Article pubs.acs.org/JAFC
High-Yield Phosphatidylserine Production via Yeast Surface Display of Phospholipase D from Streptomyces chromofuscus on Pichia pastoris Yihan Liu, Tao Zhang, Jing Qiao, Xiaoguang Liu, Jiaxin Bo, Jianling Wang, and Fuping Lu* Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, National Engineering Laboratory for Industrial Enzymes, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China ABSTRACT: The gene encoding phospholipase D (PLD) from Streptomyces chromof uscus was displayed on the cell surface of Pichia pastoris GS115/pKFS-pldh using a Flo1p anchor attachment signal sequence (FS anchor). The displayed PLD (dPLD) showed maximum enzymatic activity at pH 6.0 and 55 °C and was stable within a broad range of temperatures (20−65 °C) and pHs (pH 4.0−11.0). In addition, the thermostability, acid stability and organic solvent tolerance of the dPLD were significantly enhanced compared with the secreted PLD (sPLD) from S. chromof uscus. Use of dPLD for conversion of phosphatidylcholine (PC) and L-serine to phosphatidylserine (PS) showed that 67.5% of PC was converted into PS at the optimum conditions. Moreover, the conversion rate of PS remained above 50% after 7 repeated batch cycles. Thus, P. pastoris GS115/pKFS-pldh shows the potential for viable industrial production of PS. KEYWORDS: phospholipase D, Pichia pastoris, surface display, biotransformation, phosphatidylserine
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INTRODUCTION Phosphatidylserine (PS) is a phospholipid component. It has found many applications in the pharmaceutical and functional food sectors.1 Recent clinical studies have shown that PS supplemented in the diet plays an important role in revitalizing brain cell membranes and improving memory performance in individuals experiencing age-associated memory impairment or Alzheimer’s disease.2−4 In addition, PS can be an effective athletic nutrient supplement combating exercise-induced stress by blunting the exercise-induced increase in cortisol levels.5 Animal organs, such as bovine brains, are a potential source of PS preparations; however, these PS preparations might not be suitable for human use due to concerns about the transmission of infectious diseases, such as bovine spongiform encephalopathy. Natural PS can also be obtained from soybeans, vegetable oils, egg yolk, and biomass; nevertheless, they are unlikely to be appropriate sources for industrial-scale PS production due to their low availability. Previous studies have reported the enzymatic conversion of phosphatidylcholine (PC) to PS via phospholipase D (PLD).6,7 Among the available phospholipids, PC can be easily obtained in large amounts from various natural materials, such as soybean and egg, which are relatively inexpensive for the industrial production of phospholipids. Thus, the conversion of PC using PLD could be an efficient method of PS preparation. PLD catalyzes two reactions: (i) the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline via the cleavage of its phosphodiester bond and (ii) a transfer reaction in which the phosphatidic acid moiety is transferred to an acceptor alcohol (transphosphatidylation).6,7 The transphosphatidylation activity of PLD plays a key role in converting PC into other naturally rare phospholipids.8 Within the past decade, several PLD genes have been cloned from various species, including mammals, plants, yeasts, and bacteria.9−15 In © 2014 American Chemical Society
particular, PLDs from Streptomyces are of great interest because of their high transphosphatidylation activities compared to the activities of PLDs derived from plants and mammals.12 However, studies on the industrial applications of free PLD have been hindered by its high cost and low productivity. Moreover, transphosphatidylation is performed in acid biphasic systems, which result in alteration of protein structure and a significant reduction in PLD activity; thus, free PLD is not suitable for PS production. Immobilized enzymes are preferred over free enzymes in industrial applications due to their enhanced stability and reusability.16 One effective strategy to immobilize enzymes in inert carriers is via cell-surface display, which allows enzymes to be displayed on the surface of microorganisms, such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris, to function as immobilized enzymes.17 Yeast surface display is a powerful method for the expression of a functional protein on the cell surface based on the fusion of an enzyme of interest to an anchoring protein. Flo1p from S. cerevisiae is a lectin-like anchor protein that plays an important role in flocculation. The Flo1p anchor attachment signal sequence (FS anchor) contains the N-terminal flocculation functional domain (FFD), which can recognize and adhere noncovalently to cell wall components. Moreover, many studies have demonstrated that the FS anchor protein is efficient for the expression of recombinant proteins on the surface of P. pastoris.32,33 Cell surface engineering enables the development of a novel strategy for functionalizing yeasts to serve as whole cell biocatalysts with increasing enzyme stability and eliminate the need for enzyme Received: Revised: Accepted: Published: 5354
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purification.18−21 Recently, several enzymes, such as Yarrowia lipolytica lipases, Candida antarctica lipase B, and Enterobacter sp. FMB-1 sucrose isomerase, have been successfully immobilized onto the yeast cell surface, and their potential as biocatalysts for industrial applications has been investigated.17,21,22 In the present study, the phospholipase D gene (pld) cloned from Streptomyces chromofuscus was efficiently expressed on the cell surface of P. pastoris GS115 with FS as anchor protein. The enzymatic properties of the displayed PLD (dPLD) were systematically investigated. Importantly, the dPLD was successfully applied in the PS production so as to assess its potential in industrial applications.
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L biotin, and 15 g/L agar. The P. pastoris transformants harboring pKFS-pld (GS115/pKFS-pld) and pKFS-pldh (GS115/pKFS-pldh) were selected by incubation at 30 °C for 48 h on MD plates. Immunofluorescence Assay and Enzyme Assay. The transformants were precultured in YPD medium at 30 °C for 12 h and used to inoculate 50 mL of BMGY medium (13.4 g/L YNB, 10 g/L yeast extract, 20 g/L peptone, 20 g/L glycerol, and 4 × 10−4 g/L biotin) in a 250 mL shake flask at an initial OD600 value of 0.2. After incubation for 24 h, the cells were harvested by centrifugation at 4500 rpm for 10 min and resuspended in BMMY medium (13.4 g/L YNB, 10 g/L yeast extract, 20 g/L peptone, and 4 × 10−4 g/L biotin) containing 5 g/L methanol. To maintain the induction of the fusion protein, methanol was added to the culture every 24 h to a final concentration of 5 g/L, and the cells were incubated for 8 days at 30 °C with agitation (250 rpm). An immunofluorescence assay was performed using a mouse monoclonal antihemagglutinin (HA) tag and a fluorescein isothiocyante (FITC)-conjugated goat anti-mouse IgG. Yeast cells were harvested and washed with phosphate-buffered saline (PBS, 10 mM, pH 7.4) containing 1 mg/mL bovine serum albumin (BSA), and then the cells were incubated with the mouse monoclonal antihemagglutinin (HA) tag in a dilution of 1:100 in PBS for 30 min on ice, washed twice with PBS containing 1 mg/mL BSA, and mixed with FITC-conjugated goat anti-mouse IgG diluted 1:200 on ice for 30 min. After a washing with PBS, the cells were observed with a fluorescence microscope. The induced cells were harvested by centrifugation and washed twice with deionized water and lyophilized for activity assays.22 The PLD activity in this study, represented as the hydrolytic activity due to its convenience of determination, was determined according to the method of Uhm et al. with minor modifications.23 The PLD hydrolytic activity was determined by measuring the amount of choline released from the substrate PC. The cells were resuspended in 10 mM TrisHCl (pH 8.0) at a concentration of 100 mg dry cells/mL. The reaction was initiated by adding 100 μL of cell solution to 10 mM Tris-HCl (pH 8.0) containing 10 mM CaCl2, 20 mM TX-100, and 10 mM PC in a final volume of 1.5 mL at 37 °C for 10 min and was terminated by adding 0.5 mL of a solution containing 1 M Tris-HCl (pH 8.0) and 0.5 M ethylenediaminetetraacetic acid (EDTA). The reaction mixture was boiled for 5 min to completely arrest the reaction and then subsequently centrifuged at 6000 rpm for 5 min and mixed with 2 mL of 10 mM Tris-HCl (pH 8.0) containing 1 unit of choline oxidase, 1 unit of peroxidase, 0.4 mg of 4-aminoantipyrine, and 0.2 mg of phenol. The solution was then incubated for 20 min at 37 °C. The absorbance of the resulting mixture was measured spectrophotometrically at 500 nm. One unit (U) of hydrolysis activity of PLD was defined as the amount of enzyme that produced 1 μmol of choline per minute under these assay conditions. Enzyme Characterization. The optimal temperatures of the dPLD and secreted PLD (sPLD) were assayed by incubating the reaction mixture at various temperatures ranging from 30 to 65 °C in 50 mM sodium acetate buffer (pH 8.0). The thermostability of the enzyme was determined by incubating the enzyme at various temperatures ranging from 20 to 65 °C at pH 8.0 for 1 h. The remaining activity was then measured under standard conditions. Optimum pH values for the two forms of PLD were determined by measuring the hydrolytic activity with 1 mM PC as the substrate in the pH range of 4.0−6.0 using 50 mM sodium acetate buffer, in the pH range of 7.0−8.0 using 50 mM potassium phosphate buffer, and in the pH range of 9.0−11.0 using 50 mM Tris-HCl buffer at 37 °C. The stability of PLD at various pH values was determined by preincubating the PLD in solutions at various pH values, ranging from 4.0 to 11.0, at 25 °C for 1 h. The residual activity was then quantified under standard assay conditions. Organic Solvent Stability of the Enzyme. In this study, the conversion of PC to PS was performed in biphasic systems consisting of water-insoluble organic solvent. Diethyl ether was used as the organic solvent for the production of PS by dPLD as our previous studies demonstrated its suitability for sPLD-mediated reactions (data
MATERIALS AND METHODS
Chemicals and Enzymes. Choline oxidase (EC 1.1.3.17 from Alcaligenes sp.), peroxidase (EC 1.11.1.7 from horseradish), PC, PS, and PA were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). DNA modifying enzymes, such as restriction endonucleases, T4 DNA ligase, and La Tap DNA polymerase were purchased from Takara Co. (Dalian, China). Strains, Media, and Vectors. S. chromof uscus AS 4.331 used for amplifying the PLD gene was purchased from China General Microbiological Culture Collection Center and was used to amplify the PLD gene. E. coli DH5α [F‑ φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 λ‑ thi‑1 gyrA96 relA1] was purchased from Takara Biotechnology Co. Ltd. and used as a host for general gene manipulation. P. pastoris GS115 [ his4 ] and the pKFS vector were kind gifts from Professor Ying Lin (South China University of Technology). P. pastoris GS115 was employed as a host for the expression of the yeast cell display vector. Yeast extract− peptone−dextrose (YPD), buffered glycerol complex (BMGY), and buffered minimal methanol (BMMY) media were prepared according to the instructions of the Original Pichia Expression Kit (Invitrogen, USA). The FS anchor sequence was amplified and inserted into the vector pPIC9K without signal peptide, resulting in recombinant plasmid designated pKFS. The pKFS vector was employed as the yeast cell surface display vector. Heterologous PLD was displayed on the P. pastoris cell surface by fusion with FS via the N-terminal anchor system, which adhered to the cell surface via noncovalent interactions with the mannan chain of the cell wall.22 Construction of the PLD Expression Plasmid. The PLD gene was amplified from genomic DNA of S. chromof uscus using the following primers: P1 5′-ccgacgcgttacccatacgacgtcccagactacgctgccgaccaggcgcccgccttcct-3′ and P2 5′-ccggaattccctacacggggtcgtaggtgcgc-3′. (Recognition sites of MluI and EcoRI are underlined, and the HA tag sequence is shown in italics.) The amplified fragment was digested with MluI and EcoRI and subsequently cloned into the pKFS vector to generate the plasmid pKFS-pld. To construct the codon-optimized PLD gene (pldh), the rare codon used in the PLD sequence was replaced according to the preferred codon usage of P. pastoris. The synthesized pldh (Sangon Co., Ltd., Shanghai, China) was cloned into the pKFS vector to generate the plasmid pKFS-pldh. All plasmids were confirmed by sequencing. Yeast Transformation of pKFS-pld and pKFS-pldh. Recombinant plasmids (pKFS-pld and pKFS-pldh) were transformed into P. pastoris GS115 cells using electroporation according to the instructions provided with the Original Pichia Expression Kit. The pKFS vector without the PLD gene insertion was transformed into P. pastoris GS115 (GS115/pKFS) as control. Transformation was performed according to the manufacturer’s instructions. Ten micrograms of plasmid DNA linearized by SalI was mixed with 80 μL of electrocompetent cells and subjected to electroporation at 1500 V (25 μF, 5 ms, BTX ECM399). Next, 1 mL of 1 M ice-cold sorbitol was added to the cuvette immediately after pulse discharge. Subsequently, 200 μL aliquots were plated in MD medium containing 13.4 g/L yeast nitrogen base (YNB) without amino acids, 20 g/L glucose, 4 × 10−4 g/ 5355
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Figure 1. Schematic diagram of (a) plasmid pKFS-pld and (b) plasmid pKFS-pldh encoding the PLD protein. not shown). To determine the effect of diethyl ether on the activity of dPLD, 5 mL of the enzyme preparation (containing 40 U of dPLD) was added to 5 mL of diethyl ether in a final concentration of 50% and incubated at 37 °C for specific times at a rotor speed of 200 rpm. The residual enzymatic activity of dPLD was then determined under standard assay conditions. Conversion of PC to PS. To obtain the optimum dPLD-catalyzed transphosphatidylation conditions, the reaction was carried out in biphasic systems with various volume ratios between buffer and organic solvent (2.0:1.0, 1.5:1.0, 1.0:1.0, 1.0:1.5, 1.0:2.0), in which PC (0.05, 0.1, 0.15, 0.2, 0.25, 0.3 M) was dissolved in the organic solvent phase (diethyl ether), whereas dPLD (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 U/ mL) and serine (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 M) were dissolved in 0.2 M sodium acetate buffer phase (containing 0.1 M CaCl2). The mixture was stirred vigorously at different reaction temperatures (20, 30, 40, 50, 60, 70 °C) and pH values (3.5, 4.0, 4.5, 5.0, 5.5, 6.0) for 6, 8, 10, 12, 14, or 16 h with different rotor speeds (200, 300, 400, 500, 600, 700 rpm) to obtain a homogeneous emulsion. The optimum sPLD (from S. chromof uscus)-catalyzed transphosphatidylation conditions was obtained as control. The amount of biotransformation reaction products was determined using HPLC. The transphosphatidylation conversion rate (%) of PC to PS was defined as the percentage of PS obtained compared with the initial PC. Recycling of the dPLD. The repeated use of dPLD was investigated on the basis of the optimal reaction conditions. After one cycle of reaction finished, The yeast cells were harvested, washed with 0.2 M sodium acetate buffer (pH 4.5), and used for the next batch under the same conditions. Recyclability of dPLD was evaluated by the conversion rate (%) of PS in each batch. High-Performance Liquid Chromatography (HPLC) Analysis. The phospholipids PC, PS, and PA in the reaction mixture were analyzed via high-pressure liquid chromatography using an Agilent 1200 Infinity HPLC (Palo Alto, CA, USA) equipped with an Agilent 1260 Infinity diode array detector (wavelength = 205 nm). The column (purchased from Waters, Milford, MA, USA) was a μPorasil 10 μm 125 Å (3.9 × 300 mm), which was maintained at 25 °C. The eluting solvent was acetonitrile/methanol/85% phosphoric acid (100:8:0.8, v/v) with the flow rate of 1.0 mL/min. Prior to sampling, the agitation was stopped, and the reaction mixture was left undisturbed until the two phases were completely separated. After solvent evaporation, the fraction residue was redissolved in normal hexane/isopropanol (70:30, v/v), and 10 μL of a solution containing phospholipids was directly injected into the HPLC column. The relative concentrations of phospholipids were estimated from the peak area of the integrator. Each phospholipid peak was determined via the elution retention time using a standard phospholipid solution.
condon usage of P. pastoris. It has been shown that a correlation existed between the codon bias of a gene and its expression levels in a given organism. Codon optimization has been shown to be effective for maximizing the production of heterologous proteins in yeast.24,25 Thus, consistent with the codon usage frequency of P. pastoris using the statistical method, the pld codons were optimized. The synthesized 1533 bp pldh showed 74% homology with the wild-type pld. Compared with the pld, the synthesized pldh contains 404 nucleotide variations, involving 396 of 511 codons. Meanwhile, the overall GC content of the synthesized pldh was reduced to 49.38 from 72.02%. Construction of Yeast Strains for the Cell Surface Display of PLD. Yeast surface display is a powerful technique that is used to engineer proteins with enhanced affinity, specificity, stability, and catalytic activity. Several studies have successfully employed engineered yeast cells to display lipases on their surfaces as biocatalysts such as C. antarctica lipase B, Rhizopus oryzae lipase, and Rhizomucor miehei lipase.22,26,27 In this study, the yeast surface display system of P. pastoris was employed to immobilize PLD. The pld and pldh were ligated into the expression vector pKFS of P. pastoris to generate the plasmids pKFS-pld and pKFS-pldh (Figure 1). The recombinant plasmids were then linearized by SalI and electroporated into P. pastoris GS115 to obtain the pKFSpld/GS115 and pKFS-pldh/GS115, in which the recombinant PLD was displayed on the yeast cell surface by FS and its secretion signal peptide. Localization of dPLD in P. pastoris and dPLD Activity Assay. To examine whether the dPLD was indeed displayed on the surface of P. pastoris, the cells that displayed PLD on GS115/pKFS-pld and GS115/pKFS-pldh were examined by immunofluorescence microscopy. As shown in Figure 2, the cells containing the recombinant vectors pKFS-pld and pKFSpldh were labeled with green fluorescence on the surface, whereas the control cells containing pKFS were not fluorescent. This observation confirmed that dPLD was successfully displayed on the cell surface of the yeast. Following induction, the activity of the P. pastoris cells that displayed PLD on GS115/pKFS-pld and GS115/pKFS-pldh and the culture supernatant were examined. As shown in Figure 3, the activity of PLD displayed on GS115/pKFS-pldh was higher compared to the PLD displayed on GS115/pKFS-pld after induction, whereas the activity of the culture supernatant was negligible. Meanwhile, the activity of PLD displayed on
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RESULTS AND DISCUSSION Comparison of Wild-Type pld and pldh. The S. chromof uscus PLD possesses unusual codons compared with 5356
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Figure 2. Immunofluorescent labeling of PLD on the cell surface of Pichia pastoris cells harboring the plasmids pKFS-pld and pKFS-pldh. The cells were immunologically labeled with the anti-HA antibody as the first antibody and FITC-conjugated anti-IgG as the second antibody. Control cells (GS115/pKFS) showed no fluorescence.
Figure 4. (a) Effects of temperature on PLD. Influence of temperature on dPLD and sPLD activity was investigated by assaying the activity from 30 to 65 °C in 50 mM sodium acetate buffer (pH 8.0). (b) Effects of temperature on PLD stability. Thermostability of the dPLD and sPLD was studied by incubation at various temperatures ranging from 20 to 65 °C at pH 8.0 for 1 h. The residual activity was then measured under standard conditions. The data points represent the mean ± SD of three replicates.
incubation at 65 °C, the sPLD had no detectable activity, whereas the remaining dPLD activity was 63.0%. On the basis of these results, dPLD was active over a broader temperature ranges and had a higher thermostability than sPLD, which is beneficial to industrial applications. The activity of dPLD and sPLD at different pH values is shown in Figure 5a. The sPLD exhibited an optimal pH value at pH 7.0, whereas the dPLD had a maximum activity at pH 6.0. Interestingly, the dPLD maintained high activity at all pH ranges studied, whereas the sPLD showed dramatically decreased activity by moving 1 pH unit away from the optimal pH value. This might be related to the higher stability of the dPLD. Importantly, the yeast surface display also greatly enhanced the stability of the enzyme at the highest pH (11.0) and lowest pH (4.0) values studied (Figure 5b). Although the sPLD could not withstand incubation at pH 4.0 for 1 h, the dPLD retained >70% activity. These results suggested that the pH stability of the dPLD was significantly improved by immobilization on the yeast cell surface. Thus, dPLD showed the property of acid stability, which might serve as a basis for further application in industry. Tolerance of PLD Activity to Organic Solvents. The synthesis of PS by PLD is usually performed in biphasic systems consisting of water-insoluble diethyl ether. The tolerance of PLD to diethyl ether was investigated by determining the stability of the enzyme in diethyl ether at 37 °C for 60 h. The activity of sPLD showed a rapid decline, whereas that of dPLD was maintained. Compared with the
Figure 3. Activity of dPLD expressed on the surface of recombinant Pichia pastoris harboring pKFS-pld and supernatant at different induction times. Activity of dPLD expressed on the surface of recombinant Pichia pastoris harboring pKFS-pldh and supernatant at different induction times. The activity of dPLD on the surface of P. pastoris and supernatant was determined by sampling 10 mL of the fermentation broth and assaying the activity of cells and supernatant. The data points represent the mean ± SD (error bars) of three independent experiments.
GS115/pKFS-pldh reached a maximum of 88.4 U/g after 144 h of culture, which was 2.8 times that of PLD displayed on GS115/pKFS-pld. These results demonstrated that the PLD was all firmly immobilized on the yeast cell surface and the production of PLD was improved by codon optimization of pld. Characterization of the dPLD. The enzymatic characteristics of dPLD were determined and compared with those of sPLD from S. chromof uscus. The optimum temperature for activity was increased after the enzyme was displayed on the yeast surface. The sPLD showed the highest activity at 50 °C, whereas the dPLD showed a maximum activity at 55 °C. At a temperature of 65 °C, the sPLD was nearly inactive, whereas the dPLD maintained >90% of its optimal activity (Figure 4a). The stability of the enzyme was determined by incubating the enzyme at various temperatures, ranging from 20 to 65 °C, for 1 h. As shown in Figure 4b, the residual enzyme activity of the dPLD was 72.3% after incubation at 60 °C for 1 h, whereas the sPLD lost >80% of its initial activity. Moreover, after 1 h of 5357
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enhanced, which was beneficial to the application of PLD in the synthesis of PS. Conversion of PC to PS. In this enzymatic reaction, several factors, including the volume ratios between buffer and organic solvent, PC concentrations, dPLD concentrations, serine concentrations, pH value, reaction temperature, reaction time, and rotor speed, can affect PS yield. In view of this, the effects of these parameters on the reaction were assayed to maximize PS yield. The conversion rate of PC catalyzed by dPLD was compared with those of sPLD. Under optimum conditions (buffer/diethyl ether ratio 1.0:1.0, 0.1 M PC, 3.0 U/mL dPLD, 2.0 M serine, pH 4.5, 40 °C, rotor speed 600 rpm), 67.5% of PC in the reaction system could be converted by dPLD within 10 h, whereas the sPLD could convert 86.7% of PC in its optimum reaction conditions (data not shown). These results indicated that the dPLD on the surface of P. pastoris had the correct spatial configuration and could convert PC to PS with high efficiency. The dPLD had several advantages through display PLD on the yeast cell surface. By standard fermentation, dPLD could be readily produced as a yeast whole cell biocatalyst; thus, no further work was required to either purify or immobilize the enzymes. Furthermore, when compared with sPLD, dPLD exhibited favorable thermostability, acid stability, and organic solvent tolerance. Operational Stability of dPLD. In industrial applications, recycling of PLD is required to reduce the production costs. Thus, the operational stability of dPLD was examined. The dPLD whole cells were recovered by centrifugation after each batch reaction, followed by washing with 0.2 M sodium acetate buffer (pH 4.5), and then added to fresh reactants. The result showed that the conversion rate of PS was >50% after seven batches (Figure 7), indicating that the dPLD whole cell biocatalyst exhibits a relatively good operational stability and that it is particularly suitable for conversion of PC to PS.
Figure 5. (a) Effects of pH on PLD. Optimal pH was determined by assaying the dPLD and sPLD at various pH values (pH 4.0−6.0, 50 mM sodium acetate buffer; pH 7.0−8.0, 50 mM potassium phosphate buffer; pH 9.0−11.0, 50 mM Tris-HCl buffer), at a temperature of 37 °C. (b) Effects of pH on PLD stability. Influence of pH on stability of dPLD and sPLD was determined by preincubating the PLD in solutions at various pH values, ranging from 4.0 to 11.0, at 25 °C for 1 h. The residual activity was then measured under standard conditions. The data points represent the mean ± SD of three replicates.
sPLD, which retained only 12.4% of its initial activity after incubation for 30 h, dPLD could maintain 63.1% residual activity (Figure 6). After 40 h of incubation, the sPLD showed no detectable activity, whereas the remnant dPLD activity was 53.8%. When PLD was displayed on the cell surface of P. pastoris, the stability of dPLD in organic solvents was significantly
Figure 7. Investigation of repeated use of dPLD. After one cycle of reaction finished, the yeast cells were harvested, washed with 0.2 M sodium acetate buffer (pH 4.5), and used for the next batch under the same conditions. The conversion rate of PS in each detected batch (from the first to the eighth batch) was calculated. The data points represent the mean ± SD of three replicates.
The yeast cell surface display is a convenient method for preparing soluble, correctly folded recombinant proteins with native activity. Recently, several studies have successfully used yeast cells displaying recombinant proteins on their surfaces as whole cell biocatalysts, such as lipase from the fungus Geotrichum sp. for enriching eicosapentaenoic acid and docosahexaenoic acid in fish oil28 and linoleic acid isomerase
Figure 6. Effect of diethyl ether at a concentration of 50% (v/v) (5 mL of diethyl ether/5 mL of enzyme liquid) on dPLD and sPLD activities. The time course of the remaining PLD activity was assayed under previously described standard conditions. The data points represent the mean ± SD of three replicates. 5358
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from Propionibacterium acnes for bioconversion of linoleic acid to conjugated linoleic acids.29 Here we report successful PLD display on the cell surface of P. pastoris. Previous studies on the enzymatic synthesis of PS involved the use of sPLD. For instance, sPLD from Actinomadura sp. was used for PS synthesis using a bile salt mixed micelle system, achieving a transphosphatidylation conversion rate of 57% after 24 h.30 PS synthesis with the conversion rate up to 88% was also documented using the sPLD from Streptomyces racemochromogenes.31 However, PLD-associated costs are still a major limiting factor for a sPLD-based process for efficient commercial PS production. In this study, we have demonstrated that the dPLD on the surface of P. pastoris converted 67.5% of PC to PS within 10 h. Although the PS yield was relatively lower compared with some of the sPLD-based process, the conversion rate of PS remained >50% after seven batches. Moreover, the thermostability, acid stability, and organic solvent tolerance of the dPLD were significantly enhanced compared with the sPLD. Considering the relatively high cost of sPLD, PLD-displaying whole cell biocatalysts might be particularly suitable for costeffective PS production.
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AUTHOR INFORMATION
Corresponding Author
*(F.L.) Mail: College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin 300457, China. Phone: 086 + 022 + 60601958. Fax: 086 + 022 + 60602298. E-mail:
[email protected]. Funding
This work was supported by the National High-Tech Research and Development Plan (“863” Plan) (2013AA102106-07), the Tianjin Research Program of Application Foundation and Advanced Technology (14JCYBJC23800), the program for Changjiang Scholars and Innovative Research Team in University (IRT1166), and the National Natural Science Fund (31101219) of China. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Professor Ying Lin (South China University of Technology) for providing technical assistance. ABBREVIATIONS USED PLD, phospholipase D; dPLD, displayed phospholipase D; sPLD, secreted phospholipase D; PC, phosphatidylcholine; PS, phosphatidylserine; PA, phosphatidic acid; pld, phospholipase D gene; pldh, codon-optimized phospholipase D gene; YPD, yeast extract−peptone−dextrose; BMGY, buffered glycerol complex; BMMY, buffered minimal methanol; YNB, yeast nitrogen base; EDTA, ethylenediaminetetraacetic acid
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REFERENCES
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