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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING BIOCHEMICAL PROCESSES FOR THE PRODUCTION OF SUSTAINABLE FUELS AND CHEMICALS

Location: Renewable Product Technology Research Unit

Title: Production of Candida antaractica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell & Expression in Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production

Authors
item Hughes, Stephen
item Moser, Bryan
item Harmsen, Amanda -
item Bischoff, Kenneth
item Jones, Marjorie -
item Pinkelman, Rebecca -
item Bang, Sookie -
item Tasaki, Ken -
item Doll, Kenneth
item Qureshi, Nasib
item Liu, Siqing
item Saha, Badal
item Jackson Jr, John
item Cotta, Michael
item Rich, Joseph
item Caimi, Paolo -

Submitted to: Journal of the Association for Laboratory Automation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 5, 2010
Publication Date: May 15, 2010
Repository URL: http://hdl.handle.net/10113/48483
Citation: Hughes, S.R., Moser, B.R., Harmsen, A.J., Bischoff, K.M., Jones, M.A., Pinkelman, R., Bang, S.S., Tasaki, K., Doll, K.M., Qureshi, N., Liu, S., Saha, B.C., Jackson Jr, J.S., Cotta, M.A., Rich, J.O., Caimi, P. 2010. Production of Candida antaractica Lipase B gene open reading frame using automated PCR gene assembly protocol on robotic workcell and expression in ethanologenic yeast for use as resin-bound biocatalyst in biodiesel production. Journal of the Association for Laboratory Automation. 16(1):17-37. DOI: 10.1016/j.jala.2010.04.002.

Interpretive Summary: A rapid and reliable PCR assembly strategy for producing gene ORFs was developed that is capable of being used in an iterative fashion on an integrated robotic workcell. An automated protocol was scripted to perform the PCR assembly and subsequent DNA purification, and was used in the construction of recombinant Candida antarctica lipase B (CALB) open reading frames (ORFs) expressed in yeast to produce ethanol and biodiesel as resin-bound biocatalyst. The scripting of automated protocols for the liquid handler and scheduling of PCR assembly steps on the robotic workcell that was demonstrated in this work can be used in an iterative automated fashion for construction of any gene ORF. Rapid production of gene ORFs is essential for large-scale production of libraries of ORFs from full genome sequences or from systematically mutagenized optimized sequences based on a single gene ORF. Use of lipases as biocatalysts to accomplish transesterification would also help to decrease the costs associated with the traditional method of biodiesel production as well as to overcome some of the technical drawbacks especially if large amounts of the lipases can be expressed in the yeast used in ethanol production processes in the biorefinery.

Technical Abstract: A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was produced using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. The lycotoxin-1 (Lyt-1) C3 variant gene ORF was added in-frame with the CALB ORF to potentially improve availability of the active enzyme at the surface of the yeast. 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 novel enzymes catalyzed 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 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.

Last Modified: 10/25/2014