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-stepcharging 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. (JALA 2011;16:17-37) Keywords: Candida antarctica lipase B, transesterification, biodiesel, Saccharomyces cerevisiae, one-stepcharging resin, resin-supported biocatalyst a Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. INTRODUCTIONMost of the fuel ethanol produced in the United States is made from corn starch in either wet-mill or dry-grind ethanol facilities. Projections indicate that corn supplies will not be able to meet the increasing demand for biofuels. Lignocellulosic biomass, an abundant and renewable carbon source, has the potential to supplement or replace starch feedstocks for the production of fuel ethanol, 1 but current technology is constrained by production costs. The profitability of ethanol production from lignocellulosic biomass will be improved if high-value coproducts are also generated. Current processes for fuel ethanol production from starch yield substantial amounts of corn oil as a byproduct. The corn oil is used for the manufacture of biodiesel, thereby removing the oil from the dried distiller grain solubles to give more digestible defatted animal feed. Corn oil triacylglycerides (TAGs) are converted to fatty acid ethyl esters (biodiesel) and glycerol by transesterification with ethanol. One method of catalyzing this transesterification reaction is using lipase enzymes. 2 An integrated biorefinery combining starch ethanol and cellulosic ethanol facilities may become more cost effective if biodiesel is produced as a coproduct using lipase-catalyzed single-step column transesterifi...

No abstract

and their respective research groups at the USDA for their collaboration and insight on this project. And finally, I would like to thank my family, friends, and especially my partner, Michael, for giving me the continuous support and encouragement needed to complete this project. S.M.R. viii 22. Michaelis-Menten plot for the substrate p-NPB ranging from 40-1000 µM and the truncated CalB enzyme using the method of Blank et al. (2006). 23. Michaelis-Menten plot for the substrate p-NPB ranging from 40-6000 µM and the truncated CalB enzyme using the method of Blank et al. (2006). 24. Michaelis-Menten plot for the substrate p-NPB ranging from 40-20000 µM and the truncated CalB enzyme using the method of Martinelle et al. (1995). 25. Michaelis-Menten plot for the substrate p-NPB ranging from 40-20000 µM and the full-length CalB enzyme using the method of Martinelle et al. (1995).