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Applied and Environmental Microbiology, March 2006, p. 1873-1877, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1873-1877.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Engineering of Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae for Improved Cyclodextrin Production

Zhankun Wang, Qingsheng Qi,^* and Peng George Wang

The State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong 250100, People's Republic of China

Received 17 August 2005/ Accepted 18 December 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cyclodextrins (CDs), which have a hydrophilic outside and a hydrophobic central cavity, are used in the food, pharmaceutical, chemical, cosmetic, and agricultural industries because they are able to form inclusion complexes with a wide variety of hydrophobic compounds by partially encapsulating them in their apolar cavities (29, 33). CDs are cyclic -D-(1,4)-linked D-glucose oligosaccharides that consist of six, seven, and eight glycosyl units, known as -, ß-, and -CDs, respectively, and they are produced by the action of 1,4--D-glucan 4--D-(1,4--D-glucano)transferase or cyclodextrin glucanotransferase (CGTase) (EC 2.4.1.19) (26, 31).

The production of CDs by CGTase is an important industrial process, and it has been reviewed by many authors (2, 12, 26, 28). The CD production process is normally divided into two processes. The solvent process that is commonly used requires an organic complexing reagent to precipitate a specific CD selectively and to obtain one main product, while the nonsolvent process does not require a complexing reagent and results in a mixture of CDs. The addition of debranching enzymes (e.g., pullulanases and isoamylases) before the actual enzymatic CD production process can increase the yield by 4 to 6% (4, 7, 27). CDs are also produced by immobilized CGTase, which enables reuse of the enzyme (30). However, efficient CD production using whole-cell biotransformation has not been achieved so far. The composition of the CD products obtained by a CGTase synthesis reaction is determined primarily by the type of the enzyme employed and can be manipulated by addition of complexing agents or organic solvents to the reaction mixture (3, 27).

CGTases catalyze transglycosylation reactions, which include cyclization, coupling, and disproportionation reactions, as well as a hydrolysis reaction (10, 11, 18, 31, 34). The disproportionation and hydrolytic reactions produce glucose and maltose besides the oligosaccharides. Glucose and maltose are efficient acceptors of the CGTase coupling reaction, and they react with CDs to form linear oligosaccharides and thus affect the formation of CDs. Lima et al. (20) proposed a CD production process in which the CGTase synthesis reaction is combined with yeast fermentation. The yeast consumes the glucose and maltose, which are produced during CD production by CGTase, while the ethanol produced by the yeast increases the yield of CDs by forming inclusion complexes with CDs, which are inhibitors of CGTase (19). CGTases have also been employed to produce high yields of large-ring CDs by adjusting the reaction conditions to prevent conversion of these CDs to small CDs (25, 32).

In this study, we set up a novel system in which CGTase was anchored on the surface of Saccharomyces cerevisiae and was used as an immobilized enzyme. The yeast with CGTase on the surface could utilize starch. The final ratio for conversion of CDs from starch was also increased because of removal of glucose and maltose by the yeast.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains and media.
Escherichia coli DH5 (lacZM15 hsdR recA), obtained from Gibco-BRL Life Technology, was used as a host for recombinant DNA manipulation. S. cerevisiae EBY-100 was purchased from Invitrogen Life Technology. E. coli was grown in LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride), and S. cerevisiae was grown either in YPD medium (1% yeast extract, 2% polypeptone, 2% glucose), on minimal dextrose plates (0.67% yeast nitrogen base without amino acids, 2% glucose, 0.01% leucine, 0.01% tryptophan, 1.5% agar), or in YNB-CAA medium (0.67% yeast nitrogen base without amino acids, 0.5% Casamino Acids, 2% glucose or galactose).

Construction of the recombinant plasmid.
The DNA sequence encoding cyclodextrin glucanotransferase from Bacillus circulans 251 was amplified by PCR using Taq DNA polymerase and the following primers: CERP (5'-GCGAAT TCATGAAGAAATTTCTGAAATCG-3') and CPEP (5'-AGTCTCGAGTGG CTGCCAATTCACGTTAAT-3') (15). A 2.1-kb DNA fragment encoding cyclodextrin glucanotransferase, verified by electrophoresis, was obtained. The PCR products and plasmid vector pYD1 (Invitrogen), harboring a gene segment encoding a fusion protein, were digested with endonucleases EcoRI and XhoI. The digested products were purified and ligated with T4 DNA ligase to construct recombinant plasmid pYD/cgt for yeast surface display. The identity of the recombinant plasmid was confirmed by restriction enzyme digestion and electrophoresis. The recombinant plasmid was then transformed into S. cerevisiae EBY-100 cells by the electroporation method (6).

Expression of the target cgt gene.
The yeast harboring the pYD/cgt plasmid was incubated in 20 ml YNB-CAA medium containing 2% glucose for about 24 h at 30°C until the optical density at 600 nm was about 2 to 5. Cells were harvested and washed with phosphate-buffered saline (PBS) (10 mM, pH 7.5) and then were resuspended in YNB-CAA medium containing 2% galactose to an optical density at 600 nm of 1. The resuspended cells were then cultivated at 20°C with shaking for 36 h.

Immunofluorescence microscopy.
Immunofluorescence microscopy was performed as reported previously, with some modifications (21). Primary antibody against V5 at dilution of 1:5,000 was incubated with cells at room temperature for 30 min. After the cells were washed three times with PBS (10 mM, pH 7.5), the second antibody, fluorescent isothiocyanate-conjugated goat anti-mouse immunoglobulin G at a dilution of 1:500, was reacted with the cells at room temperature for 1 h. After washing, the cells were observed under a microscope.

Release of CGTase from immobilized yeast cell surface.
The Aga2-CGTase fusion protein was released from the cell wall by treatment with 100 µM dithiothreitol (DTT). The surface-engineered cells were washed with PBS (10 mM, pH 7.5) and resuspended in 10 mM PBS (pH 7.5) containing 100 µM DDT. The mixture was then incubated for 2 h at 30°C. The released soluble protein supernatant was obtained by centrifugation (4°C, 1,700 x g). Then the released soluble protein was dialyzed and concentrated with a freeze dryer.

CGTase activity assay.
The iodine-starch colorimetric method was used for the assay for amylolytic activity of CGTase, with some modifications. The surface-engineered cells or soluble proteins released by DTT were incubated with a 0.5% potato starch solution in 100 mM sodium phosphate (pH 9.0) at 50°C. At regular intervals, samples were taken and boiled for 10 min to end the reaction. After centrifugation (10,000 x g, 3 min), 1 ml of supernatant was added to 1 ml of an iodine solution (0.3567 g/liter potassium iodate, 4.5 g/liter potassium iodide, 0.9% HCl) and 6.2 ml distilled water. The control was added to 1 ml of a 0.5% potato starch solution in 100 mM sodium phosphate (pH 9.0) and 6.2 ml distilled water. The absorption at 660 nm was determined. The amylolytic activity was determined as follows: (AB – AU)/AB · 800, where AB is the absorption of the control and AU is the absorption of the sample. One unit of amylolytic activity was defined as the amount of enzyme that hydrolyzed 10 µg starch at 50°C in 15 min.

The cyclization activity of CGTase was determined based on the formation of a CD complex with phenolphthalein that resulted in a stable colorless compound (25). The substrate was incubated in 50 mM phosphate buffer (pH 6.0). Samples (100 µl) were mixed with 100 µl phenolphthalein in 800 µl sodium hydroxide (0.03 M). The stable colorless complex was detected by photometric measurement at 550 nm. One unit of activity was defined as the amount of enzyme that produced 1 µmol of ß-cyclodextrin/min.

Determination of CDs by HPLC.
The amounts of -, ß-, and -CDs were determined by high-performance liquid chromatography (HPLC), using a refractive index detector and a 15-cm Shim-peak CLC-NH₂ 6 µm column (Shimadzu Scientific Instruments, Inc., Maryland). Samples (1 ml) were taken at regular intervals (12 h) and boiled for 5 min. The CDs in the supernatant were obtained by centrifugation (10,000 x g, 30 min) and filtration. CDs were eluted with acetonitrile-water (60:40, vol/vol) at a flow rate of 0.5 ml/min. Standard CDs at concentration of 10 µmol/liter were dissolved in the same buffer and analyzed under the same conditions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of the surface display plasmid.
A plasmid containing the CGTase gene was constructed as described in Materials and Methods; in this plasmid the CGTase gene was fused with the Aga2 gene to allow secretion and display of the protein of interest. At the end of the target gene, a V5 epitope gene and a polyhistidine-tagged nucleotide sequence were also connected for detection and possible purification of the displayed protein. The resulting construct was introduced into S. cerevisiae EBY-100 by electroporation.

Expression of CGTase on the surface of yeast.
To detect expression of the protein on the cell surface, immunofluorescence microscopy with anti-V5 antibody was performed (Fig. 1). Cells expressing the CGTase-a-agglutinin fusion protein were clearly labeled, although not all the cells were labeled equally intensively. This indicated that the expressed CGTase-a-agglutinin fusion proteins were successfully anchored on the cell wall of the yeast. The intensity of the labeling varied from cell to cell, probably due to the different levels of expression in the individual cells.

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FIG. 1. Immunofluorescent labeling of surface-engineered S. cerevisiae EBY-100 containing pYD1/cgt (left panel) and control yeast S. cerevisiae EBY-100 with pYD1 (right panel).

Functional expression of CGTase on the cell surface was also detected by incubation of the engineered yeast on YNB-CAA (galactose-induced) agar plates supplemented with 2% soluble starch (Fig. 2). A halo was observed after iodine staining around yeast colonies harboring pYD/cgt but not around the control harboring pYD1 after 4 days of incubation, indicating that the CGTase was functionally anchored on the cell surface and gave the yeast the ability to hydrolyze starch.

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FIG. 2. Photographs of normal yeast and engineered yeast colonies incubated on YNB-CAA (galactose-induced) agar plates supplemented with 2% soluble starch for 4 days. (Left panel) S. cerevisiae EBY-100(pYD1); (right panel) S. cerevisiae EBY-100 containing pYD1/cgt.

Properties of the surface-engineered CGTase.
The optimum temperature for the immobilized CGTase was 50°C, which is similar to the optimum temperature for its wild-type counterpart (Fig. 3, top panel). However, CGTase anchored on the surface of yeast had a wider pH range (Fig. 3, bottom panel) (the optimum pH was 9, and the enzyme was quite stable from pH 6 to pH 11) than wild-type CGTase from B. circulans 251, whose optimal pH is 6 and whose activity decreases significantly at a higher or lower pH (10).

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FIG. 3. Effects of temperature (top panel) and pH (bottom panel) on the activity of surface-engineered CGTase. CD synthesis activity was measured at various temperatures or pHs (acetate, Tris-HCl, and Gly-NaOH buffer were used at different pHs). One milliliter of engineered cells (about 0.04 ± 0.002 g [wet weight]) or an equivalent amount of soluble protein was used for analysis. The results are expressed as relative activity. The activity under optimized conditions was defined as 100%. Each experiment was performed in triplicate. The CD synthesis activity of released CGTase was measured under the same conditions synchronously and was compared to the activity of surface-engineered CGTase. , surface-engineered CGTase; •, released CGTase.

The performance and product specificity of the immobilized CGTase were studied by incubation with 4% soluble starch (Table 1). At the initial stage of the reaction, the ratio of -CD to ß-CD to -CD was 1:20:3.7. However, with extended incubation, the product ratio changed to 1:58:0.67, which indicated that the ß-CD content was 75%. Compared to the ratio of the products produced by DTT-solublized CGTase after the same incubation time, the ß-CD content increased from 64% to 74%, but the -CD content decreased from 30% to 13%. The ratio of -CD to ß-CD to -CD for wild-type CGTase from B. circulans 251 CGTase (20) was 1:4.8:1.8, indicating that the -CD, ß-CD, and -CD contents were 13%, 64%, and 23%, respectively.

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TABLE 1. Product ratio for surface-engineered CGTasea

Characterization of the CGTase released from the cell surface.
To further characterize the activity of the CGTase immobilized on the cell surface, the cyclization and amylolytic activities of immobilized and free CGTase released from cell surface by DTT were compared (Table 2). The CGTase immobilized on the yeast surface had a cyclization activity of 1.88 U/ml, while the free enzyme released from the yeast surface had a cyclization activity of 3.3 U/ml, which is almost twice that of the CGTase immobilized on the cell surface. At the same time the amylolytic activity of the CGTase also changed from 112.98 U/ml to 278 U/ml due to DTT release. The optimum temperature and optimum pH for the released CGTase were similar to the values for immobilized CGTase, but they changed substantially when the incubation conditions changed (Fig. 3).

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TABLE 2. Comparison of the activities of immobilized CGTase and the released soluble enzymea

Fermentation of yeast with surface-engineered CGTase.
To determine the cyclodextrin production during fermentation with the surface-engineered yeast, the induced cells were resuspended in YNB-starch medium (30°C, pH 6.0). At the same time, a soluble enzyme released from the same volume of engineered yeast was used as a control (Fig. 4). At the initial stage of incubation, faster cyclodextrin production by the soluble enzyme than by the immobilized enzyme was observed. However, after 28 h, the cyclodextrin production by the soluble enzyme ceased, and the amount of cyclodextrin at this time was 18.6 mg/ml, which represented a starch conversion ratio of 46.5%; while cyclodextrin production by the surface-engineered cells continued until day 4, the total production of cyclodextrin by surface-engineered cells was 24.07 mg/ml, which indicated that the starch conversion ratio was more than 60%.

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FIG. 4. Time course analysis of cyclodextrin production by surface-engineered CGTase and soluble CGTase released by DTT. CDs were produced at 30°C and pH 6. The CDs produced were analyzed by HPLC as described in the text. All values are the averages of two separate experiments. , surface-engineered CGTase; •, released CGTase.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cyclodextrin production by CGTase is a starch-utilizing process (8). During this process, CGTase digests and cyclizes the starch to produce CDs, as well as glucose and other oligosaccharides. Glucose and maltose, which are products of this cyclization reaction, affect the formation of CDs by the CGTase coupling reaction and thus limit the ratio of conversion of starch to CDs in the end. Lima et al. suggested a cofermentation system in which CGTase was incubated with yeast to overcome this problem (20). The overall CD production was increased by cofermentation with yeast. However, this production process also made the downstream CD production process more complicated. Here, we anchored the CGTase on the surface of yeast, and the enzyme could be used as an immobilized enzyme. The anchoring and immobilization of CGTase avoided isolation and purification of the protein. Furthermore, the surface-engineered yeast had direct starch-utilizing ability, while wild-type S. cerevisiae itself did not.

The method of surface engineering was initially described as an example of phage display. The development of this phage display method has facilitated isolation of specific ligands, antigens, and antibodies from a complex library (5). Bacterial and yeast surfaces appeared to be more suitable for displaying large numbers of proteins with a practical purpose (1, 37). Surface display of functional enzymes as biocatalysts has several advantages, including ease of preparation and minimal mass transfer resistance, compared with conventional immobilized enzymes and immobilized whole-cell systems (14, 16). However, most of the engineered biocatalysts are used only for immobilization of the protein or simply provide the cell with a novel enzymatic function. Examples include expression of the cellulose binding domain on the cell surface of E. coli and expression of Rhizopus oryzae glucoamylase on the surface of S. cerevisiae to facilitate ethanol production (13, 36). CGTase has been expressed previously in E. coli and Bacillus, but purification of CGTase that was expressed in vivo was time-consuming and costly (9, 23, 24). CGTase on the outer membrane of E. coli was shown to be inactive because the substrate could not access the active center of CGTase (35). In our study, CGTase was anchored on the yeast surface through a-agglutinin. a- Agglutinin consists of a core subunit, which is encoded by AGA1 and is located on the cell wall, and is linked to a small binding subunit encoded by AGA2. CGTase and the Aga2 protein are connected to the Aga1 protein through disulfide bonds, which indicates that there is a relatively long distance between CGTase and the cell wall. Expression of CGTase on the cell surface of S. cerevisiae not only provided the yeast with starch-utilizing activity but also improved the production of CDs.

The formation of a complex between ethanol and cyclodextrin may increase the production of cyclodextrin. This has been proven by Blackwood and Bucke (3) and Lee and Kim (17). The continuous removal of cyclodextrins by precipitation of CD-ethanol complexes from the reaction system shifts the equilibrium in favor of CD production; on the other hand, the removal of CD-ethanol complexes may reduce the intermolecular transglycosylation reaction that causes degradation of CD products. However, the most important effect in our immobilization system is the removal of glucose, which is an efficient acceptor in the CGTase coupling reaction. Fermentation by the yeast consumes the glucose produced by the amylolytic reaction of CGTase with the starch.

The immobilization of CGTase on the surface of yeast resulted in some differences compared to the wild-type CGTase from B. circulans 251, such as higher product specificity, wider pH adaptation, etc. The fusion of CGTase with a-agglutinin and the V5 antigen polyhistidine at the N terminus and C terminus may be responsible for this; on the other hand, analysis of the CGTase amino acid sequence revealed two potential glycosylation sites in a eukaryotic expression system. Expression of CGTase from B. macerans in S. cerevisiae has been confirmed, and there is a 38% increase in molecular mass because of the glycosylation events (22). This hyperglycosylation may significantly increase the thermal stability and pH stability. This study showed that the released CGTase also has a higher optimum pH than the wild-type CGTase from B. circulans, which indicated that the shift in the optimum pH of the immobilized CGTase was caused by hyperglycosylation and protein fusion and not by immobilization.

In conclusion, we demonstrated that the yeast surface expression system is a suitable display "stage" for CGTase that can be used for production of CDs and for analysis of a reconstructed enzyme. Compared to the inactive CGTase anchored on E. coli (35), the display of CGTase on yeast was active. The surface-displayed yeast was able to produce CDs with a higher ratio of ß-CD.

 
This project was supported by a key fund from the Chinese Education Ministry (2005).

We thank L. Dijkhuizen for providing the CGTase gene from B. circulans 251.

 
* Corresponding author. Mailing address: The State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong 250100, People's Republic of China. Phone: 86-531-88365628. Fax: 86-531-88565610. E-mail: qiqingsheng{at}sdu.edu.cn.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, March 2006, p. 1873-1877, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1873-1877.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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