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

Abstract

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...

Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

“…For CALB, different synthetic variants have been generated using automated PCR, and these have been tested for biodiesel production from corn and soybean oil. In particular, Hughes and coworkers [ 210 ] fused the CALB gene with the sequence encoding the Lyt-1 peptide, to facilitate the movement of the enzyme out of the S. cerevisiae cell. As expected, the supernatant of the yeast cells expressing this CALB Lyt-1 fusion protein had a specific activity almost 4-fold higher compared to the supernatant of the yeast cells expressing CALB alone.…”

Sustainable conversion of coffee and other crop wastes to biofuels and bioproducts using coupled biochemical and thermochemical processes in a multi-stage biorefinery concept

“…E2 XI gene and the Yersinia pestis XKS gene were obtained by PCR amplification from the plasmids used previously (Hughes et al 2009b ) and placed into a SUMO-XI-XKS polyprotein expression cassette behind the TRP1 promoter. The SUMO system can also be used for the concomitant expression of a value-added protein co-product, such as an insecticidal peptide (Hughes et al 2008 ), the enzyme Candida antarctica lipase B (Hughes et al 2011 ), or the natural peptide sweetener, brazzein (Pinkelman, manuscript submitted). Cell biomass production, sugar consumption, and ethanol production by recombinant Saccharomyces cerevisiae NRRL Y-50183 grown on borra (spent coffee grounds; Mussatto et al 2011 ) acid/enzymatic hydrolysate are presented in Fig.…”

Functional Expression and Characterization of a Truncated Candida antarctica Lipase B in Yeast

“…One of the current objectives in this area of research is to prepare a highly active, stable, low-cost lipase expressed at high levels in an ethanologenic yeast strain that can be incorporated into ethanol production facilities (Hughes et al 2011). Most fuel ethanol is produced from corn starch, which yields substantial amounts of low quality corn oil as a byproduct.…”

“…Western blot analysis of the synthetic Candida antarctica lipase B sequence previously constructed (Hughes et al 2011) containing six artificial ATG start codons determined that five of the six possible truncated CalB proteins were able to be expressed in yeast grown in galactose medium, with four of these five proteins containing the most distinctive active site feature, the residues Asp187-His224-Ser105, collectively known as the catalytic triad. Only the protein expressed at 33.8 kDa, the longest ORF of the five sequences expressed, contained the other key structural features of the lipase active site;…”

“…However, highly visible protein bands on the Western blot were still present in the cell pellet samples after mixing with the detergents, showing almost no change in appearance compared to the cell pellet samples prior to mixing with detergent. In addition to the truncated CalB enzyme, a full-length version of the CalB protein(Hughes et al 2011) was assessed alongside for comparison of each the detergent samples to rule out any possibility of underlying aberrations in the truncated CalB protein sequence as a cause of preventing it from solubilizing in the cell supernatant.…”