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백 승필, 유 영제

서울대학교 응용화학부 효소 및 환경 생물공학 연구실

Development of cellobiose-utilizing yeast using surface-display of beta-glucosidase

Pack, Seung Pil, Yoo, Young Je

Enzyme and Environmental Biotechnology Laboratory

School of Chemical Engineering, Seoul National University

Introduction

Ethanol production from cellulosic materials has received increasing attention as an alternative energy source, because cellulose is the most abundant and low-cost biomass material in nature. A number of process strategies have been conducted for the efficient ethanol production from cellulose. As one of the important researches, the development of novel recombinant microbe, which can utilize cellulosic material as sole carbon source for ethanol production, has been required. Especially, Saccharomyces crevisiae, lack of the ability to utilize. cellulosic material, has been one of the most intriguing microbes, because it is known for the excellent ethanol-producing metabolism and also is a famous host strain suitable for genetic technology.

Cellobiose-utilizing Saccharomyces crevisiae has been constructed many times by the genetical introduction of heterologous gene encoding beta-glucosidase into yeast cell for secretive expression of the recombinant enzyme. However, it is still difficult to apply the recombinant Saccharomyces crevisiae to the ethanol production process because there are many genetical and physical problems in elevating and maintaining cellobioase activities during the fermentation. In order to overcome the problems, several researches has been tried. For example, chromosome-integration of heterologous gene has been researched to endow the recombinant Saccharomyces crevisiae with mitotic stability. Even still, deactivation of the secreted recombinant beta-glucosidase is main problem.

For the past several years, the expression of proteins on the surfaces of bacteriophages and bacteria has been actively studied. These systems are expected to be useful for the construction of microbial biocatalysts. To immobilize protein on the cell surface of Saccharomyces crevisiae, molecular information for the native protein anchored to the cell wall, alpha-agglutinin was utilized. Alpha-agglutinin is a mannoprotein involved in the sexual mating adhesion. Alpha-agglutinin has a glycosylphosphatidyl -inositol (GPI) anchor attachment signal, which is involved in cell wall anchoring. Therefore, the expressed fusion protein, which is genetically combined with alpha-agglutinin, is immobilized on the cell wall.

In this study, to stabilize beta-glucosidase, surface display system was newly adopted because the surface display is one of the c-terminal specific immobilization method and is expected to be effective in overcoming the recombinant protein deactivation. In order to construct novel recombinant Saccahromyces cerevisiae, which can express beta-glucosidase on the cell surface, beta-glucosidase was genetically combined with c-terminal half of alpha-agglutinin. Then, novel surface-engineered cellobiose-utilizing yeast was applied to ethanol production from cellobiose and glucose mixture.

Material and Methods

Strain and DNA

Saccharomyces cerevisiae L2612 ( leu2-3 leu2-112 ura 3-52 trp1-289 can1 cyn1 gal+) was used as a host strain for transformation. The expression cassettes of the gene consists of PGK promoter, killer toxin signal sequence, structural gene cloned from Bacillus circulans sub sp. c-termanal half gene of alpha-agglutinin and PGK transcription terminal sequence. Restriction enzyme digestion, electrophoresis, DNA ligation transformation and DNA preparation from E.coli were performed by the standard methods according to Sambrook et al..

Media and Cultivation Condition

YPD medium was composed of glucose(20 g/L),  yeast extract (10 g/L), bacto-peptone (20 g/L).  YPDC  medium was composed of glucose(10g/L), cellobiose (10g/L), yeast extract(10 g/L), bacto-peptone(20 g/L).

Batch culture of recombinant yeast was conducted in a 500 ml flask containing 100 ml medium at 30 C, 280 rpm. Seed culture was performed in a cap tube containing 5 ml YPD medium by inoculating single colony of each recombinant strains.  After cultivating for 24 hr at 30 C, 280 rpm, the seed culture was transferred to main flasks.

Isolation of cell wall

The cells were harvested by centrifugation in isolation buffer (10mM-tris-HCL, pH 7.8, 1nMPMSF). The cells, buffer, and glass beads (diameter 0.45-0.50mm) were mixed in a ratio of 1:2:1 (wet wt/vol/wt) in a glass tube and agitated vigorously, with a bench-top vortex mixer, for 5 min at 0℃.

Assays

The expressed and secreted protein was verified using SDS-Polyacrylamide gel electrophoresis.  The extracellular proteins were obtained using solvent precipitation according to Bollag and Edelstein. The proteins separated by 10% SDS-PAGE were stained with Comassie blue.

The beta-glucosidase activity was determined by measuring pNP(p-nitrophenol) concentration derived from pNPG(p-nitrophenyl -D-glucose). The reaction mixtures consisted of 450 l of 10 mM potassium phosphate buffer(pH 5.8), 50 l of 10 mM pNPG and 100 l of cultured supernatant after centrifugation. They were incubated at 45 C for 20 minutes. One unit of pNPGase activity was defined as the amount of enzyme required for releasing total reducing sugar equivalent to 1 mol of pNP per minute. The pNP concentration was measured by spectrophotometer (Pharmacia LKB - Ultraspec III) at 420 nm.

Cell growth was monitored with spectrophotometer (Pharmacia LKB - Ultraspec III) at 600 nm.  Residual glucose concentration was measured with glucose analyzer (YSI 2700, Yellow Springs, OH).  Residual cellobiose  concentration was determined  with HPLC (Waters 510 , Waters ). The  LC column was purchased from Alltech Econoshere and  RI detector (Waters 410, Waters) was used.  Ethanol concentration in the culture medium was measured with GC(Shimadzu GC-14B)-FID. The GC column was 2 feet long and 1/8 inch of diameter packed with Porapak Q(80-100 mesh). Isopropanol(10 g/L) was used as an internal standard for ethanol.

Results and Discussion

Construction of the cell surface expression plasmid

To immobilize beta-glucosidase protein on the cell wall surface of S. cerevisiae, the beta-glucosidase gene was fused with the c-terminal half of alpha-agglutinin gene (Fig. 1a). The procedure is as follows; Yeast expression plasmid pPGK-AG containing PGK promoter, alpha-agglutinin gene and PGK terminal sequence was kindly provided by Dr, Krujan. It was prepared as a vector by digestion with EcoRI and Hind III. The fragment of killer-toxin siganl sequence and beta-glucosidase gene was acquired by PCR (polymerase chain reaction) using pSK-bgl as template and inserted to the prepared pPGK-AG vector. The resulting plasmid was named as pBGL-AG (Fig.1.a). Control expression plasmid, which can secrete beta-glucosidase, was constructed by deleting alpha-agglutinin gene from pBGL-AG and named as pBGL(Fig 1.b) .

a. pBGL-AG                                b. pBGL

Fig 1. Expression vectors for surfae-display and secretion of beta-glucosidase

Transformation and Characterization

Constructed vectors, pBGL-AG and pBGL was introduced into S. cerevisiae L2612 by electroporation method. The transforments were named as L2612(pBGL-AG) and L2612(pBGL) respectively. The localization of beta-glucosidase was examined by investigating the activity of culture medium, cell pellet and cell wall fraction. The surface-displaying yeast, L2612(pBGl-AG) showed beta-glucosidase activity prevalently in cell pellet and cell wall fraction. In contrast, the secreting yeast, L2612(pBGL) showed the activity only in culture medium. The activity profile of beta-glucosidase were compared between L2612(pNBGK-AG) and L2612(pBGL). The beta-glucosidase activity of L2612(pBGL-AG) was higher and more stable than that of L2612(pBGL).

The change of relative activity was investigated using crude fraction of beta-glucosidase. The resistance and stability of beta-glucosidase from both sources were compared. The cell wall fraction of L2612(pBGL-AG) showed more stable  beta-glucoidase activity than the culture medium activity of L2612(pBGL). This means that the cell-wall immobilized enzyme is more stable and resistant to the harsh medium environment than free expressed enzyme (Fig 2.).

Fig 2. Relative activity change of surface-displyed beta-glucosidase (□)and free secreted beta-glucosidase(●) in the storage of culture medium ( pH 4, 30℃ )

Cultivation in celloiose-containing media

Cells were cultivated in YPDC media, in which cellobiose is supplied as a carbon source. The cell growth, beta-glucoisdase activity, cellobiose uasge, and ethanol production was assayed (Fig 3). The cellobiose usage of L2612(pBGL-AG) shows  about 10% increase compared with L2612(pBGL).

Fig 3. Cultivation of surface-displaying yeast L2612 (pBGL-AG)

Reference

M. T, Schreuder et al. "Targeting a Heerologous Protein to he cell wall of Saccharomyces cerevisiae", Yeast, 9, 399 (1993)

M. Ueda and Atsuo Tanaka "Cell Surface Engineering of Yeast", Journal of Bioscience and Bioengineering, 90(2) 125 (2000)