Synergistic effects of 2A-mediated polyproteins on the production of lignocellulose degradation enzymes in tobacco plants.
Cost-effective bioethanol production requires a supply of various low-cost enzymes that can hydrolyse lignocellulosic materials consisting of multiple polymers. Because plant-based enzyme expression systems offer low-cost and large-scale production, this study simultaneously expressed β-glucosidase (BglB), xylanase (XylII), exoglucanase (E3), and endoglucanase (Cel5A) in tobacco plants, which were individually fused with chloroplast-targeting transit peptides and linked via the 2A self-cleaving oligopeptideex from foot-and-mouth disease virus (FMDV) as follows: [RsBglB-2A-RaCel5A], [RsXylII-2A-RaCel5A], and [RsE3-2A-RaCel5A]. The enzymes were targeted to chloroplasts in tobacco cells and their activities were confirmed. Similarly to the results of a transient assay using Arabidopsis thaliana protoplasts, when XylII was placed upstream of the 2A sequence, the [RsXylII-2A-RaCel5A] transgenic tobacco plant had a more positive influence on expression of the protein placed downstream. The [RsBglB-2A-RaCel5A] and [RsE3-2A-RaCel5A] transgenic lines displayed higher activities towards carboxylmethylcellulose (CMC) compared to those in the [RsXylII-2A-RaCel5A] transgenic line. This higher activity was attributable to the synergistic effects of the different cellulases used. The [RsBglB-2A-RaCel5A] lines exhibited greater efficiency (35-74% increase) of CMC hydrolysis when the exoglucanase CBHII was added. Among the various exoglucanases, E3 showed higher activity with the crude extract of the [RsBglB-2A-RaCel5A] transgenic line. Transgenic expression of 2A-mediated multiple enzymes induced synergistic effects and led to more efficient hydrolysis of lignocellulosic materials for bioethanol production.
Bio-energy Research Institute, Department of Forest Products and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea.
Dae-Seok
DS
Lee
Kwang-Ho
KH
Lee
Sera
S
Jung
Eun-Jin
EJ
Jo
Kyung-Hwan
KH
Han
Hyeun-Jong
HJ
Bae
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000648:ultrastructure; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D044602:Cellulases / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008031:Lignin / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics
D020815:Polyproteins; D021382:Protein Sorting Signals; C036909:lignocellulose; D008031:Lignin; D044602:Cellulases; D002480:Cellulase; D043364:Endo-1,4-beta Xylanases