Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance
© The Author(s) 2017
Received: 8 December 2016
Accepted: 9 March 2017
Published: 15 March 2017
Resistance to deconstruction is a major limitation to the use of lignocellulosic biomass as a substrate for the production of fuels and chemicals. Consolidated bioprocessing (CBP), the use of microbes for the simultaneous hydrolysis of lignocellulose into soluble sugars and fermentation of the resulting sugars to products of interest, is a potential solution to this obstacle. The pretreatment of plant biomass, however, releases compounds that are inhibitory to the growth of microbes used for CBP.
Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an alcohol dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrations found in acid-pretreated biomass. The mechanism of detoxification of hydroxymethylfurfural was shown to be primarily reduction using NADPH as the cofactor. In addition, we report the construction of new expression vectors for homologous and heterologous expression in C. thermocellum. These vectors use regulatory signals from both C. bescii (the S-layer promoter) and C. thermocellum (the enolase promoter) shown to efficiently drive expression of the BdhA enzyme.
Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Expression of genes that reduce the effect of these inhibitors, such as furan derivatives, will serve to enable commercial processes using plant biomass for the production of fuels and chemicals.
KeywordsConsolidated bioprocessing Clostridium thermocellum Butanol dehydrogenase Furfural 5-hydroxymethyl-2-furfural
Clostridium thermocellum is a Gram-positive, thermophilic anaerobic bacterium and one of the most promising candidates for CBP because of its ability to deconstruct plant biomass and convert it directly to ethanol, lactic acid, acetic acid, formic acid, hydrogen, and amino acids including valine and alanine [1, 2]. While most metabolic engineering of C. thermocellum has focused on improving ethanol production [1, 3, 4], improving tolerance to inhibitors generated from biomass pretreatment is essential to make CBP by C. thermocellum an industrially relevant process . Furfural, 2-furaldehyde, and HMF, 5-hydroxymethyl-2-furfural, are generated during pretreatment and inhibit both growth and fermentation by microorganisms , including C. thermocellum. Saccharomyces cerevisiae , Escherichia coli , and Caldicellulosiruptor bescii  can convert furfural and HMF to the less toxic alcohols, furfuryl alcohol and furan dimethanol, respectively. Overexpression of oxidoreductases, such as alcohol dehydrogenases (ADH1, ADH6, and ADH7) [7, 10, 11], a propanediol oxidoreductase (FucO) , and a butanol dehydrogenase (BdhA)  has been shown to increase specific furfural and HMF conversion rates. Among them, Teth39_1597 encoding the BdhA enzyme from Thermoanaerobacter pseudethanolicus 39E was shown to reduce both furfural and HMF at 60 °C using NADPH as the cofactor . We recently demonstrated that heterologous expression of this heat-stable BdhA enzyme increased resistance of engineered C. bescii strains to both furfural and HMF . C. bescii is a hyperthermophilic, Gram-positive, anaerobic bacterium that has the unusual ability to grow on a variety of lignocellulosic biomass substrates without conventional pretreatment [13, 14]. We recently engineered C. bescii to produce ethanol directly from switchgrass making it a strong candidate for CBP . Pretreatment, however, increases rates of hydrolysis but releases furans that are toxic to growing cells. C. thermocellum relies primarily on pretreated biomass producing ethanol at high yield (72% of theoretical maximum) and produces ethanol as a single fermentation product [16, 17], making it perhaps the strongest candidate so far studied for CBP. To test whether BdhA from T. pseudethanolicus might also improve resistance to these compounds in C. thermocellum, we designed new expression vectors for C. thermocellum, using three different promoters, the C. bescii S-layer promoter, and the C. thermocellum Clo1313_1809 and enolase promoters. The vectors were based on the C. bescii replicon pBAS2 [18, 19]. Expression of BdhA in C. thermocellum resulted not only in increased resistance to HMF but also increased growth on cellulosic substrates and improved ethanol production. These data suggest that redox homeostasis in C. thermocellum plays an important role in its growth on cellulosic substrates.
Results and discussion
Heterologous expression of the bdhA gene from T. pseudethanolicus in C. thermocellum
Plasmid DNA was transformed into a pyrF deletion mutant of C. thermocellum  and transformants were selected for uracil prototrophy. The presence of the plasmid in transformants was confirmed by PCR analysis (Additional file 1: Figure S1A). Primers (SK04 and DC228) were used to amplify the portion of the plasmid containing the open reading frame of bdhA, annealing to regions of the plasmid outside the bdhA gene. The expected PCR product was detected for pSKW01 and pSKW02 transformants but not for pSKW04 (Additional file 1: Figure S1A) suggesting that pSKW04 might have integrated into the C. thermocellum chromosome. To test whether the plasmids were replicating autonomously, total DNA isolated from C. thermocellum transformants containing pSKW01 (JWCT06), pSKW02 (JWCT07) or pSKW04 (JWCT08) was used to back-transform E. coli. Transformants were obtained for DNA from JWCT06 and JWCT07 but not JWCT08, again suggesting that while the plasmid was present it was not autonomously replicating. Two different digestions by restriction endonucleases performed on plasmid DNA purified from two independent E. coli back-transformant colonies resulted in identical digestion patterns relative to the original plasmids (Additional file 1: Figure S2), indicating that plasmids (pSKW01 and pSKW02) were autonomously replicating in C. thermocellum and were structurally stable during transformation and replication in C. thermocellum and back-transformation into E. coli. To test whether plasmid pSKW04 had integrated into the chromosome PCR amplification using various primers inside and outside plasmid sequences was used. The enolase promoter sequence is the only region of homology between the plasmid and the chromosome and the only potential site for homologous recombination. PCR amplification using a primer set specific for 0.1 kb upstream region of enolase promoter in the chromosome (SK038) and plasmid pSKW04 (DC461) (Additional file 1: Figure S1B) confirmed pSKW04 plasmid integration into the C. thermocellum chromosome at the site of the enolase promoter region via a single crossover event.
Effects of BdhA expression on the growth and tolerance of C. thermocellum to furan derivatives
In vitro NADH- and NADPH-dependent conversion activity of strains expressing BdhA
Aldehydes are toxic to microbial cell growth and cells convert these compounds to less toxic compounds such as alcohols and carboxylic acids. Previous studies reported that reduction activity was much higher than oxidation activity for detoxification of the furan derivatives [6, 8, 27]. To investigate the mechanism of conversion of furfural and HMF by strains expressing BdhA, we examined NADH- and NADPH-dependent activities of furfural and HMF reduction. Crude extracts from the BdhA expressing strains, JWCT06 and JWCT08, and the control strain (JWCT02) were prepared and in vitro reduction of furfural and HMF was measured. While specific activities of crude extracts of all strains toward furfural were similar, BdhA expression increased the reduction of HMF by 59–69% relative to the control (Fig. 4c, d). This result is likely due to the specificity of the BdhA enzyme as this enzyme is known to have a twofold higher activity on HMF than furfural . We concluded from these data that the mechanism of improved detoxification rates of HMF by BdhA expression (Fig. 3d) results from higher reduction of HMF.
Bacterial strains, media, and culture conditions
Strains and plasmids used in this study
BL21 containing pSKW01 (ApramycinR)
BL21 containing pSKW02 (ApramycinR)
BL21 containing pSKW04 (ApramycinR)
C. thermocellum DSM 1313 ΔpyrF (ura −/5-FOAR)
LL1005 containing pDCW89 (ura +/5-FOAS)
LL1005 containing pSKW01 (ura +/5-FOAS)
LL1005 containing pSKW02 (ura +/5-FOAS)
LL1005 containing pSKW04 (ura +/5-FOAS)
E. coli/C. thermocellum shuttle vector (ApramycinR)
Intermediate vector 1 (ApramycinR)
Intermediate vector 2 (ApramycinR)
Source of Teth39_1597 (bdhA) open reading frame (ApramycinR)
Expression vector containing PS-layer -bdhA (ApramycinR)
Expression vector containing PClo1313_1809 -bdhA (ApramycinR)
Expression vector containing Penolase -bdhA (ApramycinR)
Construction and transformation of bdhA expression vectors
Plasmid DNA was isolated using a Qiagen Miniprep Kit (Qiagen, Valencia, CA, USA). Chromosomal DNA from C. thermocellum strains was extracted using the Quick-gDNA MiniPrep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. Plasmids used in this study were constructed using Q5 High-Fidelity DNA polymerase (New England BioLabs, Ipswich, MA, USA) for PCR reactions, restriction enzymes (New England BioLabs, Ipswich, MA, USA), and the Fast-link DNA ligase kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer’s instructions. Plasmid pSKW01 (Fig. 1a) was constructed in two cloning steps. First, the 2.8 kb Cthe0423 expression cassette containing the regulatory region of Cbes2303 (S-layer protein), a C-terminal 6X Histidine-tag, and a Rho-independent transcription terminator was amplified by PCR with primers DC460 (with an added PvuI site) and DC461 (with an added NotI site) using pDCW144 as template. A 7.7 kb DNA fragment containing the pSC101 replication origin for E. coli, a putative C. thermocellum replication origin, an apramycin resistance gene cassette (AprR) and a C. bescii pyrF cassette was amplified with primers DC481 (with an added PvuI site) and DC482 (with an added NotI site) using pDCW89 as template. These two linear DNA fragments were digested with PvuI and NotI, and ligated to construct an 10.5 kb intermediate vector, pDCW148. In a second step, the 7.9 kb DNA fragment was amplified with primers DC576 (with and added PstI site) and DC466 (with an added SphI site, a 6X Histidine-tag, and a stop codon) using pDCW148 as a template. A 1.2 kb DNA fragment containing the coding sequence of bdhA (Teth39_1597) was amplified with DC577 (with an added PstI site) and DC578 (with an added SphI site) using pDCW171 as template. These two linear DNA fragments were digested with PstI and SphI, and ligated to construct a 9.1 kb plasmid, pSKW01. Plasmids pSKW02 and pSKW04 are identical to pSKW01 except for the promoter regions (Fig. 1). To make this change, a 0.3 kb DNA fragment containing the regulatory region of Clo1313_1809 was amplified with primers SK07 (with an added PstI site) and SK36 (with an added AvrII site) using C. thermocellum LL1005 genomic DNA (gDNA) as template. The 9.0 kb DNA fragment of pSKW01 without the regulatory region of Cbes2303 was amplified with primers SK04 (with an added PstI site) and SK28 (with an added AvrII site). These two linear DNA fragments were digested with PstI and AvrII, and ligated to construct a 9.3 kb plasmid, pSKW02 (Fig. 1b). In the case of plasmid pSKW04 (Fig. 1c), a 0.2 kb DNA fragment containing the enolase promoter region was amplified with primers SK19 (with an added PstI site) and SK26 (with an added AvrII site) using C. thermocellum LL1005 gDNA as template. E. coli BL21 cells were transformed by electroporation in a 1-mm-gap cuvette at 1.8 kV and transformants were selected for apramycin resistance. The sequences of all plasmids were verified by Automatic sequencing (Genewiz, South Plainfield, NJ, USA).
Electrotransformation of C. thermocellum cells was performed as previously described . Cultures, electro-pulsed with plasmid DNA (~0.5 μg), were recovered in CTFUD+ C medium  at 60 °C. Recovery cultures were transferred to liquid CTFUD-NY medium  without uracil to allow selection of uracil prototrophs. Cultures were plated on solid CTFUD-NY media to obtain isolated colonies, and DNA was isolated from transformants. Taq polymerase (Sigma, St. Louis, MO, USA) was used for PCR reactions to confirm the presence of the plasmid. PCR amplification with primers (SK04 and DC228) outside the gene cassette on the plasmid was used to confirm the presence of the plasmid with the bdhA gene. In the case of the JWCT08 (ΔpyrF + pSKW04) strain, integration of plasmid pSKW04 after a single crossover in the enolase promoter region was verified by PCR amplification with primers SK038 (specific for 0.1 kb upstream region of enolase promoter in JWCT08 gDNA) and DC461 (specific for plasmid pSKW04). Primers used for plasmid constructions and confirmation are listed in Additional file 1: Table S1.
Preparation of cell lysates and western blotting
Clostridium thermocellum strains (JWCT02, JWCT06, JWCT07, and JWCT08) were grown to mid-log or stationary phase at 60 °C in 20 mL CTFUD-NY medium without uracil. Cells were harvested by centrifugation at 6000×g at 4 °C for 15 min, and cell pellets were washed using 50 mM Tris–Cl buffer (pH 8.0) and resuspended in Tris–Cl buffer to OD600 20. Cells were lysed by boiling in the presence of SDS . Cell free extracts were electrophoresed in 4–15% gradient Mini-Protean TGX gels, that were either stained using Coomassie blue or were transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus and then probed with His-tag (6xHis) monoclonal antibody (1:5000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.
To test tolerance to various compounds, cultures of JWCT02, JWCT06, or JWCT08 strains were serially passaged every 24 h in 20 mL CTFUD-NY medium without uracil. After the second transfer, cultures were inoculated to the last culture to initial optical density (OD600) of 0.01. Batch fermentations were performed at 60 °C without agitation in 10 mL CTFUD-NY medium without uracil supplemented with either furfural or HMF at 0 or 10 mM concentrations. Optical cell density was monitored using a Jenway Genova spectrophotometer, measuring absorbance at 600 nm.
Cellobiose, glucose, acetate, lactate, ethanol, HMF, and furfural concentrations were determined by high-performance liquid chromatography (HPLC, Agilent Technologies 1200 Series). Metabolites were separated on an Aminex HPX-87H column (Bio-Rad Laboratories) at isocratic temperature (50 °C) and a flow (0.6 mL/min) rate in 5.0 mM H2SO4, and then passed through a refractive index detector (Agilent 1200 Infinity Refractive Index Detector). Peak areas and retention times were compared to known standards of the same analyte.
Enzyme activity assays
To prepare protein extracts, C. thermocellum cells were grown at 60 °C in 20 mL CTFUD-NY medium without uracil to an OD600 of 0.7–0.9, harvested by centrifugation at 6000×g at 4 °C for 10 min, suspended in CelLytic B cell lysis reagent (Sigma, USA), and lysed by a combination of 4X freeze-thawing and sonication (3 times for 15 s at 40 amps with 1 min rests on ice). Samples were centrifuged to separate protein lysate from cell debris, and the supernatants were used as protein extracts. To determine reduction activity of HMF and furfural, the reaction solution was formulated with 500 μL of 100 mM potassium phosphate buffer (pH 7.2), 300 μL of 33 mM furfural or HMF, and 100 μL of the crude enzyme solution. The absorbance change at 60 °C and 340 nm wavelength was monitored by a Jenway Genova spectrophotometer after addition of 100 μL of 1 mM NAD(P)H. One unit of reduction activity was defined as the amount of enzyme oxidizing 1 μmol NAD(P)H per minute. Protein concentrations were determined using the Bio-Rad protein assay kit.
- LB broth:
Luria Bertani broth
sodium lauryl sulfate
high-performance liquid chromatography
SK, DC, JE, and JW conceived and designed the study. SK and JG performed experiments. SK, JG, DC, JE, and JW interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.
We thank Shreena Patel for technical assistance, Jordan Russell and Adam Guss for critical review of the manuscript.
The authors declare that they have no competing interests.
Availability of supporting data
Plasmids and strains generated in this study are available upon request.
This work was supported by the BioEnergy Science Center, US DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the US DOE under contract DE-AC05-00OR22725. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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