A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance

The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 113-5D. Strains overexpressing ADH6 had increased HMF conversion activity in cell-free crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity.