Method

Lignocellulose Degradation Pathway

In order to break down lignocellulose into glucose molecules, we have planned to construct three pathways, aiming at: a) lignocellulose pretreatment/lignin degradation, b) cellulose degradation, and c) hemicellulose/xylan degradation. For each pathway to work, multiple enzymes are needed to work collaboratively. We have used standard cloning methods to construct secretory vectors pPICZα-ΒBa_K500000, pPICZα-BBa_K500001 and pPICZα-BBa_K500003 for pretreatment, 9k-BBa_K805011 and ZXFα-bglX for cellulose degradation, 9k-BBa_K805012 and ZXFα-BBa_K805010 for hemicellulose degradation.

Cell Surface Codisplay

We have planned to construct two whole-cell biocatalysts with the ability to induce synergistic and sequential cellulose and hemicellulose degradation reactions through codisplay of two types of degradation enzymes on each of the Pichia pastoris cell surface. Cell surface display technique fuses foreign protein with the surface of microorganisms and can enhance the activity of enzymes by concentrating them. We utilized the cell surface display technique to simultaneously codisplay endoglucanase as fusion protein with the N-terminal-half region of ankyrin Sed1 and β-glucosidase as fusion protein with the C-terminal–half region of α-agglutinin Aga2. The other codisplay system consists of endoxylanase as fusion protein with the C-terminal–half region of α-agglutinin Aga2 and β-xylosidase as fusion protein with the N-terminal-half region of ankyrin Sed1. Cell surface display was confirmed by observation of immunofluorescence-labeled cells with an upright fluorescence microscope, which will be introduced in the detection section.

Ethanol Fermentation

In order to convert glucose into ethanol and thereby produce cellular energy, we have planned to incubate our engineered yeast cells for fermentation. Ethanol fermentation is classified as anaerobic because yeasts perform this conversion in the absence of oxygen.

Detection

In order to make sure that several of our cell display enzymes are actually displayed on the surface of yeast, we utilized Immunofluorescence technique and Flow Cytometry to detect protein location and their relative abundance. Immunofluorescence technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific protein targets within a cell, and therefore allows detection and visualization of the distribution of our target proteins. We purchased Mouse Anti HA-Tag Polyclonal antibody and Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H+L) for the detection of endoxylanase gene xyn and β-glucosidase gene bglX, and Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody for the detection of endoglucanase gene cel5A and β-xylosidase gene RuXyn. Flow cytometry was used to aid the analysis.
We have also used enzyme assay methods to detect the enzymatic activities of our secretory enzymes.


References:
Wei, Xiao-Min, Yu-Qi Qin, and Yin-Bo Qu. Molecular Cloning and Characterization of Two Major Endoglucanases from Penicillium decumbens. J. Microbiol. Biotechnol. (2010), 20(2), 265–270.
Gary Xie et al. Genome Sequence of the Cellulolytic Gliding Bacterium Cytophaga hutchinsonii. Appl. Environ. Microbiol. 2007, 73(11):3536.
Qu W, Shao W. Cloning, expression and characterization of glycoside hydrolase family 11 endoxylanase from Bacillus pumilus ARA. Biotechnol Lett. 2011 Jul;33(7):1407-16.
Jungang Zhou, Lei Bao, Lei Chang, Yufei Zhou, Hong Lu. Biochemical and kinetic characterization of GH43 b-D-xylosidase/a-L-arabinofuranosidase and GH30 a-L-arabinofuranosidase/b-D-xylosidase from rumen metagenome. J Ind Microbiol Biotechnol (2012) 39:143–152.
Dai Min. Surface display of Candida Antarctica lipase B and Thermomyces lanuginosus lipase with synergy in Pichia pastoris. Wuhan: Huazhong University of Science and Technology. 2012
Pan Xiaoxing. Surface display of Geotrichum sp. Lipase on the cell wall of Saccharomyces cerevisiae and Pichia pastoris. Wuhan: Huazhong University of Science and Technology. 2012
Yasuya Fujita et al. Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes. Appl. Environ. Microbiol. 2002, 68(10):5136.
Toshiyuki Murai et al. Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing b-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus. Appl. Environ. Microbiol. 1998, 64(12):4857.
Yasuya Fujita et al. Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme. Appl. Environ. Microbiol. 2004, 70(2):1207.