A Comparative Multidimensional LC-MS Proteomic Analysis Reveals
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Clarkson et al. Biotechnology for Biofuels 2014, 7:165 http://www.biotechnologyforbiofuels.com/content/7/1/165 RESEARCH Open Access A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E Sonya M Clarkson1,2, Scott D Hamilton-Brehm1,2,4, Richard J Giannone1,3, Nancy L Engle1,2,TimothyJTschaplinski1,2, Robert L Hettich1,3 and James G Elkins1,2* Abstract Background: Chemical 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. Results: Thermoanaerobacter 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. Conclusions: T. 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. Keywords: Thermophiles, Lignocellulosic, Biofuels, Proteomics, Inhibitor, Pretreatment, Furfural, 5-hydroxymethylfurfural, Butanol dehydrogenase * Correspondence: [email protected] 1BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341, USA 2Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341, USA Full list of author information is available at the end of the article © 2014 Clarkson et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Clarkson et al. Biotechnology for Biofuels 2014, 7:165 Page 2 of 14 http://www.biotechnologyforbiofuels.com/content/7/1/165 Background engineered strains of E. coli [12,21-23]. Interestingly, Thermophilic bacteria, such as Clostridium thermocellum members of the genus Thermoanaerobacter have been and Caldicellulosiruptor species have gained interest for shown to tolerate pretreated biomass hydrolysates [24,25], their possible use as biocatalysts for converting lignocellu- and engineered strains give improved ethanol yields from losic biomass into renewable fuels and chemicals [1-4]. both C5 and C6 sugars [26]. These properties have en- The potential advantages of thermal bioprocessing include couraged the development of several Thermoanaerobacter improved kinetics, reduced viscosities of concentrated slur- species for bioethanol production from hydrolysates ries, lower oxygen solubility, and reduced process cooling (primarily xylose) and on cellulose when paired with a requirements [2]. In addition, several bacterial phyla cellulolytic partner [27]. While surveying thermophilic include thermophiles that are able to utilize plant cell walls bacteria for intrinsic tolerance to furfural (unpublished), directly through the action of complex (hemi)cellulase we observed robust growth and rapid reduction of the systems expressed either as free enzymes, cellulosomes, or compound by Thermoanaerobacter pseudethanolicus 39E multifunctional enzymes [5]. Relying on these native (formally known as T. ethanolicus [28]). This study aims enzymes in a bioprocessing scheme could substantially to identify and characterize traits that enable this organism reduce or even eliminate the need for exogenous enzymes to grow in the presence of and to simultaneously detoxify for cellulose solubilization with a resulting improvement in furan aldehydes through reduction to less toxic alcohols. process economics [3,6,7]. To render plant material more reactive to microbial or Results and Discussion enzymatic digestion, physical and chemical pretreatment T. pseudethanolicus 39E furan aldehyde tolerance methods are generally applied, and improvement in We initially investigated the growth tolerance of T. pseu- pretreatment technologies remains a highly active field dethanolicus 39E to the furan aldehydes furfural and of research [8,9]. Pretreatment with dilute acid at high 5-hydroxymethylfurfural (5-HMF). The addition of 10 mM temperatures has the benefit of solubilizing the hemicel- and 15 mM furfural increased specific growth rates to lulose fraction of biomass, which produces fermentable 0.52 ± 0.03 h-1 and 0.49 ± 0.01 h-1, respectively, versus the C5 oligomers and monomers [9]. However, one disad- control at 0.38 ± 0.01 h-1 (Figure 1A). 5-HMF also stimu- vantage of dilute acid pretreatment is that the process is lated growth at 10 mM compared to no addition (0.51 ± non-specific and, depending on its severity, generates a 0.03 versus 0.41 ± 0.02 h-1), while growth rates were similar number of toxic by-products [10]. Inhibitory compounds to the control at 15 mM 5-HMF (0.45 ± 0.02 versus 0.41 ± generated by dilute acid pretreatment typically fall into 0.02 h-1; Figure 1B). Both 10 mM furfural and 5-HMF four categories: organic acids (acetic acid, ferulic acid), slightly increased cell yield at 12 h by approximately 11% ketones (acetovanillone), phenolics (coniferyl alcohol, and 12%, respectively (Figure 1). Higher growth rates and catechol), and aldehydes (furfural, hydroxymethylfurfural, increased cell yield from the addition of subinhibitory vanillin). Mixtures of inhibitors, especially those including concentrations of furfural and5-HMFsuggestthat39E the furan aldehyde furfural, often have a synergistic effect metabolism is constrained by electron flow, which is on inhibiting cell growth and fermentation. For example, relieved by the furan aldehydes serving as an alternative furfural increases the toxicity of acetate in yeast [11] and dissimilatory electron acceptor. The concentration resulting phenols in Escherichia coli [12,13]. Furfural is estimated to in 50% inhibition of growth (IC50)withfurfuralwas30mM be responsible for 33% of the toxic effect of sugar cane after12hand30to40mMafter24h.TheIC50 for hydrolysate on E. coli LYO1 [14]. 5-HMF was between 20 and 30 mM after both 12 and In order to compete with more robust ethanologens 24 h. As shown in Table 1, the determined values are com- such as Saccharomyces cerevisiae, several limitations parable to or slightly higher than those of other thermo- inherent to fermentative thermophilic bacteria must be philicbacteria,whiletheyarehigher than reported values overcome. These limitations include relatively low ethanol for E. coli, S. cerevisiae, and Zymomonas mobilis. Though titer and yield from mixed-acid fermentation pathways, direct comparisons are difficult due to differences in the although breakthroughs in metabolic engineering have growth conditions used in the various studies, these results improved the yield of ethanol from carbohydrates in some suggest that T. pseudethanolicus 39Ehasacomparableif thermophiles to near theoretical limits [15]. Another not higher tolerance to the furan aldehydes furfural and major hurdle for thermophilic bioprocessing is growth 5-HMF than other studied organisms. inhibition by a wide range of compounds encountered in biomass fermentations. Insights into overcoming end- Furan aldehyde reduction and glucose fermentation product inhibition in C. thermocellum have recently In order to establish the mechanism of increased furan emerged [16,17]; however, growth inhibition from other aldehyde tolerance,