ARS | Publication request: GMAX-L Saccharomyces Cerevisiae Strains for Profitable Sustainable Cellulosic Ethanol and Biodiesel Production Concurrently using Engineered Workcell

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 7, 2009
Publication Date: May 4, 2009
Citation: Hughes, S.R., Moser, B.R., Doll, K.M., Tasaki, K., Harmsen, A., Jones, M., Bang, S., Gibbons, W., Butt, T. 2009. GMAX-L Saccharomyces cerevisiae strains for profitable sustainable cellulosic ethanol and biodiesel production concurrently using engineered workcell [abstract]. Center for Process Analytical Chemisty. Abstract No. 2. p. 1.

Technical Abstract: A stable GMAX-L strain of Saccharomyces cerevisiae is being constructed using pSUMO expression cassettes that are extremely high expression level plasmids designed for use on automated workcell. This strain expresses xylose isomerase, xylulokinase, XIB1, and XIG1 for anaerobic cellulosic ethanol production. The approach that will be used to produce the GMAX-L strain expressing lipases for production of biodiesel includes: 1) Add the Candida antarctica lipase B (CALB) gene that was recently assembled at USDA, NCAUR, BBC using amino acid scanning mutagenesis technology and optimized for expression from pYES2 DEST 52 to the recently engineered GMAX cellulosic yeast strain; 2) Workcell automated assembly, optimization, and expression of the CALB enzyme outside the yeast using a cell penetrating peptide, lycotoxin-1, to increase permeability of the yeast cell membrane allowing the lipase activity to take place on the surface of the yeast cell. Variants of this enzyme will be evaluated in high throughput in a 96-well format with different oil to ethanol ratios to determine the optimum ratio; 3) Isolate the yeast identified in the high-throughput assay and use the Mitsubishi one-step charging beads to capture the yeast expressing the lipase enzyme by means of the lycotoxin-1 tag and attach it to the beads in a column; 4) Pass the corn oil from the dry grind part of the biorefinery mixed with ethanol at the optimum ratio over the beads charged with the low cost yeast expressing lipase to produce biodiesel. This objective is to construct a single yeast that will produce both bioethanol and biodiesel to increase efficiency and cost-effectiveness by sharing products from a crossover biorefinery that is a combination of a lignocellulosic ethanol production facility and a dry grind ethanol plant.