Developing riboswitch-mediated gene regulatory controls in

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Letter

Developing riboswitch-mediated gene regulatory controls in thermophilic bacteria Joan Marcano, Jonathan Lo, Ambarish Nag, Pin-Ching Maness, and Katherine Chou ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00487 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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ACS Synthetic Biology

Developing riboswitch-mediated gene regulatory controls in thermophilic bacteria Joan G. Marcano-Velazquez†, Jonathan Lo†, Ambarish Nag‡, Pin-Ching Maness†,*, and Katherine J. Chou†,* †Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United states ‡Computational Science Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United states

Graphical Abstract

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Abstract Thermophilic bacteria are attractive hosts to produce bio-based chemicals. While various genetic manipulations have been employed in the metabolic engineering of thermophiles, a robust means to regulate gene expression in these bacteria (~55°C) is missing. Our bioinformatic search for various riboswitches in thermophilic bacteria revealed that major classes of riboswitches are present, suggesting riboswitches’ regulatory roles in these bacteria. By building synthetic constructs incorporating natural and engineered purine riboswitch sequences originated from foreign species, we quantified respective riboswitches activities in repressing and up-regulating gene expression in Geobacillus thermoglucosidasius using a green fluorescence protein. The elicited regulatory response was ligand-concentration-dependent. We further demonstrated that riboswitch-mediated gene expression of adhE (responsible for ethanol production) in Clostridium thermocellum can modulate ethanol production, redirect metabolites, and control cell growth in the adhE knockout mutant. This work has made tunable gene expression feasible across different thermophiles for broad applications including biofuels production and gene-to-trait mapping. Key Words: riboswitch, inducible and repressible systems, thermophiles, Clostridium thermocellum, gene regulation


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Thermophilic and anaerobic bacteria are attractive hosts for the industrial production of highvalued chemicals. Higher fermentation temperatures (i.e., 55 – 60 °C) lead to faster feedstock conversion rates, reduce contamination in the bioreactors with ambient microorganisms, and reduce the cooling costs associated with the biomass pretreatment steps. Collectively, these merits make thermophiles favorable platform organisms in a sustainable bio-based economy1. Various genetic engineering approaches have been implemented in thermophilic bacteria to produce target chemicals at high titers. These approaches include targeted gene deletions, replacement, and insertions that aim to redirect the carbon and electron flux towards the desired product and gain basic understanding of the bacteria2-5. In addition, the expression of non-native metabolic pathways have been introduced to thermophiles to generate compounds like isobutanol6, or expand their carbon source utilization7. In all cases, reliable and characterized genetic “parts” such as promoters, ribosomal binding sites, and gene regulatory elements are important in strain engineering toward the desired phenotype. Such tools are readily available for model organisms such as Escherichia coli and Saccharomyces cerevisiae8,9. However, finely tunable gene expression is largely missing in thermophilic bacteria with limited options. Characterized regulatable promoters using native inducers like xylose10 can unintendedly regulate additional endogenous genes. Others such as the laminaribiose promoter require an expensive inducer molecule11-13. Furthermore, up- or down-regulations of a gene may be required to fundamentally understand the gene functions when knock-ins and knockouts are not viable. For these reasons, we sought to establish genetic components that could regulate gene expression in thermophilic bacteria. These tools will also assist in elucidating the physiology of thermophiles as well as enabling genome engineering approaches including CRISPR-cas9. Riboswitches are mRNA leader sequences that can regulate gene expression upon binding to a small molecule as a ligand. They are composed of two domains, the aptamer and the expression platform (Figure 1a). The aptamer domain folds into a structure that creates a high affinity binding pocket to selectively bind the ligand. The expression platform mediates gene expression by forming transcriptional terminators or preventing protein translation by secluding the ribosomal binding site14. The natural riboswitch aptamers have evolved to recognize a wide variety of ligands such as nucleotide derivatives, co-factors, amino acids, ions, and others14. Riboswitches have been repurposed in various species to alter gene expression and as biosensors to monitor the concentration of natural intracellular compounds15. Furthermore, a riboswitch can be adapted to recognize orthogonal ligands by mutagenesis of its binding pocket thereby expanding the molecules it recognizes16. These riboswitch variants can in theory regulate multiple individual genes in response to different ligands in parallel. In addition, it is possible to generate novel RNA aptamers by artificial evolution and then couple them to natural expression platform to create functionally new riboswitches17. The wide range of applications makes riboswitches attractive regulators for the development of inducible and repressible genetic systems in thermophiles.



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Figure 1. Riboswitches in thermophilic bacteria. a) Example of a transcriptional “off” riboswitch mechanism of transcription termination; aptamer – green shade, expression platform-blue shade. When the ligand binds to the aptamer domain during transcription, it allows the expression platform to fold into a transcriptional terminator that will prevent the RNA polymerase to transcribe the downstream gene. b) Distribution of 911 riboswitch sequences identified using INFERNAL in 73 representative thermophilic bacterial genomes. Previously, the only in vivo characterization on riboswitch activity at an elevated temperature is based on a fluoride riboswitch in the archaea Thermococcus kodakarensis which can grow in the range of 60°-100°C18. Since the transcription and translation machineries for archaea species is more akin to eukaryotic systems19, the characterization and engineering of riboswitch dedicated to thermophilic bacteria is necessary for its implementation. In this work we design and build plasmid-based synthetic constructs incorporating both natural and engineered riboswitch sequences originated from foreign bacteria and measure the activity of riboswitches using a thermotolerant green fluorescence protein20. We also assess riboswitchdependent tuning of a gene responsible for ethanol production (adhE) in Clostridium thermocellum (C.t.). Our work herein represents the first in vivo characterization of riboswitches at 55°C and highlights the feasibility of using mesophilic riboswitches to regulate gene expression in thermophiles. Results and Discussion Previous bioinformatics analysis had uncovered that riboswitches are present throughout the majority of bacterial clades in nature21. However, no study has focused on the diversity and distribution of riboswitch sequences specifically in thermophilic bacteria. Using 4 ACS Paragon Plus Environment

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the INFERNAL software package22, which utilizes covariance models (CMs) built with the RNA sequence and structural conservation, we identified 911 unique riboswitch sequences among 73 thermophilic genomes analyzed from the RefSeq database (Figure 1b). Only 5 of the analyzed genomes belonging to the genus of Hydrogenobacter and Hydrogenobaculum do not contain riboswitches. Of the 25 types of riboswitch searched with the CM, only 17 were detected, leaving 8 types of riboswitches not detected in any of the analyzed genomes (Supporting File R1). Notably, the riboswitches that were not found in the thermophilic genomes correspond to variants of S-adenosylmethionine and pre-quenosine-1 (preQ1) riboswitches which also occur in low frequencies among general bacterial databases21. Results showed that major classes of riboswitches are present in thermophiles and the relative abundance of each class resembles those observed for all bacterial classes21. The genomes that we characterized as “thermophilic” are for organisms that have an optimal growth temperature greater than 50°C, suggesting that riboswitches are prevalent regulatory components among thermophiles. We also have identified riboswitches in organisms classified as hyperthermophiles (e.g., Caldicellulosiruptor bescii) that grow optimally at 75°C, which suggest that riboswitches can fold into their functional conformations at higher temperatures. It is important to note that our bioinformatics analysis did not encompass all riboswitch classes known to-date but only those we could obtain a co-variance model in the Rfam database. Riboswitch classes like the tungsten, nickel, and fluoride riboswitches21 were not included in our analysis. Nevertheless, this data indicates that riboswitches play a regulatory role in the metabolic pathways of thermophilic bacteria. Among the riboswitches uncovered in thermophiles, purine riboswitches are promising candidates of synthetic regulators for gene expression as their structural elements have been characterized extensively to facilitate the rational design of these sequences for altered functionalities16. These riboswitches bind to either guanine or adenine that can be added to the culture media at relatively high concentration without toxicity. Within the purine riboswitch class, only one nucleotide difference in the binding pockets dictates whether the riboswitch binds guanine or adenine, which highlights their ligand-binding specificity23. Moreover, purine riboswitches have been utilized in synthetic biology due to their small size and modularity between the aptamer and the expression platform24. Interestingly, we only found guaninesensing purine riboswitches among the thermophilic genomes we analyzed, suggesting that thermophiles might have developed an alternative mode of adenine regulation. To use natural purine riboswitches found in thermophiles for applications, we investigated the two guanine-binding riboswitch sequences present in the genome of Thermoanaerobacterium saccharolyticum (T.s.) which is an anaerobic and thermophilic bacterium (optimal growth 60°C). The two purine riboswitches in T.s. are located upstream of the genes encoding for xanthine permease (tsac_2584) and inosine-5’-monophosphate dehydrogenases (tsac_2588), which is consistent with previous findings and strongly suggests their regulatory roles in purine metabolism25. The secondary structures of these RNAs obtained with the Mfold web server also indicate that these sequences adopt the canonical structure of purine riboswitches (Supporting Figure S1)23,26. To evaluate the regulatory activities of T.s. riboswitches, we employed a thermophilic fluorescence reporter assay using super-folder green fluorescence protein (sfGFP) previously demonstrated to be active in the aerobic thermophile, Geobacillus thermoglucosidasius (G.t.), 5 ACS Paragon Plus Environment


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at 55°C20. Both purine riboswitches from T.s. were inserted downstream of the pRp1S strong promoter respectively in plasmid pG1AK-sf such that the riboswitch will control the expression sfGFP once it is introduced into G.t. (Figure 2a, Supporting Table S1). A summary of all plasmids constructed for this study is shown in (Supporting Table S2). We observed that the G. t. cultures achieved a maximal normalized fluorescence at the mid-exponential growth stage and we consistently used this growth phase to compare the riboswitch regulatory effect at different ligand concentrations (Supporting Figure S2). To trigger the regulatory response from the riboswitches we used guanosine as the ligand, as cells can import the compound and convert it into guanine enzymatically and guanosine has been previously used to characterize guaninesensing riboswitches in gram-positive bacteria27. Under our studied conditions, the addition of the ligand did not substantially impact the growth of the cells. The extent of influence the ligands have on growth at the highest tested ligand concentrations are shown in Supporting Figures S3 and S4. Results showed that when 0.25 mM of guanosine was added, the maximal normalized fluorescence was reduced from 38 ± 1 to 15 ± 0.1 fluorescence units (~2.5-fold change) in the cultures carrying the Tsac 2588 riboswitch, and from 20 ± 2 to 2.8 ± 0.1 (~7-fold change) for the cultures carrying the Tsac 2584 riboswitch (Figure 2c). No repression is observed for cells carrying the parental pG1AK plasmid which constitutively expresses sfGFP in the absence of a riboswitch. As an additional control, we introduced a point mutation in the Tsac 2584 aptamer domain at a position (C74U) responsible for its interaction with guanine. With this mutation, we no longer observed the guanosine-dependent repression seen in the wild-type sequences (Supporting Figure S5). For the Tsac 2584 riboswitch, the fluorescence level approaches the background autofluorescence with increasing level of guanosine indicating a tight repression, while the Tsac 2588 riboswitch displays about 30% of residual fluorescence in comparison to the control pG1AK parental plasmid. Note that the insertion of either riboswitch to the system causes a basal reduction in gene expression without adding guanosine. This could be due to the intracellular guanine levels triggering a response off the riboswitch as it has been observed in previous reports27. In addition, this reduction in gene expression could also result from changing the sequence of the initial transcribed region, which can negatively impact transcription frequencies28.


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Figure 2. Quantifying the activities of riboswitches in regulating gene expression in thermophiles. a) We use sfGFP as a reporter in G.t. The promoter, RBS, and sfGFP are parts of the parental plasmid pG1AK. b) Riboswitch construct designed to regulate the adhE gene expression in C.t. The RBS* is composed of 21 nucleotides upstream of the ATG of gene clo1314_2638 in C.t. c) Regulatory response of the two T.s. purine riboswitches using sfGFP as a reporter in G.t. Guanosine was used instead of guanine due to its higher solubility and its previously shown ability to affect purine riboswitches in gram-positive bacteria27. Data is represented with n ± standard deviation (stdev) with n = 3 biological replicates. These results represent the first in vivo characterization of riboswitch sequences in thermophilic bacteria. The regulatory mode of the T.s. riboswitches in question was experimentally determined to be repression rather than activation, and the reporter system enabled the measurement of riboswitch activity at 55°C. The two T.s. riboswitches demonstrated tunable gene repression to different extents and ranges, which is useful for different applications in thermophiles. While the ability to tune down gene expression is valuable for research and biotechnological applications, riboswitches can also serve as inducers of gene expression upon binding a ligand. To establish an inducible system for thermophiles, we explored the feasibility of using riboswitch sequences from a mesophilic organism to upregulate gene expression in a thermophile. The pbuE riboswitch from Bacillus subtilis is an adenine-binding riboswitch that has been widely utilized as a biochemical and biophysical model for RNA structure-to-function relationships23. This riboswitch contains a strong transcriptional terminator in its expression platform and can tightly control the transcription of a downstream gene. When the ligand binds the aptamer, the riboswitch conformation prevents the terminator from interfering with the polymerase thereby allowing transcription of the downstream gene(s)29. 7 ACS Paragon Plus Environment


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Figure 3. 2-aminopurine (2-AP)-dependent induction of gene expression mediated by a mesophilic pbuE riboswitch tested in G.t. at 55 °C. a) Normalized fluorescence level in cultures with increasing concentrations of 2-AP. b) Regulatory response is detected at [2-AP] at 1, 3, 6, and 10 M for riboswitch mutant P1 = 10. Data is represented with n ± stdev and n = 3 biological replicates. C) The secondary structures without and with mutations elongating P1 that stabilizes the aptamer domain. We introduced the pbuE riboswitch into our construct following the same scheme illustrated in Figure 2a and quantified the regulatory response with normalized fluorescence in increasing concentrations of 2-aminopurine (2-AP) in G.t. using sfGFP at 55°C (Figure 3a). 2-AP as a ligand has been shown to elicit a response from pbuE riboswitch both in vitro and in vivo29 at 37°C. In the absence of 2-AP, all constructs containing a pbuE-derived riboswitch displayed a fluorescence level similar to the background from cells not expressing sfGFP, demonstrating a tight transcriptional repression from the riboswitch’s terminator. When 2 mM of 2-AP was added to the culture, the normalized fluorescence increased from 1.4 ± 0.02 to 23 ± 1.8 (~16fold change) which accounts for ~40% of the fluorescence observed in the culture constitutively expressing sfGFP (59 ± 8.9) without a riboswitch. While this riboswitch is responsive to 2-AP in upregulating the gene expression in G.t., high dose of 2-AP required to elicit the regulatory response is not optimal. To increase the sensitivity and dynamic range of the inducible system, we introduced rational mutations in the 5’ side of the P1 paired region to elongate P1 from 5 base pairs (wt) to 8 and 10 base pairs which favored the aptamer domain over the terminator (Figure 3c). These mutations had been previously shown to maintain a functional riboswitch at 8 ACS Paragon Plus Environment

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37°C, but the effects of the mutations in the riboswitch’s sensitivity were not explored29. Results showed that at 2 mM 2-AP, riboswitches with elongated P1 displayed an increased maximal normalized fluorescence of 39.1 ± 1.1 (~24-fold change) and 41.1 ± 0.6 (~29 fold change) for P1 = 8 bp and P1 = 10 bp, respectively. The fluorescence becomes ~80% of that observed by the constitutive promoter lacking a riboswitch, and the mutated riboswitches displayed an increased regulatory range by ~22% over the wild-type riboswitch at 2 mM 2-AP. Moreover, the riboswitch mutant (P1 = 10 bp) can be activated to 50% expression of the no riboswitch control at a concentration of 300 μM, indicating improved sensitivity to 2-AP over the wild-type sequence. Given the increased sensitivity in the riboswitch with P1 = 10 bp, we repeated the experiment but titrating 2-AP in the low micromolar range (Figure 3b). Our results indicate that the riboswitch can activate gene expression of sfGFP at a concentration as low as 1 μM. These results demonstrated the concept that the range of expression a riboswitch can regulate and its sensitivity to the ligand can be increased with rational engineering of its secondary structure. Beyond characterizing and demonstrating the activity of riboswitches activities in G.t. with a reporter protein, it would beneficial if the riboswitches can be applied to tuning the expression of a gene important in a functional pathway in vivo, thereby demonstrating its physiological relevance. We chose to test the riboswitch in a thermophilic anaerobic bacterium Clostridium thermocellum owing to its importance in biotechnological applications30 and the lack of inducible promoters besides a costly laminaribiose-inducible system13.

Figure 4. Fermentation product profile for the riboswitch-controlled expression of adhE gene in C.t. The fermentation products in the adhE complemented strain were quantified using HPLC and the percentages represent the average of 3 experiments. Ethanol production by C.t. was chosen to be the readout of the riboswitch activity. Ethanol production in C.t. relies primarily on the NADH-dependent bifunctional alcohol and aldehyde dehydrogenase encoding gene, adhE (clo1313_1798), as the deletion of adhE gene reduced ethanol production by >90%31 (data for this study, Figure 4). The production of ethanol 9 ACS Paragon Plus Environment


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provides an important NADH sink for the bacteria and adhE is one of the most highly expressed genes in C.t. Deleting the chromosomal copy of adhE resulted in a strain with impaired growth in rich media (max OD600 became ~0.4, Figure 5a red line). To assess the effect of riboswitch activity on tuning fermentation products and growth in the 2-AP induced versus uninduced states, C.t. adhE gene was transcriptionally fused to the clo1313_2638 promoter native to C.t. and expressed from a plasmid pJMK06 (Supporting Figure S6) in an adhE mutant at 55°C. In the adhE complementation strain, growth was restored to the wild-type level (max OD600 ~ 1.4, Figure 5a black line, Supporting Figure S7). The fermentation products ethanol, lactic acid, and acetic acid quantified 40 hours post inoculation were normalized by the sugar consumed. Without a riboswitch, ethanol accounted for 55% of the fermentation products while lactic acid accounted for 15 % (Figure 4) in the adhE complementation strain. This product distribution was similar to that of the wild-type strain (about 55% ethanol and 20% lactic acid, Supporting Table S3). We then introduced the pbuE-derived riboswitches including the wild-type and the two elongated P1 riboswitches into the clo1313_2638 promoter to drive the expression of adhE following the scheme shown in Figure 2b and Supporting Table S1. Ethanol production was below 22% in all the uninduced conditions, with 52 – 57 % of fermentation products as lactic acid. When 2-AP was added to the culture to induce adhE expression, ethanol production remained unchanged in the culture without a riboswitch. However, for the cultures with P1=8 and P1=10 riboswitch variants, ethanol accounted for 41% and 48% of the fermentation output, respectively (Figure 4). Note that as ethanol production increased in the 2-AP induced cultures, lactic acid production decreased. Since lactic acid production, catalyzed by lactate dehydrogenase, is also a NADH sink in C.t., its production may compensate for the removal of NADH sink when ethanol is not produced in the mutant. This data demonstrated riboswitchmediated tunable induction of adhE gene expression and consequently directed the metabolic flux between lactic acid and ethanol under this design. We also characterized the growth response to riboswitch-mediated adhE expression in the adhE complementation strain with and without 2-AP. In the absence of 2-AP, growth of the cultures expressing the adhE under the control of the riboswitches resembled that of the adhE mutant (with no plasmid), indicating that the riboswitch was indeed tightly repressing the adhE gene (Figure 5b-d). When 1 mM 2-AP was added to the media, no substantial improvement in growth was observed for the culture carrying the wild-type riboswitch plasmid (Figure 5b), but growth improvement was observed in the cell culture carrying the pbuE P1 mutants (Figure 5cd). For the riboswitch mutant P1=10, the maximal OD600 reached 0.6 without 2-AP but reached 1.4 with 1 mM 2-AP. The riboswitch activity resulted in a growth phenotype and restoring the adhE expression appeared to improve the bacterial fitness. This data is consistent with the riboswitch activities observed in the G.t. reporter assay in which higher levels of sfGFP at 1 mM of 2-AP was observed in the P1=10 mutant than that in wild-type (Figure 3a).


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Figure 5. Growth profile for the plasmid-based complementation of adhE gene under the control of pbuE riboswitch. Red line: adhE mutant, black and blue lines: complemented strain without and with 2-aminopurine. The error bars represent the standard deviation for the average of three biological replicates. In this work, we performed a bioinformatics search for riboswitch sequences present in thermophilic bacteria, which indicates their significance in regulatory roles in thermophiles. The reporter assay provides a platform that enables rapid characterization of the natural and rationally engineered riboswitches activities at elevated temperatures. By illustrating a tunable expression system applicable in the industrially relevant microbe C.t., we addressed the challenge in strain engineering when certain gene deletions are not be feasible. Our work also shows ligand-dependent (Figures 2 and 3) and riboswitch-dependent (Figure 5) gene regulations, which can be implemented to elicit a desired response in different dynamic range. By demonstrating that a set of riboswitches can function in two thermophilic bacteria (G.t. and C. t.), our data indicates that these gene-expression fine-tuning tools can be “portable” across 11 ACS Paragon Plus Environment


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different species. As our findings reveal that mesophilic riboswitches can function in thermophiles, it unlocks the potential to use various characterized mesophilic riboswitches to regulate thermophilic gene expression. In summary, inexpensive and finely tunable gene expression is needed to advance basic science and is fundamental to strain engineering but is lacking in majority of the organisms. To our knowledge, this work establishes the first tunable, inducible, and repressible gene regulatory systems in thermophiles. Materials and Methods Media, plasmids, strains All G.t. experiments were perform using the DSM 2542T strain (Geobacillus Genetic Stock Center, Colombus OH). All experiments in C.t. were performed in a DSM 1313 (DSMZ, Brunswick Germany) derived strain with the hpt (clo1313_2927) gene deleted for counterselection purposes32 and hpt is referred to as the wild-type host strain. The two purine riboswitch sequences from T.s. were amplified with PCR from the DSM 8691 strain (DSMZ, Brunswick Germany). The replicating plasmid pG1AK for G.t. was a kind gift from Dr. Tom Ellis. Plasmid pJMK06 was developed in-house for gene expression in C.t. (Supporting Figure S5). PCR and cloning were performed using Q5 High fidelity polymerase and Hi-Fi DNA assembly master mix, respectively (New England Biolabs, MA). Recipes for the defined glucose medium (DG) and CTFUD-rich medium can be found in the Supporting Methods. Both C.t. and G.t. were transformed by electroporation following previous published protocols19,32. The resulting G.t. transformants were cultivated in a defined glucose medium (DG) while the fluorescence of the culture was tracked. The adhE gene was deleted in a C.t. background strain with an altered hydrogenase (Clo1313_1791) gene expression to presumably compensate for the redox imbalance created by the lack of ethanol formation. See Supporting Methods for details. Identification of riboswitches in thermophilic bacteria The genome sequences of the thermophilic bacteria were obtained from the RefSeq database and analyzed for the presence of riboswitches using the INFERNAL software package. A full description of the bioinformatics analysis can be found in the Supporting Methods. Riboswitch activity assays in Geobacillus thermoglucosidasius. G.t. strains transformed with different plasmids were grown aerobically overnight in 5 mL TGP media (Supporting Methods) with 12.5 μg/mL kanamycin at 55°C and shaken at 200 rpm. The freshly grown overnight culture was used to inoculate 5 mL of fresh TGP media with kanamycin and incubated in the same condition for ~5 hrs. when the cultures reached OD600 ~1.0. This secondary growth culture was used to inoculate 5 mL of Defined Glucose Media (DG) with 250fold dilution and was sub-aliquoted into a 96-well plate with 200 microliters per well where different concentrations of the effector ligand was added. The plate was incubated in a Biotek Synergy Neo2 plate reader and the OD600 and the fluorescence intensity was tracked with a (440/20 excitation, 500/27 emission) filters for 24 hrs. To quantify the normalized fluorescence, 12 ACS Paragon Plus Environment

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the value of the raw fluorescence was taken when the cells were in mid-exponential phase (OD600 0.4 -0.6) and divided by the optical density to normalize the fluorescence. Clostridium thermocellum growth C.t. strains were routinely grown at 55°C and shaken at 100 rpm in sealed “Balch type” tubes containing 10mL CTFUD rich media and 0.5% (w/v) cellobiose as the main carbon source. Strains expressing adhE with and without a pbuE riboswitch upstream were initially cultured in tubes until mid-log phase (OD600~0.3-0.4) and then diluted to an OD600 ~0.1. To measure cell growth, the diluted culture was sub-aliquoted into a 96-well plate where 2-AP was added to the concentrations indicated in the experiment in triplicates. Cell growth was continuously measured in Greiner-Bio 96-well plates incubated anaerobically in a BioTek Synergy Neo2 plate reader. To measure C.t. fermentation products in strains carrying the respective plasmids expressing the adhE gene under the control of pbuE riboswitch and mutated riboswitches, cells were grown in 10mL CTFUD rich media containing 0.5% (w/v) cellobiose. After 40 hours of incubation, the fermentation products were measured by HPLC (Supporting Methods). Supporting Information: Figures S1-S7; Tables S1-S3; Supporting Methods; Supporting References Supporting File R1: Table of thermophilic bacteria included in our analysis. Acknowledgements We thank Dr. Tom Ellis for the kind gift of plasmid pG1AK. This work was supported by National Renewable Energy Laboratory Director's Fellowship Program funded by Laboratory Directed Research and Development Subtask 06271601 (J.G.M.), the US Department of Energy (DOE) Energy Efficiency and Renewable Energy (EERE) Fuel Cell Technologies Office under Contract DE-AC36-08-GO28308 (J.L, P.C.M. and K.J.C) and National Renewable Energy Laboratory Research Data Initiative (A.N) References

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