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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMICS AND ENGINEERING OF STRESS TOLERANT MICROBES FOR LOWER COST PRODUCTION OF ETHANOL FROM LIGNOCELLULOSE

Location: Bioenergy Research Unit

Title: Protein Engineering of GRE2 from Saccharomyces cerevisiae for Enhanced Detoxification of 5-Hydroxymethyl Furfural

Authors
item Moon, Jaewoong
item Liu, Zonglin

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 1, 2010
Publication Date: N/A

Technical Abstract: Furfural and 5-hydroxymethylfurfural (HMF) are representative inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds using tolerant Saccharomyces cerevisiae through NAD(P)H-dependent aldehyde reductions. GRE2 is a commonly recognized up-regulated gene by stress conditions with reductase activities. Using a directed enzyme evolution approach, we engineered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for Y62-C11 and Y62-G6, respectively. Clones of these mutants showed better growth rates and were able to establish viable cultures under 30mM HMF challenges as an initial inoculum when compared with a wild type GRE2 clone on a synthetic medium. The improved mutants displayed approximately 3- to 4-fold increase of specific enzyme reduction activity toward HMF, and a 3- to 9-fold increase to furfural compared with their parental wild type gene. Derivatives of S. cerevisiae NRRL Y-50049 transformed with these mutated genes demonstrated significantly higher levels of HMF-to-FDM conversion. In purified Y62-G6, aldehyde reductase activity toward furfural and HMF using NADPH was 13- to 15-fold increased compared with a wild type GRE2. Site directed mutagenesis and subsequent enzyme assay showed that the amino acid substitution of Asp at 285 affected cofactor preference for enhanced reduction activities.

Last Modified: 7/30/2014
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