Protein engineering of GRE2 from Saccharomyces cerevisiae for enhanced detoxification of 5-hydroxymethylfurfural

Authors: Moon, Jaewoong; Liu, Zonglin

Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2011
Publication Date: 7/17/2012
Citation: Moon, J., Liu, Z. 2012. Protein engineering of GRE2 from Saccharomyces cerevisiae for enhanced detoxification of 5-hydroxymethylfurfural. Enzyme and Microbial Technology. 50:115-120.

Interpretive Summary: Lignocellulosic biomass conversion to ethanol requires a pretreatment of the biomass prior to enzymatic hydrolysis in order to be utilized for microbial growth and fermentation. Dilute acid hydrolysis is among the commonly applied biomass pretreatment procedures but generates numerous inhibitory compounds. Furfural and 5-hydroxymethylfurfural (HMF), derived from dehydration of pentoses and hexoses during the biomass pretreatment, are representative inhibitors in hydrolysates that inhibit microbial growth and interfere with subsequent fermentation. Inhibitors furfural and HMF can be detoxified in situ by tolerant yeast strains. GRE2 of yeast is a stress tolerant core gene possessing moderate aldehyde reduction activities. Using directed enzyme evolution and reverse engineering methods, we modified the genetic code of the enzyme and generated mutations displaying improved detoxification capability. We also demonstrated that specific genetic codes affect different cofactor preference for the enzyme reduction activities. This is the first example of application of enzyme evolution for improved detoxification of furfural and HMF by yeasts. Outcomes of this study will aid efforts in dissection of mechanisms of inhibitor-stress tolerance and tolerant strain development.

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 interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds using tolerant Saccharomyces cerevisiae by aldehyde reductions. YOL151W (GRE2) is a commonly recognized up-regulated gene by stress conditions with reductase activities toward furfural and HMF using cofactor nicotamide adenine dinucleotdie hydride (NADH). Applying directed enzyme evolution approach, we engineered genetic code of the 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 a better growth rate and were able to establish a viable culture under 30mM HMF challenges as an initial inoculum compared with a wild type GRE2 clone on a synthetic medium. Compared with the wild type control, crude cell extracts of the mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural, respectively. While remaining its aldyhyde reduction activities using cofactor NADH, mutant Y62-G6 displayed significantly higher reductase activities using an additional cofactor NADPH with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Confirmed by using reverse engineering and site directed mutagenesis, we conclude that the amino acid substitution of the Asp285 is responsible for the increased enzyme activities by utilizing the additional cofactor NAD phosphate hydride (NADPH).