Cellulosic ethanol production by combination of cellulase-displaying yeast cells
Highlights
► Two fungal and two bacterial endoglucanases were expressed on the yeast surface. ► Enzyme activities of the surface-displayed endoglucanases were analyzed. ► Ethanol was produced from PASC by consortium of cellulase-displaying cells. ► Ethanol production was optimized by adjusting the ratio of each cell type.
Introduction
Cellulosic biomass is considered as one of the most promising alternatives to fossil fuels for the production of biofuels and other useful chemicals [1]. Hydrolysis of crystalline cellulose to glucose requires the sequential reactions of three groups of cellulases, endoglucanase (EG), exoglucanase (mostly cellobiohydrolase, CBH), and β-glucosidase (BGL), which are produced in cellulolytic fungi and bacteria [2], [3]. EG randomly cleaves internal β-1,4-glycosidic bonds in the amorphous regions of crystalline cellulose. From the reaction products of EG, CBH produces cellodextrins such as cellobiose and cellotriose, which are finally hydrolyzed to glucose by BGL [4].
The high cost of cellulase is the main obstacle for the utilization of cellulosic biomass. Therefore, a great deal of effort has been made to screen and develop cellulases with high enzymatic activity and stability [5], [6]. On the other hand, consolidated bioprocessing (CBP), combining cellulase production, cellulose degradation, and fermentation in a single step, is considered as the most cost-effective way to utilize cellulosic biomass [7], [8]. One of the key requirements for cellulosic ethanol production by CBP is to develop microbial strains equipped with the properties of efficient cellulose degradation as well as ethanol production. Saccharomyces cerevisiae, having a high capacity of ethanol production, is one of the strong candidates for CBP. So far, to develop cellulolytic yeast strains, various fungal or bacterial cellulases have been expressed in yeast as secreted forms [9], [10] or displayed on the yeast surface [11], [12], [13], [14], [15]. Especially, the surface display of cellulases enables to mimic cellulosome structure found on the surface of anaerobic cellulolytic bacteria such as Clostridium thermocellum, Clostridium cellulovorans and Ruminococcus flavefaciens [16], [17]. Cellulosome is generated by assembly of multiple dockerin-containing cellulases and xylanases into a scaffoldin, an integrating protein containing cohesion domains, through interaction between dockerin and cohesion domains [18]. Such a multifunctional cellulosome structure allows highly efficient cellulose degradation through synergistic effects of various enzymes. To date, several fungal cellulases such as Trichoderma reesei EGII and CBHII, and Aspergillus aculeatus EGI and BGLI were expressed on the yeast surface [11], [12], [19]. In addition, some bacterial cellulases such as C. thermocellum CelA and BglA, and Clostridium cellulolyticum CelE and CelG were purified from Escherichia coli or secreted from yeast cells, and then attached to a chimeric scaffoldin expressed on the yeast surface [17], [20].
To generate efficient cellulolytic yeast strains, it is important to select cellulases based on their enzymatic properties when displayed on the yeast surface. However, comparative analysis of various cellulases expressed on the yeast surface has not yet been conducted. Among the three types of cellulases, EG shows the highest diversity, which might be necessary to degrade various types of cellulose in nature. Therefore, as an effort to search for enzymes suitable to develop cellulolytic yeast strains, we compared the activities of fungal and bacterial EGs expressed on the yeast surface. For fungal EGs, we selected T. reesei EGII, which has been widely studied on account of its high enzymatic activity, and EGI from a thermophilic fungus, Thermoascus aurantiacus. For bacterial EGs, we selected CelA and CelD from C. thermocellum, anaerobic and thermophilic bacteria. Except for EGII, other EGs have not yet been directly expressed on the yeast surface. Based on the characterized enzymatic properties, we chose T. aurantiacus EGI for ethanol production from PASC in combination with T. reesei CBHII and A. aculeatus BGLI. Previously, multiple cellulases were co-displayed in a single cell for ethanol production [11], [14]. However, in this study, we adapted a new strategy of combining three types of yeast cells each displaying different cellulase, which allows convenient optimization of ethanol production by adjusting the combination ratio of each cell type.
Section snippets
Strains and culture conditions
S. cerevisiae EBY100 strain (MATa GAL1-AGA1::URA3 ura3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL) containing pCTCON plasmid were grown in a selective SD-CAA medium (20 g/l dextrose, 6.7 g/l yeast nitrogen base, 5 g/l casamino acids, 5.4 g/l Na2HPO4 and 8.56 g/l NaH2PO4·H2O). For the induction of cellulase gene cloned in pCTCON vector, OD600 of 1 cells grown in SD-CAA medium were harvested and resuspended in SG-CAA medium containing galactose instead of glucose, and further incubated for
Expression of cellulases on the yeast surface
Two bacterial endoglucanases (EGs), C. thermocellum CelA and CelD, and two fungal EGs, T. aurantiacus EGI and T. reesei EGII, were expressed under the control of Gal1 promoter as Aga2-fusion proteins (Fig. 1). Aga2-fused EGs were displayed on the surface of S. cerevisiae EBY100 through a pair of disulfide bond between Aga2 and Aga1 [22], a yeast cell wall protein. CelA and CelD contain dockerin domains involved in the generation of cellulosome complex. The catalytic domains of CelA and CelD are
Discussion
In this study, we developed an ethanol production system in which yeast cells displaying different types of cellulases are combined in an optimized ratio. We first selected an EG suitable for the yeast surface display based on the characterization of the surface-immobilized enzyme activities, rather than those of free enzymes. We analyzed enzymatic properties of four fungal and bacterial EGs expressed on the yeast surface. The pH optimums of all the tested EGs attached on the yeast surface were
Acknowledgements
We are grateful to Drs. Y.-S. Kim and H. Zhao for providing plasmids. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-2010-C1AAA001-0028801).
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