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標題: PGASO: A synthetic biology tool for engineering a cellulolytic yeast
作者: Chang, Jui-Jen
Ho, Cheng-Yu
Ho, Feng-Ju
Tsai, Tsung-Yu
Ke, Huei-Mien
Wang, Christine H-T
Chen, Hsin-Liang
Shih, Ming-Che
Huang, Chieh-Chen
Li, Wen-Hsiung
關鍵字: Consolidated bioprocess;Synthetic biology;Yeast;Cellulolytic enzymes;Bio-ethanol
Project: Biotechnology for Biofuels, Volume 5, Issue 53
To achieve an economical cellulosic ethanol production, a host that can do both cellulosic
saccharification and ethanol fermentation is desirable. However, to engineer a noncellulolytic
yeast to be such a host requires synthetic biology techniques to transform
multiple enzyme genes into its genome.
A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression
(PGASO), that employs overlapping oligonucleotides for recombinatorial assembly of gene
cassettes with individual promoters, was developed. PGASO was applied to engineer
Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a
recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase
and endoglucanase (both of Trichodermareesei ), a beta-glucosidase (from a cow rumen
fungus), a neomycin phosphotransferase, and a green fluorescent protein. High
transformation efficiency and accuracy were achieved as ~63% of the transformants was
confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon
source for growth and can directly convert cellobiose and beta-glycan to ethanol.
This study provides the first example of multi-gene assembly in a single step in a yeast
species other than Saccharomyces cerevisiae . We successfully engineered a yeast host with a
five-gene cassette assembly and the new host is capable of co-expressing three types of
cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression
of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing
synthetic biology tools.
ISSN: 1754-6834
DOI: 10.1186/1754-6834-5-53
Appears in Collections:生命科學系所

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