Sunday, August 11, 2013
Pavilion (Sheraton San Diego)
As obesity increases and the health benefits of artificial sugars are being questioned, a low- to no-calorie substitute is needed. Brazzein produced as a recombinant, value-added product would fulfill the need for a low- to no-calorie sweetener while being produced from a yeast strain grown on agricultural wastes in turn lowering the environmental impact of producing brazzein. Brazzein is a water soluble, heat and pH stable sweet protein isolated from the West African plant Pentadiplandra brazzeana Baillon, which is 2,000x sweeter than a 2% (w/v) sucrose solution on a weight basis. The specific aim of this study is to genetically engineer an ethanologenic yeast strain expressing brazzein in a yeast artificial chromosome (pYAC) to check the feasibility of expressing brazzein in pYAC and Saccharomyces cerevisiae. The brazzein gene has been amplified and ligated into the vector pRS305 behind the LEU2 promoter sequence. The brazzein cassette containing the sequences for brazzein and the LEU2 promoter and terminator was ligated with the two arms of pYAC and subsequently transformed and expressed in S. cerevisiae PJ69-4, an auxotrophic strain with mutations in the uracil, histidine, leucine, and tryptophan genes. The expression level of brazzein from the recombinant strains has been compared. We have also modified the yeast artificial chromosome with a multiple cloning site (pYAC-MCS), which can be used for cloning lignocellulolytic enzymes such as cellulases, xylanases, and laccases, to meet our long-term goal of developing an industrial yeast strain capable of producing ethanol and brazzein based on seamless fermentation of lignocellulosic waste.