF102 LDs with similar mobility was efficiently produced in rice seeds when fused to Ole18, to the synthetic PAF102 (sPAF102) added to the EV LDs, even to higher levels than PAF102 (Montesinos et al., 2016). or to the sPAF102 alone (Figure 6C). This band was not Recombinant protein production is not only dependent on present in Ole18 LDs or in non-digested Ole18PAF102 LDs, gene expression and protein synthesis but also on its stability indicating that the fusion protein was correctly processed and the in host cells to allow its accumulation to significant amounts. PAF102 released. Therefore, the lack of Ole18-CecA accumulation to detectable To characterize the released PAF102 peptide, we tested the levels by immunoblot analysis might be due to instability in antifungal activity of the LDs fractions before and after TEV P. pastoris cells. Indeed, our results show that CecA is highly protease digestion. For that, we monitored the growth of susceptible to the yeast proteases. In contrast, PAF102 seems F. proliferatum in the presence of LDs at different concentrations. to be more resistant to Pichia proteases and accumulates to We did not detect any fungal growth inhibitory activity in high levels. Reports have been already published on the high the intact LD fractions, regardless of whether they were from susceptibility to proteolytic degradation of CecA by fungal EV, Ole18 or Ole18-PAF102 strains (Figure 6D), whereas the proteases (Bland and Lucca, 1998) or of CecA-derived peptides by sPAF102 completely inhibited F. proliferatum growth at the very tobacco plant proteases (Cavallarin et al., 1998). However, CecA low concentration of 2 µM. This data indicates that the fusion stably accumulated in transgenic rice plants, suggesting limited protein Ole18-PAF102 was not active, at least when embedded proteolysis by rice proteases (Coca et al., 2006; Bundó et al., into LDs. However, TEV protease digested Ole18-PAF102 LDs 2014; Montesinos et al., 2016). This data indicates differential clearly inhibited the growth of F. proliferatum, and the inhibitory degradation of CecA by host proteases, with its stability varying activity was equivalent to that of EV LDs supplemented with from one system to another: the stability of CecA to protein sPAF102 and incubated with TEV protease (Figure 6E). It is extracts derived from the host system should first be tested when worth noting that the synthetic PAF102 was more active in the planning to produce CecA in a new system. Still, the use of presence of EV LDs and TEV protease, completely inhibiting protease-deficient strains might improve production of CecA fungal growth at the even lower concentration of 0.25 µM. in P. pastoris. These strains have been used for the production Frontiers in Microbiology | www.frontiersin.org 9 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts FIGURE 6 | Biologically active PAF102 is recovered from yeast cells. (A) Immunoblot of total protein extracts (25 µg) and LD proteins from P. pastoris cell cultures transformed with empty vector (EV) or the indicated gene constructs, using anti-Ole18 or anti-PAF102 antibodies as indicated. (B) Immunoblot analysis of LDs fractions before (–) or after (+) TEV protease digestion using anti-PAF102 or anti-Ole18 antibodies as indicated. LDs were from Ole18 and Ole18PAF102/Dgat1 (#1 and #2 independent colonies) strains. (C) Immunoblot analysis of PAF102 peptide released from the fusion Ole18-PAF102 protein. Proteins from LDs were precipitated with acetone after TEV protease digestion and separated in Tricine-SDS-PAGE. Synthetic PAF102 (sPAF102, 25 or 50 ng), alone or in combination with the EV proteins, was run in parallel for comparative purposes (D,E) F. proliferatum growth inhibitory assays by indicated LD fractions in comparison to synthetic PAF102 (sPAF102) at varying concentrations (D) or by TEV digested LDs fractions in comparison to EV LDs supplemented with sPAF102 (E). of protease-sensitive proteins with variable success depending membranes containing neutral lipids, associating spontaneously on the proteases involved in the degradation events (Ahmad with them and assisting their sequestration into domains and et al., 2014). Our results suggest that the efficiency of the ultimately in the formation of a particle filled with neutral production system here developed would be dependent on the lipids. Yeast LDs do not have similar structural proteins, but peptide to be produced. perform their functions properly (Koch et al., 2014). However, Oleosin proteins have an important structural role in oleosins from different plant species produced in S. cerevisiae facilitating the formation and stability of plant LDs. They are cells target LDs and induce the formation of LDs (Ting et al., small lipophilic proteins with a conserved central hydrophobic 1997; Jacquier et al., 2013; Jamme et al., 2013; Vindigni et al., domain, inserted within the phospholipid bilayer of LDs, and two 2013). In agreement with these reports, rice Ole18 targets and variable amphipathic N and C terminal domains covering the LD stably accumulates in LDs in P. pastoris. The fusion of PAF102 surface (Abell et al., 1997). Oleosins have an intrinsic affinity to to the Ole18 in the C-terminal also targets LDs, with the Frontiers in Microbiology | www.frontiersin.org 10 June 2019 | Volume 10 | Article 1472 Popa et al. Plant Oleosins as Carriers of AMPs in Yeasts hydrophobic domain of Ole18 embedding into the TAG matrix systems is the reduced production time. Oleosin technology in and the PAF102 remaining on the surface. There were more plants requires completion of the plant life cycle, since LDs are and larger LDs in the cells accumulating the Ole18-PAF102 mainly accumulated in seeds: depending on the species, this fusion protein than in those accumulating only the Ole18. It could take several months whereas production in P. pastoris only seems that the presence of PAF102 stabilizes the recombinant requires several days. The production of other proteins should be LDs and promotes their formation, probably due to the cationic evaluated to determine the robustness of the developed system. and amphipathic character of PAF102 and its phospholipid affinity. This suggests that the oleosin fusion technology could be successfully applied to other AMPs with a similar cationic and DATA AVAILABILITY amphipathic character. A valuable addition to the production system is the All datasets generated for this study are included in the coexpression of AtDagt1 and Ole18-PAF102 genes. AtDag1 manuscript and/or the Supplementary Files. expression enhances the TAG content in yeast cells (Bouvier- Navé et al., 2000; Aymé et al., 2014). This high content of TAG leads to overaccumulation of the Ole18-PAF102 fusion AUTHOR CONTRIBUTIONS protein, resulting in high yields of the recombinant protein. This is consistent with the requirement of TAGs for the stable MC conceived and designed this study. PF contributed to the accumulation of the A. thaliana Oleosin 1 in yeast cells (Jacquier design. CP and TR prepared the gene constructs and carried out et al., 2013). Oleosin 1 was easily degraded in mutant strains the yeast transformation experiments. CP and XS characterized of S. cerevisae deficient in TAGs that were not able to produce the yeast transformants and the accumulation of AMPs in yeast and to accumulate neutral lipids. In contrast, cells with a high cells. CP set up the LD isolation protocol. XS set up the recovery content of TAGs offer sufficient anchoring surface for the Ole18- and evaluation of PAF102 from LDs. MC coordinated the study PAF102, providing stability and allowing the accumulation of and prepared the manuscript. All authors read and approved the very large amounts of the fusion protein. The yield could be final manuscript. further improved by using modified Dagt1 genes to enhance the storage lipid content in yeast (Greer et al., 2015). One of the main advantages of the oleosin fusion technology FUNDING is the simplicity of extraction and purification of the recombinant This work was supported by the BIO2015-68790- proteins accumulated in LDs from plant material. Similarly, C2-2-R and RTI2018-101115B-C22 grants, and the P. pastoris LDs with the Ole18-PAF102 can be separated “Severo Ochoa Program for Centers of Excellence in from other cellular components by simple flotation in density R&D” (SEV-2015-0533) from the Spanish Ministerio de gradients. The yeast isolated LDs containing the Ole18-PAF102 Ciencia, Innovación y Universidades (co-financed FEDER had no activity against fungal targets, consistent with previous funds), the CERCA Program/Generalitat de Catalunya, results with LDs from rice seeds (Bundó et al., 2019). PAF102 is and the Reference Network in Biotechnology (XRB; not active while immobilized in the LDs, requiring release from Generalitat de Catalunya). the Ole18 for activity. We speculate that this cell penetrating peptide cannot enter the fungal target cell while attached to LDs, a process that is necessary for its antifungal action (Muñoz et al., ACKNOWLEDGMENTS 2013). Once released from Ole18, PAF102 produced in P. pastoris was biologically active and indistinguishable from the synthetic We thank Dr. Günther Daum (Institute of Biochemistry, Graz, peptide. PAF102 has potent activity against F. proliferatum, a Austria) for kindly providing us the anti-Erg6 antibodies. We plant pathogen that contaminates cereal grain with mycotoxins, also acknowledge support of the publication fee by the CSIC reducing grain quality and causing severe economic losses Open Access Publication Support Initiative through its Unit of (Vismer et al., 2019). Here we demonstrate that biologically Information Resources for Research (URICI). active PAF102 peptide can be produced quickly and efficiently in P. pastoris using the rice oleosin protein as a carrier to LDs. Our results suggest that the oleosin fusion technology can SUPPLEMENTARY MATERIAL be transferred to yeast cells for the production of recombinant proteins other than the AMPs here investigated. Particularly, The Supplementary Material for this article can be found we also successfully produced the GFP as an Ole18 fusion online at: https://www.frontiersin.org/articles/10.3389/fmicb. protein in P. pastoris. The main advantage of yeast over plant 2019.01472/full#supplementary-material REFERENCES Adelantado, N. (2016). Lipidomics Studies of Recombinant Pichia pastoris for Improved Recombinant Protein Secretion Through Cell Abell, B. M., Holbrook, L. A., Abenes, M., Murphy, D. J., Hills, M. J., and Moloney, Engineering. Doctoral thesis Available at: https://www.tdx.cat/handle/10803/ M. M. (1997). Role of the proline knot motif in oleosin endoplasmic reticulum 384229 topology and oil body targeting. Plant Cell 9, 1481–1493. doi: 10.1105/tpc.9.8. Ahmad, M., Hirz, M., and Pichler, H. (2014). Protein expression in Pichia pastoris: 1481 recent achievements and perspectives for heterologous protein production. 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