ARS | Publication request: Protein Engineering of GRE2 from Saccharomyces cerevisiae for Enhanced Detoxification of 5-hydroxymethylfurfural

Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: May 27, 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 nicotinamide adenine dinucleotide (phosphate) hydride (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 Arg and Phe283 to Leu; and Ile107 to Val, Gln261 to Arg, and Val285 to Asp for strains Y62-C11 and Y62-G6, respectively. Clones of these mutants showed a better growth rate and were able to establish a viable culture when challenged with 30 mM HMF 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 toward 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-furandimethanol conversion. The commonly shared amino acid substitution of Arg at 261 and the additional substitutes of Leu283 and Asp285 could affect cofactor NADH- and/or substrate-binding for the enhanced reduction activities.