A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

Abstract

BackgroundChemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria are important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may also enable improvements in candidate CBP microorganisms.ResultsThermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold by furfural and were targeted for further investigation. Teth39_1597 encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.ConclusionsT. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0165-z) contains supplementary material, which is available to authorized users.

Deletion of nfnAB in Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism

“…Interestingly, many Thermoanaerobacter species also carry a predicted adhB gene nearby as well, which encodes a Zn-dependent bifunctional alcohol/aldehyde dehydrogenase that uses NADPH instead of NADH as a cofactor (28)(29)(30) and is believed to be important for ethanol formation in T. pseudethanolicus (28,31). In a proteomic study of T. pseudethanolicus, the products of nfnAB, adhA, and adhB were among the top 50 proteins detected (32). In this study, these proteins were significantly downregulated in response to furan aldehyde addition and coincided with reduced ethanol yields, which may be due partially to downregulation of these proteins.…”

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

“…Thermoanaerobacter pseudethanolicus 39E is an extremely thermophilic, fermentative bacterium that displays tolerance to 20–30 mM concentrations of furan aldehydes and rapidly reduces these compounds in situ during growth [ 19 ]. Proteomics analyses have previously shown that a butanol dehydrogenase (BdhA) was upregulated sixfold in response to furfural exposure and suggested the enzyme may be involved in furan aldehyde reduction.…”

Horizontal transfer of a pathway for coumarate catabolism unexpectedly inhibits purine nucleotide biosynthesis

“…In particular, inhibition of microbial growth by aldehydes is commonly observed, though the mechanisms of toxicity can rarely be traced to a specific interaction (Mills et al , ; Clarkson et al , ; Yi et al , ). Enzymatic pathways have frequently evolved to limit the release of free aldehydes, for example, through enzymatic channeling (Huang et al , ).…”