ARS | Publication request: RAPID SELECTION OF ALPHA-AMYLASE MUTANTS IN YEAST LIBRARIES FOR RAW STARCH ACTIVITY

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 28, 2004
Publication Date: September 21, 2004
Citation: Wong, D., Batt, S.B., Lee, C.C., Robertson, G.H. Rapid selection of alpha-amylase mutants in yeast libraries for raw starch activity. 2003. Society of Biomolecular Screening Annual Conference, Portland, Oregon.

Technical Abstract: The production of ethanol in the United States reached a capacity of 2.7 billion gallons in 2002. The current process of ethanol production requires starch solubilized at temperatures of 120-150 degree C, prior to enzymatic conversion to dextrins. Using directed evolution, we aim to engineer alpha-amylase for the breakdown of raw starch to eliminate the need for heat treatment. The initial strategy for high-throughput screening of yeast libraries of mutants was based on activity assays using synthetic dyed starch substrates. The use of the natural substrate, untreated raw starch, however, was impeded by the interference of reducing agents, particularly the glucose commonly used in yeast growth media. Recently, we discovered that glycerol could be used as an effective alternative carbon source for yeast and resulted in unexpected significant increase in the synthesis and secretion of the recombinant alpha-amylase while suppressing cell growth. We further adapted this system to develop a rapid, direct method for screening libraries of yeast clones for the specific activity of recombinant alpha-amylases on raw starch hydrolysis.