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PGASO: A synthetic biology tool for engineering a cellulolytic yeast
BACKGROUND: To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462719/ https://www.ncbi.nlm.nih.gov/pubmed/22839502 http://dx.doi.org/10.1186/1754-6834-5-53 |
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author | 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 |
author_facet | 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 |
author_sort | Chang, Jui-Jen |
collection | PubMed |
description | BACKGROUND: To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. RESULTS: 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. CONCLUSIONS: 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. |
format | Online Article Text |
id | pubmed-3462719 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-34627192012-10-03 PGASO: A synthetic biology tool for engineering a cellulolytic yeast 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 Biotechnol Biofuels Research BACKGROUND: To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. RESULTS: 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. CONCLUSIONS: 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. BioMed Central 2012-07-27 /pmc/articles/PMC3462719/ /pubmed/22839502 http://dx.doi.org/10.1186/1754-6834-5-53 Text en Copyright ©2012 Chang et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research 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 PGASO: A synthetic biology tool for engineering a cellulolytic yeast |
title | PGASO: A synthetic biology tool for engineering a cellulolytic yeast |
title_full | PGASO: A synthetic biology tool for engineering a cellulolytic yeast |
title_fullStr | PGASO: A synthetic biology tool for engineering a cellulolytic yeast |
title_full_unstemmed | PGASO: A synthetic biology tool for engineering a cellulolytic yeast |
title_short | PGASO: A synthetic biology tool for engineering a cellulolytic yeast |
title_sort | pgaso: a synthetic biology tool for engineering a cellulolytic yeast |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462719/ https://www.ncbi.nlm.nih.gov/pubmed/22839502 http://dx.doi.org/10.1186/1754-6834-5-53 |
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