Heterologous overexpression and biochemical characterization of the (galactophospho)lipase from Fusarium solani in Pichia pastoris that is expressed in planta

International Journal of Biological Macromolecules 84 (2016) 94–100

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Heterologous overexpression and biochemical characterization of the (galactophospho)lipase from Fusarium solani in Pichia pastoris that is expressed in planta Raida Jallouli a , Madiha Bou Ali a , Mariam Charfeddine b , Radhia Gargouri-Bouzid b , Youssef Gargouri a , Sofiane Bezzine a,∗ a b

University of SFAX, Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra, BPW 3038 Sfax, Tunisia University of SFAX, Enzymes et Bioconversion, ENIS route de Soukra, BPW 3038 Sfax, Tunisia

a r t i c l e

i n f o

Article history: Received 4 October 2015 Received in revised form 29 November 2015 Accepted 2 December 2015 Available online 7 December 2015 Keywords: F. solani (galactophospho)lipase P. pastoris Heterologous expression Phytopathology

a b s t r a c t High-level extracellular production of Fusarium solani (galactophospho)lipase, named FSL, was achieved using a Pichia pastoris X33 expression system. The (galactophospho) lipase encoding gene was cloned into pGAPZ␣A with the Saccharomyces cerevisiae ␣-factor signal sequence by two different ways. The two constructs consist of an additional sequence of a (His)6 -tag of the vector fused to the N-terminus of this enzyme (tFSL) while the other expression vector was constructed without any additional sequence (rFSL). Compared to the native enzyme (nFSL) (18.75 mg/L), a high level secretion of rFSL (310 mg/L) and tFSL (240 mg/L) was achieved providing an important improvement in enzyme production. Biochemical characterization showed that pure recombinant proteins (rFSL and tFSL) presented similar behaviour towards triglycerides, phospholipid and galactolipid. Like the nFSL, rFSL and tFSL are active at high concentration of bile salts (4 mM) and calcium ions enhanced lipase activity. During plant infection, transcripts of this fungal lipase gene were detected 3, 7 and 10 days post infection. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Lipases or triacylglycerol hydrolases (E.C. 3.1.1.3) are soluble enzymes that catalyse the hydrolysis of triacylglycerols into fatty acids and glycerol at the water–lipid interface and its reverse reaction in non-aqueous solvents [1,2]. Lipases show low activity towards monomeric substrate molecules whereas the presence of aggregate substrates is usually associated with an important increased activity. This phenomenon, referred to as interfacial activation [3]. These hydrolytic enzymes are widely distributed in nature including animals, plants and microorganisms. Much attention is paid to lipases of microbial origin, such as Candida sp., Pseudomonas sp., Bacillus sp. and Rhizopus sp. for their unsurpassed roles in the field of biotechnology [4]. The most commercially important field of application is their use in food, detergent, paper

Abbreviations: nFSL, native Fusarium solani lipase; rFSL, recombinant Fusarium solani lipase; tFSL, tagged recombinant Fusarium solani lipase; NaTDCs, odium taurodeoxycholic acid; TC4t, ributyrin; TC8t, rioctanoin. ∗ Corresponding author. Tel.: +216 74675055; fax: +216 74675055. E-mail address: sofiane [email protected] (S. Bezzine). http://dx.doi.org/10.1016/j.ijbiomac.2015.12.005 0141-8130/© 2015 Elsevier B.V. All rights reserved.

industry and flavour development for dairy products. A large number of lipase-producing filamentous fungi have been characterised, including those from Rhizopus oryzae [5], Rhizopus homothallicus [6], Penicillium aurantiogriseum [7], Rhizomucor miehei [8] and Thermomyces lanuginosus [9]. Extracellular lipases produced by the phytopathogenic fungi Fusarium solani are well studied [10,11]. Some of them were described as virulent factor involved in the phytopathology [12]. Recently, a Tunisian F. solani strain was isolated in our laboratory. A novel lipase, tentatively named FSL, was purified from the culture medium [13]. This lipase contains 317 amino acids. Biochemical and kinetic properties were studied using emulsified system and monomolecular film techniques [13,14]. The FSL was specific among the microbial lipases by having important activities on various lipids: triglycerides, phospholipids [13] and interestingly galactolipids [15]. In fact, enzymes with galactolipase activity might soon play an important role for recovering fatty acids from galactolipids using hydrolysis, alcoholysis, or transesterification reactions. Moreover, it is known that an effective strategy to increase the productivity of an enzyme bioprocess production and sometimes to simplify the downstream process is through the cloning and expression of its corresponding gene into a foreign host.

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Different heterologous systems were successfully used for expression of hydrolytic enzymes from F. solani like Echerchia coli [16], Saccaromyces cerevisiae [17] as well as the methylotrophic yeast Pichia pastoris [10]. In the present study, we describe for the first time the expression of functionally active F. solani (galactophospho)lipase (FSL) in P. pastoris cells. A comparison of biochemical properties between recombinants lipases (rFSL and tFSL) and the native enzyme was made. In addition, we follow the expression of FSL during plant infection with F. solani. 2. Materials and methods 2.1. Strain, media and growth conditions The fungi F. solani was isolated in our laboratory. Plasmid PGapZ␣-A was maintained and propagated in E-coli host XL1bleu cultured in law salt Lauria–Bertani medium (LBLS) (% w/v) (0.5% yeast extract, 1% tryptone and 0.5% NaCl) at 37 ◦ C. P. pastoris host X-33 was used as the expression host. YPD (1% yeast extract, 2% peptone, 2% dextrose) media was used for P. pastoris growth; cultures were done in baffled flasks at 30 ◦ C and 100 rpm. E-coli transformants and P. pasoris recombinant strains were selected in LBLS and YPD agar medium, respectively, supplemented with zeocine (purchased from invitrogen, USA). The recombinant plasmids were confirmed by restriction analysis and sequencing. Taq DNA polymerase, restriction endonucleases, T4 DNA ligase, plasmids extraction, PCR purification and gel elution kits were obtained from promega (Madison, WI). Endoglycosidase H (Endo Hf ) was from New England Biolabs (MA, USA). 2.2. Cloning cDNA coding the Fusarium solani lipase “FSL” The cDNA encoding FSL was amplified with the FSLF primer (5 -gatcctcgagaaaaga GCCATCACCGCCTCTCAAC-3 ) which includes a cleavage site for XhoI and the FSLR primer (5 gatctctagaCTACATCTTATTGCTCTCAACG- 3 ), which contains a XbaI site. To construct the NH2 -terminal tagged FSL gene, the sequence of the forward primer (FSLF) was substituted with 5 -gatcctcgagaaaagaCATCATCATCATCATCATGCCATCACCGCCTCT CAAC-3 . Polymerase chain reaction (PCR) was performed in 50 ␮L reaction mixtures containing 20 pmol of each primer, 0.5 U of GoTaq with 10× taq buffer (Promega, USA), 2 mM dNTP mix and 0.2 ␮g cDNA as a template. The PCR program including denaturation, annealing and extension was performed 94 ◦ C for 1 min, at 56 ◦ C for 1 min and at 72 ◦ C for 1 min, respectively for 35 cycles. The amplified gene was cloned directionally using XhoI and XbaI restriction sites in the vector pGapZ␣-A. Both the vector and the gene were double-digested and purified. Ligation reaction was set in the ratio of 3:1 (insert: vector) and the E. coli transformants were selected at the zeocin concentration of 25 ␮g/mL.

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2.4. Production of FSL in P. pastoris The best clone showing the most important activity level was selected for the production of the recombinant protein. A liquid preculture step was performed for 24 h in a 250 mL erlenmeyer flask containing 50 mL of YPD medium. This pre-culture was further used to inoculate larger yeast cell cultures. The dilution was such that an initial absorbance (600 nm) of 1 was obtained, in order to start the culture directly in the exponential growth phase, as well as to establish reproducible cell culture conditions. The yeast was further grown at 30 ◦ C with orbital agitation at a rate of 100 rpm. The cell cultures were usually performed in a 1-L erlenmeyer flask containing 200 mL of YPD medium without any ZeocinTM and the growth was stopped after 48 h to limit the proteolysis of the recombinants FSL secreted into the culture medium. 2.5. Purification of recombinant FSL (rFSL) and recombinant tagged FSL (tFSL) Two liters of yeast culture medium were collected, and cells were pelleted by centrifugation at 3000 × g for 30 min at 4 ◦ C. All subsequent purification steps were performed at 4 ◦ C. The culture supernatant was concentrated over a Filtron 10 kDa Ultrasette membrane (Pall Corporation) to about 100 mL. The concentrated sample was then lyophilised overnight. 1 g of the powder containing crude enzyme was then dissolved in 2 mL buffer A (10 mM Tris, pH 8). The concentrate sample was then centrifuged at 15,000 × g for 20 min, an insoluble pellet was discarded. In the case of rFSL, the resulting clear supernatant was loaded onto a Sephadex G75 26/60 gel filtration column (Amersham Biosciences) (flow rate 2.5 mL/min, fraction volume 1.5 mL), connected to an ÄKTA explorer chromatographic device (Amersham Biosciences) and equilibrated with buffer A. Eluted fractions with lipase activity were pooled and then loaded onto a mono-Q SepharoseTM anion exchanger (GE Healthcare) column (2.5 × 12 cm, flow rate 1 mL/min, fraction volume 1 mL) equilibrated with the same buffer A. The pressure was kept below 1 MPa and the protein elution profile was recorded spectrophotometrically at 280 nm using AKTA explorer device. After a washing step with 20 mM NaCl, a linear NaCl concentration gradient ranging from 20 to 500 mM was applied for 1 h to elute the bound proteins. Fractions containing pure enzyme were pooled and concentrated to about 2 mL using a 10,000 MWCO PES Vivaspin centrifugal concentrator (Sartorius). To purify the tagged FSL (tFSL), a single step was used; a nitrilotriacetate (Ni-NTA) resin equilibrated with buffer A. tFSL was eluted with a linear imidazole gradient (100 mL of 0–500 mM in buffer A). 2.6. Analysis of rFSL and tFSL expressed in P. pastoris The yeast culture medium and fractions containing rFSL and tFSL during purification steps were analyzed by electrophoresis on 12% polyacrylamide gels in the presence of SDS as described by Laemmli [19]. The concentration of the purified recombinant protein was measured using the BCA kit (Pierce) and BSA as standard.

2.3. Transformation of P. pastoris 2.7. N-terminal sequencing To enable gene integration, the recombinant plasmids were linearized with Bgl2 enzyme and purified. Ten to twelve nanograms of each linearized plasmid were electroporated into electrocompetent X33 cells prepared using standard method [18], with an eppendorf gene pulser (1500 V). Electroporation was carried out in a 0.2 cm cuvette according to the standard protocol. After pulsing, the cells were resuspended in 1 M sorbitol, incubated for 3 h at 30 ◦ C and plated on YPD agar containing 0.1 mg/mL zeocin. The recombinants colonies obtained after 3 days were confirmed by PCR using gene-specific primers.

For N-terminal sequence analysis, rFSL and tFSL protein were gel purified and electroblotted to nitrocellulose membrane. Sequencing was carried out at the proteomics plateform of Mediterranean institute of microbiology (Marseille). 2.8. Lipase activity measurement Lipase activity was assayed potentiometrically by automatically titrating the free fatty acids released from mechanically stirred

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triacylglycerol emulsions using 0.1 N NaOH and a pH- stat. Each assay with short-chain (TC4), medium-chain (TC8) and long-chain triacyglycerols (olive oil) was performed in a thermostated vessel containing 0.5 mL triglyceride in 15 mL of a 1 mM Tris–HCl, pH 8.5, 3 mM CaCl2 and 2 mM NaTDC. To measure recombinants FSL activities in the absence of CaCl2 , EDTA (10 mM) was added to the assay system. One unit of lipase activity corresponds to 1 ␮mol of fatty acid released per minute under the assay conditions used. Specific activities are expressed here in international units (U) per milligram of protein. 2.9. Enzyme characterization 2.9.1. Study of the glycosylation of rFSL Deglycosylation of rFSL produced in P. pastoris using endoglycosidase H (Endo Hf , New England Biolabs, MA, USA) was carried out according to the manufacturer’s instructions. Ten micrograms of lipase solution was incubated with 1 ␮L of 10× Glycoprotein Denaturing Buffer at 100 ◦ C for 10 min. Then 2 ␮L of 10× G5 Reaction Buffer and 3 ␮L of Endo Hf were added to the mixture and incubated at 37 ◦ C for 1 h. 2.9.2. Substrate specificity Lipase substrate specificity was analysed using different substrates: short chain triacyglycerol (TC4), medium chain triacyglycerol (TC8), long chain triacylglycerol (emulsion of olive oil), phospholipid (egg yolk PC) and galactolipid (monogalactosyl dioctanoylglycerol: DiC8-MGDG). 2.9.3. Effect of temperature and pH on lipase activity and stability The thermal profile of enzyme activity was measured at temperatures between 25 ◦ C and 60 ◦ C. The incubation was performed in a temperature-controlled cuvette holder. In order to determine the thermal stability of recombinant lipases, aliquots of the pure enzymes solution were incubated 30 min at 30 ◦ C, 37 ◦ C, 40 ◦ C, 45 ◦ C, 50 ◦ C and 55 ◦ C. Immediately after incubation, residual activities were determined, after centrifugation, under standard assay method. The optimum pH for lipase activity was determined at different pHs ranging from 6 to 11. In order to determine the pH stability, pre-incubation was performed at room temperature for 1 h in the following buffers: 50 mM sodium acetate buffer (pH 4.0–6.0), 50 mM potassium phosphate buffer (pH 6.0–8.0), 50 mM Tris–HCl buffer (pH 7.0–9.0) and 50 mM glycine–NaOH buffer (pH 8.0–12.0). The residual activities were determined after centrifugation, under standard assay method. 2.9.4. Effect of bile salts and calcium ions on the enzyme activity In order to study the bile salt effect on the rFSL, tFSL and nFSL activities, lipase activity was measured using emulsified olive oil as substrate at 37 ◦ C and pH 8.5 in the presence of increasing concentration of Sodium Tauro Deoxy Cholic acid (NaTDC). To study the calcium dependence of the two recombinant enzymes, lipase activity was measured using emulsified olive oil as substrate at 37 ◦ C and pH 8.5 in the presence of 2 mM NaTDC. 2.9.5. Plant infection To study FSL gene expression during plant infection, potato plants (cv. Spunta) grown in vitro were used. Fifteen day-old vitroplants were infected with 102 conidia of F. solani and incubated in a culture room at 25 ◦ C with a photoperiod of 16 h/day and a luminosity of 62 ␮E/m2 s. Infected samples (roots, stem and leaves) were collected after 1, 3, 7 and 10 days post infection and directly frozen in liquid nitrogen. Total RNAs were isolated using Trizol Reagent and analyzed by RT-PCR using FSL gene-specific primers listed above.

The elongation factor gene (ef1a) (GenBank ID: AB061263) was used as constitutive gene marker. PCR products were visualized on ethidium bromide stained gels using the Gel DocXR Gel Documentation System (BioRad). The average intensity of the bands was quantified using Quantity One 1-D Analysis Software (BioRad) and graphed using Microsoft Excel. The error bar was determined from three separate biological replicates. 3. Results 3.1. Generation of recombinant Pichia clones expressing untagged and N-terminal tagged FSL The FSL gene, tagged (tFSL) and untagged (rFSL) forms, containing XbaI and XhoI sites upstream of its first codon and downstream of its Stop codon, respectively, was digested by these two restriction enzymes and inserted into the pGAPZ␣ A vector previously digested by XbaI and XhoI. The recombinant vector was then linearized by the BglII restriction enzyme and the GAP promoter-driven constitutive expression of FSL was achieved by integrating the linearized recombinant plasmid into the X-33 wild type P. pastoris genome at the GAP locus. P. pastoris tranformants generated by electroporation with the recombinant vectors (pGAPZ␣ A-rFSL and pGAPZ␣ A-tFSL) were selected on YPD plates containing Zeocin and incubated at 30 ◦ C for 3 days. The presence of FSL insert in the Zeo-resistant transformants was checked by PCR. After 48 h of yeast growth, a large amount of the recombinant lipases was reached (310 mg/L for rFSL and 240 mg/L for tFSL). Compared to the native FSL (nFSL) which was produced at a level of 18.75 mg/L by F. solani strain [13], we can conclude that heterologous expression improves the production of FSL, 16 and 12 times, respectively. This important yield of expression was comparable to previously expressed fungal lipases in P. pastoris. As can be shown in Fig. 1A and B, cells expressing the N-terminal tagged FSL were denser than those expressing the untagged FSL. Similar results were reported by Svensson et al. [20] and Horchani et al. [21]. They have justified the difference on the morphology to the hydrophilic character of the tagged lipase which could be more compatible with the host cell. However, the lipase activity measured was less important with the tagged FSL (230 U/mL on tributyrin compared to 280 U/mL for the rFSL). 3.2. Purification of the rFSL and the tFSL The rFSL was purified according to the procedure described in the experimental section with a final yield of about 34%. On the other hand the tFSL was purified in a single stage of Ni2+ nitrilotriacetate (NTA) affinity column equilibrated with buffer A and eluted by a linear gradient of imidazol with a final yield of about 55%. SDSPAGE analysis of the purified rFSL and tFSL are shown in Fig. 2A and B, respectively. These two purified enzymes exhibit a single band corresponding to a molecular mass of about 30 kDa. The Nterminal amino acid sequence of the rFSL and tFSL allowed the identification of 10 residues A-I-T-A-S-Q-L-D-Y-E and H-H-H-H-HH-A-I-T-A respectively proving the correct cleavage of the ␣-factor signal sequence at the Kex2 cleavage site at the N-terminal of the expressed lipases. Post-translational glycosylation influence sometimes some important enzymatic properties, including structural folding, solubility, catalytic activity and thermal stability [22]. For this reason, the N-linked glycosilation of FSL was checked according to procedure described in experimental section. As shown in Fig. 2C, there’s no difference in the molecular mass of rFSL before and after treatment with endoglycosidase Hf indicating that FSL is not glycosilated.

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Fig. 3. substrate specificity of nFSL, rFSL and tFSL. Lipase activity of nFSL and rFSL on tributyrine (TC4), trioctanoine (TC8) and olive oil emulsions was measured in the presence of 3 mM CaCl2 and 2 mM NaTDC. Lipase activity of tFSL on these substrates was measured in the presence of 1 mM CaCl2 and 2 mM NaTDC. Activity of nFSL, rFSL and tFSL on phospholipids was measured in the presence of 7 mM CaCl2 and 4 mM NaTDC. Specific activity of nFSL, rFSL and tFSL on monogalactosyldiacylglycerol (DiC8-MGDG) was measured in the presence of 3 mM CaCl2 and 13.33 mM NaTDC. Measurements were carried out at 37 ◦ C and pH 8.5. Data: mean ± SD.

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Fig. 1. Time-course profile production of the rFSL (A) and the tFSL (B) by recombinant P. pastoris in 250 mL shaking flasks. (Closed circles) Lipase activity (U/mL), (empty circles) cell growth (OD 600 nm). Lipase activity was measured using tributyrin as substrate at pH 8.5 and 37 ◦ C under standard conditions.

3.3. Chain length selectivity of the rFSL and the tFSL The specific activities of the two recombinant lipases (rFSL and tFSL) were measured using short (tributyrin), medium (trioctanoin), long chain triacylglycerols (olive oil), phospholipids (egg-yolk PC) and galactolipids (1,2-octanoyl-3-O-␣-d-galactosylsn-glycerol (DiC8-MGDG) as substrates and compared to those obtained with the native one (nFSL). Fig. 3 shows that the substrate preferably hydrolysed by expressed enzymes was the DiC8-MGDG with specific activities of 4700 U/mg for the nFSL, 4440 U/mg for the rFSL and 4150 U/mg for the tFSL. Using olive oil as substrate, the specific activity of the rFSL (1700 U/mg) and the tFSL (1725 U/mg) are similar to the nFSL (1650 U/mg). An increase in the lipase activity was observed with rFSL (2350 U/mg against 1600 U/mg for nFSL)

Fig. 2. SDS/PAGE (12%) analysis of the purified proteins: (A) rFSL, Lane 1: molecular mass markers; lane 2: Culture supernatant, lane 3: active fractions from Sephadex G-75 chromatography, lane 4: active fractions from Mono-Q chromatography (20 ␮g); (B) tFSL, Lane 1: molecular mass markers; lane 2: Culture supernatant, lane 3: 20 ␮g of the purified tFSL obtained after Ni-NTA affinity chromatography; (C) SDS-PAGE analysis of the rFSL treated with Endo Hf , Lane 1: molecular mass markers, lane 2: 20 ␮g of rFSL before treatment, lane 3: 20 ␮g of rFSL after treatment.

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Temperature (°C) Fig. 4. Effects of temperature on nFSL, rFSL and tFSL activities and stabilities. (A) Effect of temperature on nFSL, rFSL and tFSL activities. (B) Effect of temperature on nFSL, rFSL and tFSL stabilities. The enzymes are incubated at different temperatures for 30 min. Residual activities were measured with the pH-stat method using olive oil emulsions as substrate. Experiments were carried out in triplicates.

when using TC8 as substrate. However, the activity of rFSL was decreased on TC4 (780 U/mg) compared to the nFSL (1200 U/mg). Thus, with respect to chain length selectivity, the rFSL and the tFSL maintained the same behaviours compared to the native one. In fact, there’s a preference for long and medium chain triacyglycerols and an important activity on phospholipids and galactolipids. 3.4. Effects of pH and temperature on rFSL and tFSL activity and stability The activities of recombinant enzymes (rFSL and tFSL) were measured at different pH and temperatures. Results show that both rFSL and tFSL present an optimum activity at 37 ◦ C using olive oil emulsions as substrate similarly to the nFSL (Fig. 4A). At 50 ◦ C, the nFSL and tFSL keep about 54% of their optimal activity contrary to rFSL which preserve 82% of its activity. The thermostability of the rFSL and the tFSL was also evaluated by measuring the residual activity after 30 min incubation of the pure enzymes at various temperatures (Fig. 4B). As can be seen, there’s no significative difference in the thermal stability of rFSL and tFSL compared to the nFSL. Moreover, the activities of the rFSL and tFSL were measured at different pH using olive oil emulsions as substrates. Our results show that the behaviour of these two recombinant lipases is similar to the native one [13]. The maximal activity was measured at pH 8.5 similar to the nFSL. The rFSL and tFSL are stable in the pH

Fig. 5. Effect of increasing concentrations of bile salt (NaTDC) (A) and calcium ions (Ca2+ ) (B) on the rate of hydrolysis of olive oil emulsion by the nFSL, rFSL and tFSL. Lipolytic activity was measured at pH 8.5 and 37 ◦ C. The stars indicate the lipase activity measured in the absence of CaCl2 and in the presence of 10 mM EDTA.

range of 8.0–10 after pre-incubation at room temperature for 1 h (data not shown). 3.5. Effect of bile salts and calcium ions on the rFSL and tFSL activity The effect of varying concentrations of NaTDC on the rFSL and tFSL activities shows that, similarly to the nFSL, the maximum activity was detected at 2 mM NaTDC. The rFSL and the tFSL retain respectively 88% and 71% of their activities at 4 mM NaTDC (Fig. 5A). In order to investigate the calcium dependence of the rFSL and tFSL, we measured the hydrolysis rates of olive oil emulsion by the two enzymes in the presence of various CaCl2 concentrations. Our results showed that, the addition of Ca2+ increase the lipase activity to reach a maximum at 3 mM CaCl2 for the nFSL and rFSL and at 1 mM CaCl2 for the tFSL (Fig. 5B). 3.6. Expression of FSL in planta To determine whether FSL is expressed by F. solani during infection of its host plant, total RNA was isolated from infected potato tissues at four different times post inoculation (1, 3, 7 and ten days post infection), and analysed by semi quantitative RT-PCR. Analysis of the RT-PCR products showed the expression of FSL gene at 3, 7 and 10 days post inoculation in the roots and stems of infected plants and only at 7 and 10 days post infection in the case of leaves (Fig. 6A). There’s no fragment amplified from the RNA of uninoculated controls. To ensure the specificity of the amplified fragment, internal FSL specific primers were used and a 250 pb amplification product was obtained.

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proteins [21,27]. In this study, the specific activity and the biochemical properties were almost identical between the N-terminal tagged and the non-tagged FSL compared to the native one. In addition, there’s an improvement in thermal activity of expressed enzyme. This attitude is different from that reported by Horchani et al. [21]. These authors found that heterologous expression induces a negative effect on thermal activity of Staphylococcus aureus lipase expressed in E-coli system. However, our results are in accordance with those found with R. oryzae lipase expressed in P. pastoris [28]. Several studies reported the importance of secreted lipases for virulence of the phytopathogenic fungus [29,30]. Here, we found the expression of the FSL gene during plant infection. These results justify probably the direct involvement of the FSL on the phytopathological process of F. solani having important activities on phospholipids [14] and galactolipids [15]. 5. Conclusion In this study, we reported for the first time the cloning and the high-level expression of the F. solani lipase gene into P. pastoris. The recombinant lipases (tagged and untagged) showed almost the same properties as the native lipase purified from F. solani. Moreover, the expression of FSL gene during plant infection by F. solani may emphasises its potential involvment in determining pathogenicity. Acknowledgements This work received financial support from the “Ministère de l’Enseignement Supérieur et de la Recherche” granted to the “Laboratoire de Biochimie et de Génie Enzymatique des Lipases”. Fig. 6. (A) RT-PCR analyses of FSL mRNA expression in Spunta potato plants inoculated with F. solani at different treatment periods: 1, 3, 7 and 10 days post infection. (a) Root tissue, (b) stem tissue and (c) leaf tissue. (d) Elongation factor 1␣ (ef1␣) specific primers were used as internal control. NI: Non Infected. Standard error (SE) was determined from three independent biological replicates, and band densities were calculated by the Quantity One 1D analysis software (B) Apparition of degradation symptoms on infected plant after 10 days of infection (2) compared to the non infected one (1).

Moreover, since the third day post infection, plants show spots of degraded and necrotic tissues and after ten days post infection, symptoms of a complete degradation were very clear (Fig. 6B). However, we can correlate the expression of the FSL gene to the appearence of infection symptoms in plants. 4. Discussion P. pastoris has great potential for production of heterologous proteins such as fungal and bacterial enzymes. For this reason, we have tested the suitability of a P. pastoris system for high-level production of the F. solani (galactophospho)lipase having a large broad substrate specificity. In this study we succeeded in expressing a large amount of the extracellular tagged and non-tagged enzyme. There have been earlier examples of fungal enzymes that are well expressed and correctly folded. In fact, the Lip2 of Yarrowia lipolytica was successfully expressed in P. pastoris with an expression level of around 630 mg/L [23]. Moreover, the Candida rugosa lipase Lip4 was expressed with a production level of 100 mg/L [24]. These important yields of fungal lipases expression were related to the heterologous system (P. pastoris) which performs post-transductional modifications of higher eukaryotic organisms. The small tags extension offer many advantages like the easy purification and detection of recombinant protein and the increase of solubility [25,26]. However, some reports describe the modification of the biochemical properties of expressed tagged

References [1] K.E. Jaeger, T. Eggert, Lipases for biotechnology, Curr. Opin. Biotechnol. 13 (2002) 390–397. [2] R.K. Saxena, P.K. Ghosh, R. Gupta, W.S. Dvidson, S. Bradoo, R. Gulati, Microbial lipases: potential biocatalysts for the future industry, Curr. Sci. 77 (1999) 101–115. [3] R. Verger, Interfacial activation of lipases: facts and artifacts, Trends Biotechnol. 15 (1997) 32–38. [4] A. Pandey, S. Benjamin, C.R. Soccol, P. Nigam, N. Krieger, V.T. Soccols, The realm of microbial lipases in biotechnology, Biotechnol. Appl. Biochem. 29 (1999) 119–131. [5] R. Ben Salah, H. Mosbah, A. Fendri, A. Gargouri, Y. Gargouri, H. Mejdoub, Production of wild-type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains, FEMS Microbiol. Lett. 260 (2006) 241–248. [6] M.J.C. Diaz, J.A. Rodríguez, S. Roussos, J. Cordova, A. Abousalham, F. Carrière, J. Bratti, Lipase from the thermotolerant fungus Rhizopus homothallicus is more thermostable when produced using solid state fermentation than liquid fermentation procedures, Enzyme Microb. Technol. 39 (2006) 1042–1050. [7] V.M.G. Lima, N. Krieger, D.A. Mitchell, J.D. Fontana, Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents, Biochem. Eng. J. 18 (2004) 65–71. [8] Z.S. Derewenda, U. Derewenda, G.G. Dodson, The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 A˚ resolution, J. Mol. Biol. 227 (1992) 818–839. [9] R. Fernandez-Lafuente, Lipase from Thermomyces lanuginosus: uses and prospects as an industrial biocatalyst, J. Mol. Catal. B: Enzym. 63 (2010) 17–22. [10] M.A. Kwon, H.S. Kim, T.H. Yang, B.K. Song, J.K. Song, High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris, Protein Expr. Purif. 68 (2009) 104–109. [11] M.M.D. Maia, A. Heasley, M.M. Camargo de morais, E.H. Melo, M.A. Morais Jr., W.M. Ledingham, J.L. Lima Filho, Effect of culture conditions on lipase production by Fusarium solani in batch fermentation, Bioresour. Technol. 76 (2001) 23–27. [12] A. Nasser Eddine, F. Hannemann, W. Schafer, Cloning and expression analysis of NhL1, a gene encoding an extracellular lipase from the fungal pea pathogen Nectria haematococca MP VI (Fusarium solani f. sp. pisi) that is expressed in planta, Mol. Genet. Genomics 265 (2001) 215–224. [13] R. Jallouli, F. Khrouf, A. Fendri, T. Mechichi, Y. Gargouri, S. Bezzine, Purification and biochemical characterization of a novel alkaline (Phospho)lipase from a newly isolated Fusarium solani strain, Appl. Biochem. Biotechnol. 168 (2012) 2330–2343.

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R. Jallouli et al. / International Journal of Biological Macromolecules 84 (2016) 94–100

[14] R. Jallouli, A. Fendri, T. Mechichi, Y. Gargouri, S. Bezzine, Kinetic properties of a novel Fusarium solani (phospho)lipase: a monolayer study, Chirality 25 (2013) 35–38. [15] R. Jallouli, H. Othman, S. Amara, G. Parsiegla, F. Carrière, N. Srairi-abid, Y. Gargouri, S. Bezzine, The galactolipase activity of Fusarium solani (phospho)lipase, Biochim. Biophys. Acta 1851 (2015) 282–289. [16] K.E. Griswold, N.A. Mahmood, B.L. Iverson, G. Georgiou, Effects of codon usage versus putative 50-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm, Protein Expr. Purif. 27 (2003) 134–142. [17] C.R. Calado, M. Mannesse, M. Egmond, J.M. Cabral, L.P. Fonseca, Production of wild-type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains, Biotechnol. Bioeng. 78 (2002) 692–698. [18] J.M. Cregg, K.A. Russell, Transformation methods, Mol. Biol. 103 (1998) 27–39. [19] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [20] J. Svensson, C. Anderson, J.E. Reseland, P. Lyngstadaas, L. Bülow, Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli, Protein Expr. Purif. 48 (2006) 134–141. [21] H. Horchani, S. Ouertani, Y. Gargouri, A. Sayari, The N-terminal His-tag and the recombination process affect the biochemical properties of Staphylococcus aureus lipase produced in Escherichia coli, J. Mol. Catal. B: Enzym. 61 (2009) 194–201. [22] R.J. Sola, J.A. Rodriguez-Martinez, K. Griebenow, Modulation of protein biophysical behavior by chemical glycosylation: biochemical insights and biomedical implications, Cell. Mol. Life Sci. 64 (2007) 2133–2152.

[23] M. Yu, S. Lange, S. Richter, T. Tan, R.D. Schmid, High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization, Protein Expr. Purif. 53 (2007) 255–263. [24] S.J. Tang, J.F. Shaw, K.H. Sun, G.H. Sun, T.Y. Chang, C.K. Lin, Y.C. Lo, G.C. Lee, Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity and stability, Arch. Biochem. Biophys. 387 (2001) 93–98. [25] H. Chen, Z. Xu, N. Xu, P. Cen, Efficient production of a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free system, J. Biotechnol. 115 (2005) 307–315. [26] S. Nallamsetty, D.S. Waugh, Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners, Protein Expr. Purif. 45 (2005) 175–182. [27] M. Bou Ali, Y. Ben Ali, I. Aissa, Y. Gargouri, Eukaryotic expression system Pichia pastoris affects the lipase catalytic properties: a monolayer study, PLoS ONE 9 (2014) e104221. [28] M. Guillén, M.D. Benaiges, F. Valero, Comparison of the biochemical properties of a recombinant lipase extract from Rhizopus oryzae expressed in Pichia pastoris with a native extract, Biochem. Eng. J. 54 (2011) 117–123. [29] L.M. Rogers, M.A. Flaishman, P.E. Kolattukudy, Cutinase gene disruption in Fusarium solani f sp pisi decreases its virulence on pea, Plant Cell 6 (1994) 935–945. [30] C.A. Voigt, W. Schäfer, S. Salomon, A secreted lipase of Fusarium graminearum is a virulence factor required for infection of cereals, Plant J. 42 (2005) 364–375.