Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis
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- Su, G., Huang, D., Han, S. et al. Appl Microbiol Biotechnol (2010) 86: 1493. doi:10.1007/s00253-009-2382-0
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Two alternative cell-surface display systems were developed in Pichia pastoris using the α-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins α-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with α-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with α-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using α-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with α-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.
KeywordsYeast surface displayPichia pastorisCandida antarctica lipase BFlavor ester synthesis
In recent years, considerable progress has been made in the development of expression systems that display heterologous functional proteins on the surfaces of bacteria, phage, and yeast. Yeast surface display is a powerful method for the expression of functional proteins and can be used to increase the affinity, specificity, and stability of the expressed proteins (Boder and Wittrup 1997). Major advantages of yeast surface display are that the protein-folding and secretory machineries of yeast are strikingly homologous to those of mammalian cells. In this system, foreign proteins are displayed on the yeast cell surface in the form of a fusion with an anchoring protein. Quantitative comparisons can be performed by the use of fluorescence-activated cell analysis, and this allows the effectiveness of different anchor proteins for the expression of a particular protein to be compared. Recently, some yeast cell-surface display systems were developed in S. cerevisiae. Several functional proteins were displayed on yeast cell surface in the form of fusion proteins linked to the C-terminal half of α-agglutinin and N-terminal portion of Flo1p (FS; Kondo and Ueda 2004; Ueda 2004; Kato et al. 2006). Subsequently, Kato et al. (2007) expressed Candida antarctica lipase B (CALB) using the S. cerevisiae display system by α-agglutinin anchor protein. Tanino et al. (2007) used the FS anchor protein to display CALB on the S. cerevisiae cell surface. Yeast cell-surface engineering has been used to date in various industrial applications. CALB displayed on yeast has been used to catalyze many reactions successfully, for example, the alcoholysis of oil, including biodiesel production and the synthesis of esters (Tanino et al. 2009). We have used α-agglutinin to anchor CALB on the cell surface of S. cerevisiae and applied to the synthesis of ethyl hexanoate (Han et al. 2009). However, industrial applications are often limited by the cost of enzyme and efficiency of synthesis. To improve the efficiency of yeast cell-surface display, several groups have described the use of the yeast P. pastoris for the surface display of heterologous proteins, such as Rhizopus oryzae lipase, in conjunction with a FS anchor protein (Tanino et al. 2006; Ren et al. 2007). Two groups have used α-agglutinin from S. cerevisiae to anchor their protein of interest on the cell surface of P. pastoris (Mergler et al. 2004; Wang et al. 2007). We have used α-agglutinin to anchor CALB on the cell surface of S. cerevisiae. However, there have been no previous reports of the use on the commercially available α-agglutinin and FS systems to display CALB on the cell surface of P. pastoris.
Lipases (EC 188.8.131.52) are used as efficient catalysts in numerous industries, such as the food industry, detergent production, manufacture of paper, and pharmaceutical processing (Sharma et al. 2001). Lipases catalyze the hydrolysis of triacylglycerols at the interface between water and a hydrophobic substrate. In addition, they can catalyze the synthesis of esters due to their broad substrate specificity, in particular their specificity for different lengths of fatty acid chains. CALB is one of the most important lipases because it retains its enzymatic activity in organic media and possesses high enantioselectivity towards secondary alcohols.
In this study, to prepare highly active CALB and extend its application, we have developed and compared the use of α-agglutinin and the flocculation functional domain of FS as anchor proteins in the host P. pastoris. Furthermore, we compared the reaction characteristics of CALB displayed on the cell surface of P. pastoris and S. cerevisiae, especially with respect to the synthesis of flavor esters.
Materials and methods
Strains and materials
The gene-encoding FS domain was cloned from S. cerevisiae ATCC 60715. The coding sequences for α-agglutinin and CALB were subcloned from the plasmid pICAS-CALB (Han et al. 2009). The yeast display vector pICAS and S. cerevisiae strain MT8-1 (MATa, ade, his3, leu2, trp1, ura3), which was used to display CALB, were kind gifts from Professor Mitsuyoshi Ueda (Ueda and Tanaka 2000). S. cerevisiae MT8-1 was used for transformations and was cultured in SD medium (0.67% yeast nitrogen base [YNB] without amino acids, 2% glucose, 30 mg/L Leu, 20 mg/L His, 20 mg/L adenine, and 20 mg/L uracil) at 30°C for 96 h. P. pastoris GS115 was purchased from Invitrogen (Carlsbad, CA, USA) and cultured in complete medium (YPD: 1% yeast extract, 2% peptone, 2% glucose), selective medium (MD: 1.34% YNB, 4 × 10−5% biotin, 2% dextrose, 1.5% agar), BMGY (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4 × 10−5% biotin, 1% glycerol), or BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4 × 10−5% biotin, 0.5% methanol). The vector pPIC9K was purchased from Invitrogen. A mouse anti-FLAG monoclonal antibody was obtained from Sigma (St. Louis, MO, USA). Alexa Fluor 488 conjugated goat antimouse IgG was purchased from Molecular Probes (Eugene, OR, USA).
Construction of recombinant expression vectors for P. pastoris
The coding sequences for α-agglutinin and CALB were amplified from the plasmid vector pICAS-CALB by polymerase chain reaction using the following primers: EcoRI-CALB 5′TGTAGAATTCCTGCCTTCCGGTTCGGACCCTG and NotI-α-agglutinin 5′ATTAGCGGCCGCTTAGAATAGCAGGTACGACAAAAG.
P. pastoris GS115 was transformed with pKFS-CALB and pKNS-CALB that had been linearized with SacI using a Pichia Easy-Comp Transformation Kit (Invitrogen) according to the manufacturer's instructions. The transformants were isolated by incubation at 30°C for 48 h on MD plates.
Culture conditions and assay for CALB activity
The yeast transformants were precultured in BMGY medium at 30°C for 16 h and used to inoculate 50 ml of BMGY medium in a 500-ml baffled flask at an initial OD600 value of 0.1. After incubation for 24 h, the cultures were centrifuged at 6,000×g for 10 min and resuspended in BMMY medium that contained 1.0% methanol. To maintain the induction of the fusion proteins FS-CALB and CALB-α-agglutinin, methanol was added to the culture every 24 h to the final concentration mentioned above.
The activity of CALB was determined spectrophotometrically using p-nitrophenyl butyrate (pNPB).The substrate, pNPB, was emulsified by sonication in 50 mM potassium phosphate buffer (pH 8.0) that contained 1% Triton X-100. Yeast cells were collected by centrifugation at 6,000×g at room temperature for 10 min. After the cells had been washed twice with 20 ml of 50 mM potassium phosphate buffer (pH 7.5), the cell suspension was adjusted to an OD600 of 3.0. A 100-µl aliquot of the cell suspension and an equal volume of the substrate were mixed and allowed to react at 45°C for 5 min. The reaction was stopped by the addition of 1 ml of acetone. Then the reaction mixture was centrifuged at 16,000×g at room temperature for 1 min. A 200-µl aliquot of the resulting supernatant was placed in a 96-well plate, and the activity of CALB was assayed by measuring the absorbance of liberated p-nitrophenol (pNP) at 405 nm using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The activity was measured by the absorbance of liberated pNP at 405 nm. One unit of activity was defined as the amount of enzyme required to release 1 µmol pNP/min.
Preparation of lipase-displaying yeast cells
Yeast cells that displayed the lipase were prepared for use as a whole-cell biocatalyst in the following manner. S. cerevisiae that had been transformed with pICAS-CALB, as MT8-1/pICAS-CALB, was cultured in SD medium at 30°C for 96 h, whereas P. pastoris that had been transformed with pKNS-CALB or pKFS-CALB was cultured in BMMY medium at 30°C for 120 h as described above. The cells were then collected, washed twice with distilled water, and lyophilized for 24 h.
Immunofluorescence microscopy and flow cytometer analysis
The yeast cells were analyzed by immunofluorescence microscopy according to the method of Kobori et al. (1992). Induced cells were washed twice in ice-cold water and resuspended at 4°C in phosphate-buffered saline (PBS; pH 7.4) supplemented with 1 mg/ml bovine serum albumin. Immunostaining was carried out as follows: an antibody against FLAG was used as the primary antibody at a dilution of 1:1,000 in a total volume of 1 ml. The cells were incubated with the antibody on a rotator at room temperature for 2 h. The cells were then washed with PBS (pH 7.4) and exposed to the secondary antibody, Alexa Fluor™ 488 goat antimouse IgG (H + L), which was diluted 1:300 in a total volume of 300 µl, for 1 h at room temperature. After three washing steps, the cells were examined using a fluorescence microscope (IX71, Olympus, Tokyo, Japan) or a Cell Lab Quanta™ SC Flow Cytometer (Beckman-Coulter, Fullerton, CA, USA). For the latter, a total of 10,000 cells were analyzed for each sample, and the data were analyzed using the EXP032 software (Beckman-Coulter). Cells that had been transformed with pPIC9K or pICAS were also processed in the above manner to serve as the negative controls. The ratio between the intensity of the signal obtained from CALB-displaying and control yeast cells was calculated for each individual sample. A sample was considered to be positive if this ratio was ≥2.
Synthesis of flavor esters using yeast cells displaying CALB
All the reaction media for ester synthesis were dehydrated by gentle shaking with 3 Å molecular sieves overnight before being used. In a typical experiment for ethyl hexanoate synthesis, 10 ml of n-heptane containing 4 mmol of hexanoic acid and 6 mmol of ethanol were added to a 50-ml Erlenmeyer shaking flask capped with a septum. Then, the reaction was initiated by the addition of 0.1 g of lyophilized yeast cells displaying lipase or the commercial CALB Novozyme 435 to the reaction system and the mixture was incubated at 200 rpm and 55°C for 9 h. Samples of 50 µl were withdrawn periodically and centrifuged (10,000×g, 10 min), and 20 µl of the resulting upper layer were mixed with 1 ml of n-heptane that contained n-butyl acetate for analysis by gas chromatography (Agilent 7890A), which a hydrogen flame ionization detector and a DB-FFAP silica capillary column (0.25 mm × 30 m; Agilent, Santa Clara, CA, USA) were used. The column temperature was held at 50°C for 1 min, then increased to 60°C, 68°C, 95°C, and 200°C at a rate of 10°C/min, 40°C/min, 20°C/min, and 40°C/min, respectively, and kept at each of these temperatures for 2 min. The injector and detector temperatures were both set at 250°C. The average error for this determination was <0.7%. For the synthesis of ethyl acetate and ethyl propionate, it was carried out under the same reaction conditions. All reported data were the means of experiments performed at least in triplicate.
Construction of a CALB yeast surface display system using P. pastoris
Comparison of the α-agglutinin and FS anchor proteins for the display of CALB on P. pastoris
Synthesis of flavor esters by yeast cells that displayed CALB
P. pastoris as a cellular host for the expression of recombinant proteins has provided a great potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. In recent studies (Jiang et al. 2007), it has been indicated that the fermentation characteristics of P. pastoris, which include growth on an economical carbon source and a very high density of cell culture, are superior to those of the widely used yeast S. cerevisiae. Therefore, P. pastoris is well suited to applications that require large-scale fermentations. It is important to develop a P. pastoris cell display system for practical application. Recently, several groups described the use of P. pastoris for surface display of heterologous proteins and used the S. cerevisiae α-agglutinin to anchor their protein or S. cerevisiae FS as an anchor protein. And a novel P. pastoris cell-surface display system was based on the Pir1 cell wall protein of S. cerevisiae by Wang et al. (2008). In this study, we have demonstrated that the P. pastoris for displaying CALB on cells surface gives a higher efficiency of expression with both the α-agglutinin and FS anchor proteins than that of the S. cerevisiae system. The expression amount of CALB on P. pastoris cells surface was by indirect immunofluorescent labeling using the FLAG tag and flow cytometry analysis (Fig. 5). The results of the lipase assay showed that CALB-α-agglutinin was expressed at a lower level than that of FS-CALB, 200 and 270 U/g dry cell, respectively. The activity of CALB was tenfold higher when it was displayed on P. pastoris compared with S. cerevisiae.
Many functional enzymes have been genetically immobilized on the cell surface of yeast as a whole-cell biocatalyst. The industrial use of whole-cell biocatalysts with surface-displayed lipase has grown in recent years owing to unique advantages such as improved enzyme stability, simpler product purification, and cost-effective downstream processing. CALB has been displayed on S. cerevisiae cells surface as a whole-cell biocatalyst and used to synthesis reaction (Tanino et al. 2007, 2009). We applied P. pastoris displaying CALB as a whole-cell biocatalyst to catalyze the esterification of short-chain flavor esters in n-heptane. When the synthesis of ethyl hexanoate was analyzed using the yeast as whole-cell biocatalysts, the synthetic activity of CALB that was fused with α-agglutinin and displayed on P. pastoris was threefold higher than that of CALB fused with FS. In addition, the activity of the former was close to that of the commercial CALB Novozyme 435 (Fig. 6). The same results were obtained for the synthesis of other flavor esters under the same reaction conditions. Interestingly, for the catalytic synthesis reaction of some short-chain esters, the synthetic activity of P. pastoris that displayed CALB fused with α-agglutinin surpassed that of Novozyme 435 (Fig. 7). A number of explanations for this behavior in a non-aqueous system have been reported and include an increase in the stability of the enzyme structure and the improved solubility of lipophilic substrates and products. For example, Schilke and Kelly (2008) used long hydrophobic linkers to immobilize CALB and thus increased the activity of the enzyme in organic media. The hydrophobicity of every amino acid of the two anchor proteins was analyzed by the ExPASy ProtScale tool (http://www.expasy.org/cgi-bin/protscale.pl). These results indicated that the hydrophobicity of the α-agglutinin anchor protein was higher than that of the FS anchor protein. It may result to the hydrolytic side reactions that interfere with syntheses, which are restricted in organic media. CALB does not possess a hydrophobic “lid” covering the catalytic site, which does not exhibit “interfacial activation” caused by large structural changes. Therefore, the higher hydrophobicity of the α-agglutinin anchor protein could increase the synthetic efficiency in short-chain esters synthesis by affecting the active sites with the higher hydrophobic tethers.
In conclusion, CALB that was displayed on P. pastoris retained its original hydrolytic activity after lyophilization of the yeast and efficiently catalyzed the synthesis of flavor esters in non-aqueous media. Therefore, enzymes displayed on P. pastoris cells are expected to be effective whole-cell biocatalysts. Further studies to develop the application of yeast cells that display CALB to the synthesis of various flavor esters are underway.
This work was supported by grants from the National Natural Science Foundation of China (No. 30670053) and the Ministry of Science and Technology of the People's Republic of China (National “863” Project No. 2006AA020203).