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

Abstract

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 cellfree 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.

Metabolic engineering of Saccharomyces cerevisiae for improvement in stresses tolerance

“…12,15,29 Researchers stated that, overexpression of the dehydrogenase/reductase genes (ari1, adh7, ald6 and adh6) increased enzyme activities for furfural and/or HMF reduction and simultaneously improved the tolerability of yeast against inhibitors. [12][13][14]30 Quantitative real-time PCR data confirmed the overexpression of tps1 and ari1 in engineered strain SCTADN. As compare with wild strain (SC), SCTADN exhibited significantly higher cell survival under stresses of ethanol, furfural and HMF.…”

“…[10][11][12] Many researches demonstrated that overexpression of the aldehyde dehydrogenase/reductase genes (ari1, ald6 and adh6) in S. cerevisiae not only could eliminate the toxicity of aldehydes but also improve tolerability. [12][13][14][15] Trehalose, a non-reducing disaccharide, works as an energy source in yeasts, bacteria, fungi, invertebrates, insects and plants. 16,17 Many research demonstrated that the higher trehalose accumulated in the yeast cell the higher tolerance against various environmental stresses, for instance ethanol stress, 18 heat stress, 19 saline stress, 20 and various other environmental stresses.…”

Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14

“…Thus, HmfH provides an alternative to the nonspecific dehydrogenases that also perform these upper pathway oxidations in C. basilensis HMF14. Such nonspecific "furanic dehydrogenases" were also observed in P. putida S12, as well as in other microorganisms that cannot utilize furanic aldehydes for growth, probably serving to detoxify furanic aldehydes (31)(32)(33).…”

- Synthetic Biology: A Promising Technology for Biofuel Production

“…Trialkylamine [79] Liquid-solid extraction Activated carbon [80] Ion exchange [38,81] Lignin [82] Microbial treatment Coniochaeta ligniaria [83,84] Trichoderma reesei [33,85] Ureibacillus thermosphaericus [86] a The phenolics [89,90], furan aldehydes [91,92], and aliphatic acids [93,94]. Furthermore, overexpression of a transcription factor, Yap1 [95], and of multidrug-resistance proteins [95] has also generated hyperresistant S. cerevisiae transformants.…”