Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production
A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was constructed, and the lycotoxin-1 (Lyt-1) C3 variant gene ORF, potentially to improve the availability of the active enzyme at the surface of the yeast cell, was added in frame with the CALB ORF u...
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Online Access: | http://dx.doi.org/10.1016/j.jala.2010.04.002 http://journals.sagepub.com/doi/pdf/10.1016/j.jala.2010.04.002 http://journals.sagepub.com/doi/full-xml/10.1016/j.jala.2010.04.002 |
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crsagepubl:10.1016/j.jala.2010.04.002 2024-06-23T07:46:08+00:00 Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production Hughes, Stephen R. Moser, Bryan R. Harmsen, Amanda J. Bischoff, Kenneth M. Jones, Marjorie A. Pinkelman, Rebecca Bang, Sookie S. Tasaki, Ken Doll, Kenneth M. Qureshi, Nasib Saha, Badal C. Liu, Siqing Jackson, John S. Robinson, Samantha Cotta, Michael C. Rich, Joseph O. Caimi, Paolo 2011 http://dx.doi.org/10.1016/j.jala.2010.04.002 http://journals.sagepub.com/doi/pdf/10.1016/j.jala.2010.04.002 http://journals.sagepub.com/doi/full-xml/10.1016/j.jala.2010.04.002 en eng SAGE Publications http://journals.sagepub.com/page/policies/text-and-data-mining-license JALA: Journal of the Association for Laboratory Automation volume 16, issue 1, page 17-37 ISSN 1535-5535 journal-article 2011 crsagepubl https://doi.org/10.1016/j.jala.2010.04.002 2024-06-11T04:30:53Z A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was constructed, and the lycotoxin-1 (Lyt-1) C3 variant gene ORF, potentially to improve the availability of the active enzyme at the surface of the yeast cell, was added in frame with the CALB ORF using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. Saccharomyces cerevisiae strains expressing CALB protein or CALB Lyt-1 fusion protein were first grown on 2% (w/v) glucose, producing 9.3 g/L ethanol during fermentation. The carbon source was switched to galactose for GAL1-driven expression, and the CALB and CALB Lyt-1 enzymes expressed were tested for fatty acid ethyl ester (biodiesel) production. The synthetic enzymes catalyzed the formation of fatty acid ethyl esters from ethanol and either corn or soybean oil. It was further demonstrated that a one-step-charging resin, specifically selected for binding to lipase, was capable of covalent attachment of the CALB Lyt-1 enzyme, and that the resin-bound enzyme catalyzed the production of biodiesel. High-level expression of lipase in an ethanologenic yeast strain has the potential to increase the profitability of an integrated biorefinery by combining bioethanol production with coproduction of a low-cost biocatalyst that converts corn oil to biodiesel. Article in Journal/Newspaper Antarc* Antarctica SAGE Publications JALA: Journal of the Association for Laboratory Automation 16 1 17 37 |
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Open Polar |
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SAGE Publications |
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crsagepubl |
language |
English |
description |
A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was constructed, and the lycotoxin-1 (Lyt-1) C3 variant gene ORF, potentially to improve the availability of the active enzyme at the surface of the yeast cell, was added in frame with the CALB ORF using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. Saccharomyces cerevisiae strains expressing CALB protein or CALB Lyt-1 fusion protein were first grown on 2% (w/v) glucose, producing 9.3 g/L ethanol during fermentation. The carbon source was switched to galactose for GAL1-driven expression, and the CALB and CALB Lyt-1 enzymes expressed were tested for fatty acid ethyl ester (biodiesel) production. The synthetic enzymes catalyzed the formation of fatty acid ethyl esters from ethanol and either corn or soybean oil. It was further demonstrated that a one-step-charging resin, specifically selected for binding to lipase, was capable of covalent attachment of the CALB Lyt-1 enzyme, and that the resin-bound enzyme catalyzed the production of biodiesel. High-level expression of lipase in an ethanologenic yeast strain has the potential to increase the profitability of an integrated biorefinery by combining bioethanol production with coproduction of a low-cost biocatalyst that converts corn oil to biodiesel. |
format |
Article in Journal/Newspaper |
author |
Hughes, Stephen R. Moser, Bryan R. Harmsen, Amanda J. Bischoff, Kenneth M. Jones, Marjorie A. Pinkelman, Rebecca Bang, Sookie S. Tasaki, Ken Doll, Kenneth M. Qureshi, Nasib Saha, Badal C. Liu, Siqing Jackson, John S. Robinson, Samantha Cotta, Michael C. Rich, Joseph O. Caimi, Paolo |
spellingShingle |
Hughes, Stephen R. Moser, Bryan R. Harmsen, Amanda J. Bischoff, Kenneth M. Jones, Marjorie A. Pinkelman, Rebecca Bang, Sookie S. Tasaki, Ken Doll, Kenneth M. Qureshi, Nasib Saha, Badal C. Liu, Siqing Jackson, John S. Robinson, Samantha Cotta, Michael C. Rich, Joseph O. Caimi, Paolo Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production |
author_facet |
Hughes, Stephen R. Moser, Bryan R. Harmsen, Amanda J. Bischoff, Kenneth M. Jones, Marjorie A. Pinkelman, Rebecca Bang, Sookie S. Tasaki, Ken Doll, Kenneth M. Qureshi, Nasib Saha, Badal C. Liu, Siqing Jackson, John S. Robinson, Samantha Cotta, Michael C. Rich, Joseph O. Caimi, Paolo |
author_sort |
Hughes, Stephen R. |
title |
Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production |
title_short |
Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production |
title_full |
Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production |
title_fullStr |
Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production |
title_full_unstemmed |
Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production |
title_sort |
production of candida antarctica lipase b gene open reading frame using automated pcr gene assembly protocol on robotic workcell and expression in an ethanologenic yeast for use as resin-bound biocatalyst in biodiesel production |
publisher |
SAGE Publications |
publishDate |
2011 |
url |
http://dx.doi.org/10.1016/j.jala.2010.04.002 http://journals.sagepub.com/doi/pdf/10.1016/j.jala.2010.04.002 http://journals.sagepub.com/doi/full-xml/10.1016/j.jala.2010.04.002 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
JALA: Journal of the Association for Laboratory Automation volume 16, issue 1, page 17-37 ISSN 1535-5535 |
op_rights |
http://journals.sagepub.com/page/policies/text-and-data-mining-license |
op_doi |
https://doi.org/10.1016/j.jala.2010.04.002 |
container_title |
JALA: Journal of the Association for Laboratory Automation |
container_volume |
16 |
container_issue |
1 |
container_start_page |
17 |
op_container_end_page |
37 |
_version_ |
1802644140582764544 |