|Hector, Ronald - Ron|
Submitted to: Plasmid Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/5/2008
Publication Date: 10/30/2008
Citation: Hughes, S.R., Sterner, D.E., Bischoff, K.M., Hector, R.E., Dowd, P.F., Qureshi, N., Bang, S.S., Grynavyski, N., Chakrabarty, T., Johnson, E.T., Dien, B.S., Mertens, J.A., Caughey, R.J., Liu, S., Butt, T.R., Labaer, J., Cotta, M.A., Rich, J.O. 2009. Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system. Plasmid Journal. 61(1):22-38. Interpretive Summary: Fuel ethanol production from biomass at the industrial level using Saccharomyces cerevisiae shows great promise for satisfying future energy demands, but the limited range of materials that can be fermented remains an obstacle to cost-effective bioethanol production. Although several genetically engineered strains of S. cerevisiae have been developed that will ferment xylose to ethanol, further optimization is needed. This 3 vector set of plasmids described used on the automated platform offers the possibility of expressing pentose-utilization enzymes and commercially important peptides and proteins in industrial yeast strains currently used in the production of fuel ethanol from lingnocellulose hydrolysates.
Technical Abstract: A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was adapted for use on a fully automated plasmid-based robotic platform to screen for improved strains of S. cerevisiae engineered to express a peptide of potential commercial value in addition to xylose utilization and metabolic correction enzymes. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae strain. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into the XI-yeast. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the Lyt-1-XI-yeast construct. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to XI-yeast.