BioChem - DOS

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

Journal of laboratory automation

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.; Saha, B.C.; Liu, S.; Jackson, J.S.; Robinson, S.; Cotta, M.C.; Rich, J.O.; Caimi, P.

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 9.3g/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.

2011

Association for Laboratory Automation

2211-0682

2211-0690

16

1

pp.17-37

10.1016/j.jala.2010.04.002

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART55250643

Candida antarctica lipase B; transesterification; biodiesel; Saccharomyces cerevisiae; one-step-charging resin; resin-supported biocatalyst