Applied Microbiology and Biotechnology

, Volume 75, Issue 6, pp 1319–1325

Development of yeast cells displaying Candida antarctica lipase B and their application to ester synthesis reaction

Authors

  • Takanori Tanino
    • Division of Molecular Science and Material Engineering, Graduate School of Science and TechnologyKobe University
  • Takumi Ohno
    • Department of Chemical Science and Engineering, Faculty of EngineeringKobe University
  • Tohru Aoki
    • Central Research LaboratoriesDainippon Ink and Chemicals
  • Hideki Fukuda
    • Division of Molecular Science and Material Engineering, Graduate School of Science and TechnologyKobe University
    • Department of Chemical Science and Engineering, Faculty of EngineeringKobe University
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-007-0959-z

Cite this article as:
Tanino, T., Ohno, T., Aoki, T. et al. Appl Microbiol Biotechnol (2007) 75: 1319. doi:10.1007/s00253-007-0959-z

Abstract

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.

Keywords

Cell surface displayCandida antarctica lipase BEster synthesis

Introduction

Yeast cell surface display systems have been widely studied, and many heterologous proteins have been displayed on yeast cell surfaces using several anchor systems. Yeast cells displaying functional proteins and peptides on their cell surface can be used in several applications, such as the adsorption of heavy metal ions (Kuroda et al. 2001), protein isolation and purification (Kato et al. 2005), the high-through-put screening of combinatorial protein libraries (Shiraga et al. 2002), and in yeast whole-cell biocatalyst systems (Murai et al. 1998; Matsumoto et al. 2002). Yeast whole-cell biocatalysts displaying enzymes on their cell surface can be produced at a low cost and show a high enzymatic activity without permeabilization treatment. Using enzyme-displaying yeast whole-cell biocatalysts for novel reaction processes to produce chemicals or pharmaceuticals is one of the promising application areas.

Previously, our laboratory successfully demonstrated that a yeast whole-cell biocatalyst displaying the lipase from Rhizopus oryzae (ROL) with a Pro sequence (ProROL) using the Flo1p anchor system could effectively catalyze the methanolysis reaction in the solvent-free system (Matsumoto et al. 2002) and the enantioselective transesterification reaction (Matsumoto et al. 2004). ROL is one species of lipase, and the yeast whole-cell biocatalyst displaying the lipase on its cell surface has been shown to be useful for bioconversion reactions.

Among the lipases, the lipase B from Candida antarctica (CALB) is one of the most famous and versatile lipase. In many studies, CALB has successfully catalyzed many reactions, for example, the oil alcholysis reaction including biodiesel production (Torres et al. 2004; Modi et al. 2006), the kinetic resolution (Fransson et al. 2006; Lou and Zong 2006), and the ester synthesis reaction (McCabe and Taylor 2002; Larios et al. 2004). However, the high cost of the enzyme often becomes a problem in the industrial scene.

In this study, we constructed a novel yeast whole-cell biocatalyst displaying CALB on its cell surface using the Flo1p short (FS) anchor system (Matsumoto et al. 2002). Furthermore, we applied the CALB-displaying yeast whole-cell biocatalyst to the ester synthesis reaction using adipic acid and n-butanol, the model of the condensation reaction, at the relatively high temperature of 60°C.

Materials and methods

Strains and media

Escherichia coli NovaBlue (Novagen, Madison, WI, USA) was used as the host strain for recombinant DNA manipulation. Saccharomyces cerevisiae MT8-1 (MATa ade his3 leu2 trp1 ura3) was used as the host strain for the yeast cell-surface display system. E. coli was cultivated in Luria–Bertani (LB) medium [1% (w/v) tryptone, 0.5% yeast extract, and 0.5% sodium chloride] containing 100 μg/ml ampicillin. Yeast was cultivated in yeast–peptone–dextrose (YPD) medium [1% (w/v) yeast extract, 2% peptone, and 2% glucose] or SD medium [0.67% (w/v) yeast nitrogen base supplemented with appropriate amino acids and nucleotides and 2% glucose). For solid media, 2% (w/v) agar was added to the media described.

Cloning of CALB

The C. antarctica lipase B gene with a Pro region (ProCALB) was isolated from C. antarctica CBS 6678 chromosomal DNA by polymerase chain reaction (PCR) with the following primers containing a part of the sequence of the lipase B from C. antarctica LF058 (Z30645; Uppenberg et al. 1994): BglII–pmCALB (5-ATCGAGATCTGCCACTCCTTTGGTGAAGCGTCTACCTTCC-3) and CALB–XhoI (–5CGATCTCGAGTCAGGGGGTGACGATGCCGGAGCAGGTCCT-3). PCR was carried out using Pyrobest DNA polymerase (Takara Bio, Otsu, Japan). The amplified fragment was digested with BglII and XhoI and inserted between the BamHI and SalI sites of pUC19 (Takara Bio). The resulting plasmid vector was named pUC19-ProCALB CBS6678.

Substitution of amino acid residues by the site-directed mutagenesis

Figure 1a shows the scheme of the gradual substitution of amino acid residues in CALB. Four steps of substitution were carried out. The substitutions of T25A, T28S, and T31S in CALB CBS6678 (A25T, S28T, S31T, Q46G, A89T, N97R, and V286I) were achieved by PCR with two complementary mutagenic oligonucleotide primers incorporating the desired mutation using pUC19–ProCALB CBS6678 as a template. The thermal-cycle reaction mixture containing the mutated plasmid vector was treated with DpnI to digest the template plasmid pUC19–ProCALB CBS6678. The resulting mutated CALB and plasmid vector were named CALB1 (Q46G, A89T, N97R, and V286I) and pUC19–ProCALB1, respectively.
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Fig. 1

Scheme of gradual substitution of amino acids in CALB (a) and plasmid vector for yeast cell surface display of two types of CALB# (# = CBS6678, 1, 2, 3, and LF058; b). The substitutions of amino acids were accomplished by PCR with two complementary mutagenic oligonucleotide primers incorporating the desired mutations. The ProCALB# and CALB# amplified by PCR were inserted downstream of the FS anchor in pWIFS. The resulting plasmid vectors were named pWIFS–ProCALB# and pWIFS–CALB#, respectively

The gradual substitution of the amino acids at positions 46, 89, 97, and 281 in CALB was achieved in the same manner as that mentioned above, and the resulting CALBs were named CALB2 (A89T, N97R, and V286I), CALB3 (V286I), and CALB LF058. In addition, the plasmid vectors containing these CALBs were named pUC19–ProCALB2, pUC19–ProCALB3, and pUC19–ProCALB LF058, respectively.

Construction of expression plasmids

Figure 1b shows two types of CLAB# (# = CBS6678, 1, 2, 3, and LF058) that were used for the display on the yeast cell surface. ProCALB# and CALB# were amplified from pUC19–ProCALB# by PCR with the following primers: BglII–pmCALB and CALB–XhoI BglII–mCALB (5-ATCGAGATCTCTACCTTCCGGTTCGGACCCTGCCTTCTCC-3) and CALB–XhoI, respectively. The amplified fragments were digested with BglII and XhoI, and inserted between the BglII and XhoI sites of pWIFS (Matsumoto et al. 2002). The resulting plasmid vectors were named pWIFS–ProCALB# and pWIFS–CALB#, respectively.

Yeast transformation

The expression plasmids prepared as described above and pWIFSProROL, which is the plasmid to display the lipase from R. oryzae with a Pro sequence (ProROL; DQ489719; Matsumoto et al. 2002), were transformed into S. cerevisiae MT8-1 cells using Yeast Maker™ (Clontech Laboratories, Palo Alto, CA, USA) according to the protocol specified by the supplier.

Growth conditions

Transformants were preincubated in SD medium at 30°C for 16 h with shaking at 150 strokes/min, were used as starter to inoculate synthetic dextrose complete (SDC) medium [0.67% (w/v) yeast nitrogen base supplemented with appropriate amino acids and nucleotides, 0.5% glucose, and 2% Casamino acids] to give an initial optical density at 600 nm (OD600) of 0.03 and incubated at 30°C with shaking at 150 strokes/min.

Measurement of lipase hydrolytic activity

The lipase hydrolytic activity in the yeast whole cells was measured by spectrometric method using p-nitrophenyl butyrate (PNPB) as a substrate. The final concentration of PNPB in a substrate solution containing 0.5% (v/v) ethanol was 0.05 mM. The assay mixture, with a total volume of 2.1 ml, contained 1.5 ml of the substrate solution, 400 μl of 20 mM potassium phosphate buffer (pH 7.0) and 100 μl of the yeast cell suspension. Yeast cells collected from the culture were washed twice with distilled water and resuspended in distilled water. The cell density was determined by measuring the OD600 and an appropriate amount of the yeast cell suspension was used for lipase activity measurement. The assay mixture was incubated at 30°C for 10 min with shaking at 170 strokes/min, and the enzymatic reaction was stopped by adding 100 μl of 5% (w/v) trichloroacetic acid. The activity was assayed by measuring the absorbance of liberated p-nitrophenol (PNP) at 400 nm. One unit (U) of activity was defined as the amount of enzyme required to release 1 μmol PNP/min from PNPB at 30°C.

Protease accessibility assay

For whole-cell papain treatment, yeast cells collected from the culture were washed twice with distilled water and resuspended in 50 mM potassium phosphate buffer (pH 7.0) containing 2 mM l-cystein to give an OD600 of 4. The cell surface displayed CALBs were cleaved by incubating the suspension at 37°C for 3 h with papain at a final concentration of 37.4 U/ml. After the treatment, the cells were washed twice with distilled water by gentle centrifugation to remove the papain, and enzyme activity was measured as described above.

Preparation of lipase-displaying yeast cells

To prepare the lipase-displaying yeasts as a whole-cell biocatalyst, the transformants were cultivated in SDC medium at 30°C for 120 h. After cultivation, the cells were collected, washed with distilled water twice, lyophilized with FreeZone FZ-1 (Labconco, Kanzas, MO, USA) for 36 h, and sieved to homogenize cell-particle size.

Ester synthesis reaction using yeast cells displaying CALB

The reaction mixture, consisting of 450 mg (3.08 mmol) of adipic acid, 9 ml (98 mmol) of n-butanol (dehydrated), 0.36 ml of distilled water, and 150 mg of lyophilized cells, was incubated at 60°C with stirring at 400 rpm in ChemiStation PPW (Tokyourikagakukiki, Tokyo, Japan). An aliquot of the reaction mixture was centrifuged at 16,000×g and filtered with the 0.45-μm-filter unit Millex-LH (Millipore, Bedford, MA, USA) to remove the yeast cells. Subsequently, 20 μl of the reaction mixture was mixed with 980 μl of n-butanol (dehydrated). A 1.0-μl aliquot of the treated sample was injected into Shimadzu GC-2014 gas chromatograph (Shimadzu, Kyoto, Japan) connected to an InterCap 5 capillary column (0.32 × 30 mm; GL Sciences, Tokyo, Japan) for the determination of monobutyl adipate (MBA) and dibutyl adipate (DBA) concentrations in the reaction mixture. The column temperature was maintained at 120°C for 4 min, increased to 300°C at a rate of 20°C/min, and maintained at 300°C for 10 min. The temperatures of the injector and detector were set at 300 and 350°C, respectively.

Results

Sequence analysis and substitution of amino acid residues in CALB

We isolated CALB from C. antarctica CBS 6678 (CALB CBS6678) and obtained additional three types of the CALB, namely, CALB1 (Q46G, A89T, N97R, and V286I), CALB2 (A89T, N97R, and V286I) and CALB3 (V286I), by the four steps of substitution of amino acids in CALB CBS6678 (A25T, S28T, S31T, Q46G, A89T, N97R, and V286I; see “Materials and methods”).

Enzyme activity

pWIFS–ProCALB#, pWIFS–CALB#, and pWIFSProROL were transformed into S. cerevisiae MT8-1. Expression experiments were performed by flask cultivation, and the time courses of the lipase hydrolytic activity of the whole cells of transformants displaying ProCALB CBS6678 and CALB CBS6678 are shown in Fig. 2. In both transformants, the lipase hydrolytic activities of the yeast whole cells increased with cultivation time, and no detectable lipase hydrolytic activity was determined in the culture medium. The yeast whole cells displaying only mature CALB CBS6678 on the yeast cell surface exhibited a higher activity than those displaying ProCALB CBS6678. The lipase hydrolytic activity of the yeast whole cells reached a plateau in almost all the transformants displaying each type of CALB after 120 h of incubation (data not shown). Figure 3 shows a comparison of the lipase hydrolytic activities of the yeast whole cells at this time. The yeast whole cells displaying only mature CALB on the yeast cell surface exhibited a higher activity than those displaying ProCALB in all the CALBs. Furthermore, the lipase hydrolytic activity of the yeast whole cells displaying CALB CBS6678 (20.4 U/g-dry cell) was the highest of the activities of all the transformants and was higher than that of yeast whole cells displaying CALB LF058.
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Fig. 2

The time course of lipase hydrolytic activities of yeast whole cells displaying both types of CALB CBS6678. Yeast cells displaying ProCALB (diamonds) and CALB (squares) were grown at 30°C in SDC medium. The data points represent the average of three independent experiments

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Fig. 3

Comparison of the lipase hydrolytic activities of the yeast whole cells displaying ProCALB# (# = CBS6678, 1, 2, 3, and LF058), CALB#, and ProROL that were grown at 30°C in SDC medium after a 120-h incubation. Yeast cells harboring pWIFS were used as a control. The data bars represent the average of three independent experiments

Localization of CALB fusion proteins

To determine the localization of the CALB fusion proteins, a protease accessibility assay using papain as a protease was carried out. The results of the protease accessibility assay using yeast cells displaying both types of CALB CBS6678 are shown in Fig. 4. After papain treatment, the lipase activities of the yeast whole cells decreased to approximately 25% of those of the untreated yeast whole cells. These results indicate that a large part of the CALB was localized on an easily accessible part of the yeast cell surface.
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Fig. 4

Protease accessibility assay using yeast whole cells displaying both types of CALB CBS6678 at 37°C for 3 h with papain as protease. The vertical axis indicates the remaining lipase hydrolytic activity of the treated yeast whole cells (open bars), calculated relative to the lipase hydrolytic activity of untreated yeast whole cells (closed bars) that was defined as 100%. The data bars represent the average of three independent experiments

Ester synthesis reaction using yeast cells displaying CALB

The ester synthesis reaction of adipic acid and n-butanol using the lyophilized lipase displaying yeast whole cells was carried out at 60°C. The concentrations of MBA and DBA obtained from the GC analysis of the reaction mixture after 3 h of reaction are shown in Fig. 5. Significant differences were shown between the results of the reaction using the lyophilized lipase displaying yeast whole cells and those of the reaction using the control yeast whole cells (MT8-1/pWIFS cells). In the reaction mixture using the control cells, a small amount of MBA was detected, but DBA was not detected. In contrast, both MBA and DBA were detected in the reaction mixtures using the lyophilized lipase-displaying yeast whole cells. Moreover, in the reaction using the yeast whole cells displaying ProROL, the concentration of MBA was the highest among all the reactions, but the proportion of DBA in the total product was not high in relation to that in the reactions using the yeast whole cells displaying CALBs.
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Fig. 5

Comparison of lipase ester synthesis activities of yeast whole cells displaying ProCALB# (# = CBS6678, 1, 2, 3, and LF058) and CALB#. Yeast cells harboring pWIFS and PWIFSProROL (Matsumoto et al. 2002) were used as a control and ProROL displaying yeast, respectively. The ester synthesis reaction of adipic acid and n-butanol using the lyophilized lipase-displaying yeast whole cells was carried out at 60°C. MBA (open bars) and DBA (closed bars) concentrations in the reaction mixture were determined by gas chromatography. The data bars represent the average of three independent experiments

The reactions using the yeast whole cells displaying only mature CALBs produced better results than those achieved using the yeast whole cells displaying ProCALBs for each CALB (Fig. 5). In the reaction using the yeast whole cells displaying CALB1 and CALB CBS6678, concentrations of DBA in the reaction mixtures were high.

Figure 6 shows the time course of the ester synthesis reaction at 60°C using the lyophilized yeast whole cells displaying only mature CALB CBS6678. Adipic acid concentration decreased with reaction time, and MBA concentration remained almost constant after 24 h of reaction. On the other hand, DBA concentration increased with reaction time, and a large percentage of the adipic acid was converted to DBA until reaching 144 reaction hours. The lyophilized yeast whole cells displaying only mature CALB CBS6678 successfully catalyzed the ester synthesis reaction at 60°C and retained ester synthesis activity for more than 144 h.
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Fig. 6

The time course of ester synthesis reaction at 60°C using lyophilized yeast whole cells displaying CALB CBS6678. Adipic acid (circles), MBA (diamonds), and DBA (squares) concentrations in the reaction mixture were determined by gas chromatography. The data points represent the average of three independent experiments

Discussion

In the present study, we cloned CALB CBS6678 and successfully constructed CALB-displaying yeast whole-cell biocatalysts. The amino acid sequence of CALB CBS6678 was 97.8% identical to CALB LF058 (Uppenberg et al. 1994) with seven unique amino acids, A25T, S28T, S31T, Q46G, A89T, N97R, and V286I. To compare the activity of CALB CBS6678 with that of CALB LF058, we carried out four steps of substitution of the amino acids in CALB CBS6678 to obtain the amino acid sequence of CALB LF058. In addition to CALB LF058, we obtained three additional types of CALB (CALB1, CALB2, and CALB3) by substitution (Fig. 1a). We also compared the activities of these CALBs with that of CALB CBS66878 to investigate the possibility that CALB mutants show a higher activity than the two above-mentioned types of CALB. These five CALBs were displayed on the yeast cell-surface by two displaying types (with/without Pro region), and a protease accessibility assay revealed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface (Fig. 4). This accessibility is almost the same as that detected for CALB displayed on the cell surface of E. coli (Narita et al. 2006). A Western blot analysis using sodium dodecyl sulfate (SDS)-extracted fractions of cells displaying a CALB derivative [fused with the FLAG peptide tag (DYKDDDDK) at the N terminus] revealed that the fusion proteins are noncovalently attached to the cell wall and highly glycosylated (data not shown).

The lipase hydrolytic activity of the yeast whole cells increased with cultivation time and varied with the displaying type (Fig. 2). A comparison of the lipase hydrolytic activities of the yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) in all CALBs after 120 h cultivation revealed that only mature CALB is preferable for yeast cell-surface display using the Flo1p anchor system (Fig. 3). The Pro region contains the KEX2 protease site (-2K-1R), and this might have an effect on the post-translational modification of the CALB molecule during the transport to the cell surface and the lipase hydrolytic activity of yeast whole cells.

The lipase hydrolytic activity of the yeast whole cells displaying CALB CBS6678 (20.4 U/g-dry cell) was the highest of the activities of all the transformants and was higher than that of yeast whole cells displaying CALB LF058. This result suggests that CALB CBS6678 is superior to CALB LF058 for the display of CALB on the yeast cell surface using the Flo1p anchor system. CALB CBS6678 contains three unique sites (A25T, S28T, and S31T) compared with other CALBs; however, these do not exist in important secondary structures (α-helix and β-strand) in CALB (Uppenberg et al. 1994). These sites are located near the N terminus of CALB, which was fused to the Flo1p anchor; a structural change of the N terminus of CALB attributed to these three unique amino acids might have affected the activity of CALB displayed on the yeast cell surface. The mechanism of this phenomenon is still to be investigated.

The yeast whole cells displaying CALBs and ProROL were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. In analogy, with the measurement of lipase hydrolytic activity, the yeast whole cells displaying only mature CALB on the yeast cell surface also exhibited a higher ester synthesis activity than those displaying ProCALB for all the CALBs, and although the ratio of hydrolytic activity to ester synthesis activity is a little different, the tendencies in hydrolytic and ester synthesis activities between each CALB are almost the same (Fig. 5). Moreover, the yeast whole cells displaying CALB CBS6678 exhibited a higher ester synthesis activity than those displaying CALB LF058, and this result also indicates that CALB CBS6678 is superior to CALB LF058 for the display of CALB on yeast cell surface using the Flo1p anchor system. The monoester synthesis activity of the yeast whole cells displaying only mature CALB CBS6678 was lower than that of the yeast whole cells displaying ProROL; however, the diester synthesis activity of the former was almost threefold over that of the latter. These results indicate that a function of mature CALB CBA6678 molecule, efficiently catalyzing the ester synthesis reaction, was shown to occur even the enzyme was displayed on the yeast cell surface. These differences between CALB CBS6678 and ProROL could enable lipase-displaying yeast whole cells to become more efficient in the various application areas.

As shown in Fig. 6, the DBA concentration increased with reaction time until 144 h; this indicated that mature CALB CBS6678 displayed on the yeast cell surface retained activity under the reaction condition, which was a relatively high temperature (60°C) and contained alcohol, for at least 144 h. The enzyme stability of the yeast whole cell displaying CALB is favorable for use in bioconversion processes. These results indicated that the yeast whole cell displaying CALB can be used for condensation reactions. However, the MBA concentration remained almost constant after adipic acid was almost completely consumed. This indicates that the hydrolysis and synthesis of DBA reached an equilibrium state and that the control of the water activity of the reaction might be necessary.

To conclude, we constructed CALB-displaying yeast whole cells that exhibited favorable enzymatic activity and stability under the relatively severe reaction conditions. Further studies to improve the activity and productivity of yeast whole cells displaying CALB are under investigation.

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© Springer-Verlag 2007