Negatively Supercharging Cellulases Render Them Lignin-Resistant

May 22, 2017 - ... Crowley, Michael F.; Decker, Stephen R.; Himmel, Michael E.; Vinzant, Todd B. .... Gitlin, Irina; Carbeck, Jeffrey D.; Whitesides, ...
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Research Article pubs.acs.org/journal/ascecg

Negatively Supercharging Cellulases Render Them Lignin-Resistant Timothy A. Whitehead,*,†,‡ Chandra K. Bandi,§ Marissa Berger,∥ Jihyun Park,§ and Shishir P. S. Chundawat*,§ †

Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan 48824, United States Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, Michigan 48824, United States § Department of Chemical & Biochemical Engineering, Rutgers University, Piscataway, New Jersey 08854, United States ∥ Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey 08854, United States ‡

S Supporting Information *

ABSTRACT: Nonspecific adsorption of cellulases to lignin hinders enzymatic biomass deconstruction. Here, we tested the hypothesis that negatively supercharging cellulases could reduce lignin inhibition. Computational design was used to negatively supercharge the surfaces of Ruminoclostridium thermocellum family 5 CelE and a CelE-family 3a carbohydrate binding module fusion. Resulting designs maintained the same expression yield, thermal stability, and nearly identical activity on soluble substrate as the wild-type proteins. Four designs showed complete lack of inhibition by lignin but with lower cellulose conversion compared to original enzymes. Increasing salt concentrations could partially rescue the activity of supercharged enzymes, supporting a mechanism of electrostatic repulsion between designs and cellulose. Results showcase a protein engineering strategy to construct highly active cellulases that are resistant to ligninmediated inactivation, although further work is needed to understand the relationship between negative protein surface potential and activity on insoluble polysaccharides. KEYWORDS: Protein design, Lignocellulosic biomass, Lignin inhibition, Cellulase, Protein supercharging, Carbohydrate binding domain, Extractive ammonia pretreatment

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INTRODUCTION Cellulases are used industrially in the deconstruction of lignocellulosic biomass for the downstream conversion to biofuels and biochemicals. Lignocellulosic biomass contains hydrophobic and negatively charged aromatic polymers called lignin, which inactivate cellulases via nonproductive binding interactions.1 This nonproductive interaction is one key reason for high enzyme loading requirements and, therefore, high deconstruction costs for cellulosic biorefineries. Methods to minimize lignin-mediated enzyme inactivation include lignin removal prior to deconstruction,2 chemically modifying lignin or adding excipients to prevent enzyme adsorption,3 or increasing pH and decreasing temperature.4 An alternative solution is to re-engineer the amino acid sequences of cellulases to prevent lignin adsorption and inactivation. The advantage of this redesign strategy is that there may be no additional process costs, and reoptimization of process conditions may be unnecessary. Many fungal cellulases contain a catalytic domain (CD) and a family 1 carbohydrate-binding module (CBM1); the CBM has been mostly implicated in driving adsorption of full-length cellulases to lignin.5,6 Recently, a lignin-resistant 33-amino acid fungal cellulase CBM1 was engineered by screening single- and double-point mutants.7,8 Mutations of hydrophobic to acidic residues were strongly predictive of low lignin-binding CBM1,9 suggesting a possible mechanism of unproductive lignin © 2017 American Chemical Society

binding. Extension of the striking results from Strobel et al. to other cellulases not containing a CBM1 has not been demonstrated. Yet this is needed as, for example, β-glucosidases (which lack a CBM1) have also been shown to bind preferentially to lignin in complex mixtures.10 To enable a more comprehensive understanding of the relationship between protein surface potential and lignin adsorption, we computationally designed, constructed, and evaluated relative lignin binding for a set of protein designs where the hydrophobicity, net charge, and total charge density were varied within the physiological range of naturally occurring proteins.9 Results from this study showed that high net negative charge (−20 to −24 net charge) is the largest predictor of low lignin-protein adsorption. This finding mirrored a previous study where higher net negative charge for a Trichoderma reesei cellulase cocktail was also shown to correlate with higher Avicel conversion in the presence of lignin.11 In additional support, negatively supercharging proteins, either by incorporation of charge ladders12 or by genetically encoding multiple carboxylic acid-containing amino acids on surface-exposed residues,13 has previously been shown to render proteins aggregation resistant.14 On the basis of all of Received: April 18, 2017 Revised: May 12, 2017 Published: May 22, 2017 6247

DOI: 10.1021/acssuschemeng.7b01202 ACS Sustainable Chem. Eng. 2017, 5, 6247−6252

Research Article

ACS Sustainable Chemistry & Engineering the above findings, we tested a supercharging strategy to design lignin-resistant cellulases.

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slurry (3.5%) was weighed out on a dwb along with deionized water and sodium azide stock to a final PASC concentration of 10 g/L and 0.2% (w/v) sodium azide. PASC solution was prepared and stored at 4 C ahead of time. Zeta potential of PASC in 20 mM sodium acetate buffer, pH 6.5 was determined using a Zetasizer Nano ZSP from Malvern (Malvern, Worcestershire) according to Haarmeyer et al.9 Lignin Inhibition on PASC Hydrolysis Activity Assay. PASC slurry was warmed up to room temperature and shaken well before suspension with a magnetic stir bar and plate. The slurry was allowed to stir for 10 min to ensure homogeneous suspension prior to use. The PASC activity assay was based of a microplate based method developed previously.24 Briefly, 80 μL of suspended 10 g/L PASC slurry was added to select wells of a 96-well polypropylene roundbottom plate (0.3 mL total volume). Lignin slurry (5 g/L stock concentration), stirred and suspended in the same way, and 100 μL of suspended slurry was then added to each corresponding well. For assays conducted in the absence of lignin (or enzyme/substrate controls) 100 μL of the corresponding 50 mM MOPS buffer was added instead. Protein dilutions of CelE and CelE-CBM3a, wild type and respective designs, were prepared on ice in 10 mM MOPS buffer of appropriate pH, typically for a final concentration of 0.2 mg/mL (for a 5 mg enzyme/g cellulose loading). These dilutions were transferred in to a PCR plate on ice for subsequent pipetting using a multichannel pipet, and 20 μL of enzyme dilutions were added to make up the 200 μL total reaction volume. All assays were run in replicates of four on at least two separate days. The microplates were sealed with sealing mats and their contents gently suspended before placing the plates in an end-overend Hybridization mixing oven (5 rpm). The plates were incubated at 60 °C for a total reaction time of 48 or 120 h. Total reducing sugar concentrations was estimated using the dinitrosalicyclic acid (DNS) colorimetric assay method using glucose as standard.25 Briefly, the microplates were removed from the incubator and spun down at 3900 rpm for 10 min at 4 °C. The supernatant (30 μL) was removed and added into 60 μL of DNS reagent in PCR plates. The plates were sealed with aluminum foil covers and incubated in an Eppendorf Mastercycler at 95 °C for 5 min and 10 °C for 10 min. Next, 36 μL of incubated sample was mixed and pipetted in to 160 μL of deionized water in a clear flat-bottom 96-well polystyrene plates. The absorbance of the solution was measured at 540 nm using a SpectraMax M5e microplate reader (Molecular Devices, CA). Effect of NaCl concentration on PASC Hydrolysis. The effect of salt concentration on the activity of enzymes was studied for three different salt concentrations (0, 10, and 100 mM) at three different pH conditions. Briefly, in a 96 well microplate, 4 μg of protein was added to 800 μg of suspended PASC (dwb). The pH of reaction mixture was adjusted using 7.5 μL of 1 M MOPS buffer at the respective pH. Next, 0, 2, or 20 μL of 1 M NaCl was added to achieve the desired salt concentration in each reaction volume. The total reaction volume was adjusted to 200 μL using deionized water. The microplates were sealed and placed in and end-overend mixing incubator at 60 °C for 48 h. The percentage conversion of PASC to reducing sugars was measured via DNS assay method as described earlier. Kinetics of p-Nitrophenyl-Cellobioside Hydrolysis by WildType and Mutant Enzymes. Negatively supercharging of CelE CD or appended CBM3a domains is expected to impact binding to both lignin and cellulose because of the slight negative zeta-potential for the two biopolymers.9,26 To study the effect of supercharging only on the active site of the CD, the kinetics of the hydrolysis reaction on a chromogenic substrate analogue was studied. Briefly, in a 96 well microplate, 10 μg of protein was added to 100 μL of 2 mM paranitrophenol cellobioside (or pNPC). The pH of the reaction wells was adjusted to 7.5 using 7.5 μL of 1 M MOPS buffer and the total volume of 150 μL was adjusted using deionized water. The 96-well microplate was placed in incubator chamber of SpectraMax M5e spectrophotometer set at 25 °C and the absorbance of pNP (at 410 nm) in the reaction wells was taken every 15 min for a total duration of 4 h.

EXPERIMENTAL SECTION

Reagents. All chemicals were purchased from Sigma-Aldrich, unless otherwise noted. All DNA primers were ordered from IDT. Genetic constructs were sequence verified by Genewiz. Plasmid constructs have been deposited in the AddGene plasmid repository (www.addgene.org). Computational enzyme redesign. Proteins were designed from solved structures for CBM3a (PDB ID 1NBC) and CelE-CD (PDB ID 4IM4) and computationally characterized exactly according to Haarmeyer et al.,9 with the following modifications: surface residues were identified and computationally mutated to aspartate and glutamate within the Rosetta macromolecular software package.15 Residues were kept if they were at least 10 Å from any active site atom, and mutation did not change the Rosetta score by more than 2 Rosetta energy units. Designs were constructed from this list of residues, with final designs passing the filtering protocol detailed in Haarmeyer et al.9 Plasmid Construction. pEC-CelE-CD and pEC-CelE-CBM3a were gifts from B.G. Fox and used as base plasmids to express CelECD and CelE-CBM3a constructs, respectively.16,17 Designs were ordered as gBlocks (IDT) and cloned into parental plasmids using standard restriction enzyme molecular cloning procedures (NcoI/ KpnHF and KpnHF/PmeI restriction enzymes used for CelE-CD and CelE-CBM3a designs, respectively). All designs were as ordered except for D2-CBM3a, which contained an 8-aa in-frame insertion (VEGLRGAG) in the glycine-rich linker region connecting the CD and CBM3a domains. GFP and GFP labeled CBM3a proteins (pECGFP-CBM3a plasmid) were prepared for lignin binding assays as described elsewhere.17 Protein Purification and Characterization. CelE-CD, CelECBM3a, and assorted variants were expressed at the 100 mL scale and purified as previously described.18 Protein concentration was assessed by A280 using theoretical extinction coefficients of 85 750 and 120 150 cm−1 M−1 for CelE-CD and CelE-CBM3a variants, respectively. Apparent melting temperatures of protein variants were assessed using a modified SYPRO Orange thermal-shift assay19 exactly according to Klesmith et al.20 All samples were tested at least in triplicate. Preparation of Lignin Stocks. Lignin was extracted from corn stover using established extractive ammonia pretreatment process as outlined elsewhere.21 Briefly, extractive ammonia or EA pretreatment was conducted on milled corn stover using an ammonia-to-biomass (NH3:biomass) ratio of 6:1 (dry weight basis or dwb), a residence time of 30 min, and 10% dwb (w/w) biomass moisture. The untreated biomass lignin content was estimated using the NREL protocols (NREL/TP-510-42618 and NREL/TP-510-42620) to be approximately 14% Klason lignin and 2% acid-soluble lignin, on a dwb. The ammonia-soluble EA extractives recovered after removal of ammonia represented ∼45% of the original acid-insoluble lignin content. These extractives were further fractionated by sequential precipitation using ethanol and then water at room temperature, as discussed elsewhere.22 The ethanol-soluble and water-insoluble fraction (called fraction 3 or F3) containing majority of the lignin extracted during EA pretreatment (∼40 wt % of the total lignin present in the untreated corn stover), was lyophilized and stored at 4 C until needed for activity assays. Composition of the extracted lignin fraction determined using the NREL LAP protocol was estimated to be ∼93% (Klason acid insoluble lignin) on dry weight basis. Lignin stock solutions were fresh prepared before each binding or activity assay. Lignin powder was first weighed out and washed in 50 mM MOPS buffer of the required pH value. Washes were done at 12.5% dwb (w/v), where lignin was vortexed for 2 min and then centrifuged out at 10 000 rpm for 10 min at room temperature each wash step. Supernatant was removed after each wash step (for a total of five wash steps), and lignin solution volume was adjusted to 5 g/L. Preparation and Characterization of PASC Substrate Stocks. PASC (phosphoric acid swollen cellulose) or amorphous cellulose preparation details are provided elsewhere.23 Briefly, stock PASC 6248

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RESULTS AND DISCUSSION We tested our hypothesis that negatively supercharging cellulases renders them lignin resistant on the catalytic domain (CD) from a family 5 glycoside hydrolase (CelE-CD; gene ID Cthe0797) and a family 3a carbohydrate binding domain (CBM3a) from Ruminoclostridium thermocellum. These model proteins were chosen as solved crystal structures of the CD and the CBM are available,27 both protein domains can be expressed solubly in Escherichia coli at high yield, and the multifunctional fusion enzyme (CelE-CBM3a) has activity on both cellulose and hemicellulose.16,27 In our model system we chose to test lignin from extractive ammonia-pretreated corn stover (EA CS lignin) as lignin from ammonia-pretreated biomass has been shown to more closely resemble native lignin in structure with no significant degradation of carbohydrates into pseudolignin during ammonia pretreatment.21−23 We produced N-terminal His-tagged CelE-CD and the CelECBM3a fusion in E. coli, purified the enzymes using immobilized Ni-metal affinity chromatography, and tested their activity using simulated lignocellulosic biomass composed of phosphoric acid swollen cellulase (PASC) in the presence and absence of EA CS lignin. Figure 1a shows that both CelE-

tolerate substitutions to glutamate (GLU) or aspartate (ASP) and that are at least 10 Å away from active or binding sites. Supercharged designs were constructed where, for a subset of the positions identified above, GLU or ASP replaced the native amino acid. In total, two designs were created for CelE-CD (D1-CD, D2-CD) and three for CBM3a in the context of the CD-CBM3a fusion (D1-CBM3a, D2-CBM3a, D3-CBM3a). Compared to the wild type sequences, designs contained between 10 and 15 mutations and had 13−20 additional net negative charges (Table 1, Figure 2a−b). For this initial set of Table 1. Properties of Original and Supercharged Proteinsa variant CelE-CD D1-CD D2-CD CelECBM3a D1CBM3a D2CBM3a D3CBM3a

yield (mg/L)

Tm,app1 (°C)

Tm,app2 (°C)

net charge

± ± ± ±

no. mutations

90 200 220 180

57.1 60.7 58.0 44.7

0.0 0.0 0.0 0.3

n.a. n.a. n.a. 65.3 ± 0.3

−10 −23 −25 0

200

44.5 ± 0.4

65.0 ± 0.0

−16

12

170

44.6 ± 0.4

65.0 ± 0.0

−20

15

200

44.5 ± 0.5

65.0 ± 0.0

−18

14

n.a. 10 12 n.a.

a

n.a. not applicable; measurement error represents 1 s.d. of at least 3 independent measurements.

Figure 1. Lignin inhibits wild-type CelE-CD and CelE-CBM3a. (a) % cellulose hydrolysis yield in the presence or absence of 2.5 g/L EA CS lignin. Assays were conducted at 60 °C in MOPS buffer pH 6.5 with 5 g/L PASC and 5 mg enzyme/g cellulose loading for either 48 or 120 h. (b) Partition coefficients measured for adsorption of protein to EA CS lignin determined in MOPS buffer, pH 6.5 at 25 °C. For both graphs, error bars indicate standard error of the mean for at least 4 replicates and may be smaller than the symbol.

CD and CelE-CBM3a are inhibited in the presence of EA CS lignin. Higher pH partially removes the inhibitory effect of lignin but at the cost of lower overall conversion (Figure S1). The nonproductive binding of CBM3a to lignin was confirmed by determining the partition coefficient of a Green Fluorescent Protein (GFP) tagged CBM3a to lignin, which was 3.6-fold higher than GFP alone (Figure 1b). This suggests, as with other cellulases,26 that adsorption to lignin is correlated with inhibition in catalytic activity. Next, we used the Rosetta macromolecular software suite15 to construct negatively supercharged CBM3a and CelE-CD proteins. We identified positions on the protein surface able to

Figure 2. Structures and activity of supercharged designs. Predicted structures of (a) CD and (b) CBM3a designs. In the top row in each panel, locations of ASP or GLU mutations are shown in colored spheres, whereas active site (CD) or cellulose binding face (CBM3a) positions are shown in gray spheres. Only the CBM3a domain is shown for the fusion proteins. The bottom row in each panel shows electrostatic potential maps at ±2 kT/e. Maps were generated with Adaptive Poisson−Boltzmann Solver (APBS) and visualized using the APBS plugin in PyMol. (c) Relative activity for wild-type and designed proteins. Relative activity is determined as the initial velocity for 1.33 mM pNP-cellobioside normalized to the wild-type enzyme. Error bars indicate 1 s.d. of 3 independent measurements. 6249

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Figure 3. (a) Relative conversion of CelE-CD and designs (D1-CD, D2-CD) in the absence or presence of lignin. Relative conversion was determined by PASC conversion % at 120 h normalized by conversion % in the absence of lignin. (b) Conversion of PASC in the absence of lignin as a function of NaCl concentration at 48h. Panels c and d show the same assays for the CelE-CBM3a designs except that % PASC conversion was determined at 120 h. Error bars indicate 1 s.e.m. of 4 independent measurements.

designs we chose a negative net charge between −20 to −25 for the CD and −16 to −20 for the smaller CBM3a as our previous study showed that this portion of the phase space had the lowest nonspecific protein-lignin binding.9 Additional changes included removing an 18-amino acid unstructured N-terminal stretch on the CelE-CD designs and an inadvertent 8-amino acid insertion in the linker region for D2-CBM3a generated during the cloning process (see Methods; full sequences are provided in Notes S1−2). Genes encoding designs were custom synthesized and cloned into the parental plasmids. Designs were produced in E. coli and purified using the same procedure as the wild-type proteins. Expression yields were comparable for all CelE-CBM3a constructs, whereas CD designs yielded approximately 2-fold higher yields than that for CelE-CD (Table 1). Apparent melting temperatures, assessed by a SYPRO Orange thermalshift assay,19,20 were nearly equivalent between the designs and wild-type proteins (Table 1). CelE-CD and its designs exhibited one thermal transition, whereas CelE-CBM3a and its designs showed two thermal transitions presumably from separate unfolding of the two domains. Additionally, the relative catalytic activity of designs was tested using the soluble substrate p-nitrophenyl-cellobioside (pNPC), showing little difference for both CD redesigns and two of the three CBM3a variants (Figure 2c). Thus, with the exception of D2-CBM3a the designs were essentially indistinguishable from wild-type proteins based on thermal stability, expression, or catalytic activity on soluble substrates. We next tested the ability of the CD designs to resist ligninmediated inactivation. In contrast to CelE-CD, D2-CD had the same relative conversion on PASC in the presence or absence of AFEX CS lignin, while the relative error in activity for D1CD was too high to draw conclusions (Figure 3a). However, both designs showed much lower conversion of PASC under the same assay conditions (Figure 3b). Given that the enzymes had the same thermal stability and catalytic activity on soluble substrates, we hypothesized that cellulase-cellulose surface repulsion effects were likely responsible for the lower conversion. In particular, cellulose derived from lignocellulosic biomass is known to be negatively charged at pH 4−10 owing to

irregularly spaced carboxyl groups.28,29 In support of this finding we determined that the zeta potential of PASC at pH 6.5 was −3.5 ± 0.2 mV (n = 8, error 2 s.e.m.). Thus, if electrostatic repulsion between cellulose and supercharged proteins were responsible for the lower observed conversion we reasoned that increasing the salt concentration would rescue activity by dampening these repulsive interactions. Therefore, we tested PASC conversion of the cellulases in increasing NaCl concentration and in the absence of lignin. Whereas CelE-CD shows no difference in PASC conversion with increasing NaCl concentrations, supercharged designs showed strong increases in PASC conversion at both 10 mM and 100 mM NaCl (Figure 3b). In additional support of this hypothesis, all three of the CelE-CBM3a supercharged designs showed no inhibition in the presence of lignin (Figure 3c) and showed modest but progressive PASC conversion with increasing NaCl molarity (Figure 3d). For D3-CBM3a, the increase in activity in the presence of lignin is very intriguing and warrants investigation in future studies. In summary, we used computational protein design to test the hypothesis that negatively supercharging cellulases prevents inhibitory effect of lignin on cellulose conversion. Results support the hypothesis, although the supercharged designs had overall lower conversion on cellulose than wild-type enzymes. Evidence supports a mechanism of electrostatic repulsion between supercharged enzymes and cellulose contributing to this lower activity, as designs were as stable, nearly as active on soluble substrates, and increasing salt concentrations result in recovery of the majority of the activity. For these designs we have shown reduced lignin inhibition for only EA CS lignin. Thus, it remains to be seen whether our results hold for other lignin sources and extraction conditions. However, we have shown previously26 that nearly all cellulases are adsorbed irreversibly to lignin present in corn stover pretreated using three distinct pretreatment chemistries (dilute acid, ammonia, and ionic liquids). Mostly lignin enriched residue remaining after saccharification for dilute acid pretreated corn stover was found to irreversibly adsorb a slightly higher fraction of fungal cellulases (∼80−90%) compared to the other two pretreated substrates (∼50−80%). This can be partly explained due to the higher lignin content of dilute acid 6250

DOI: 10.1021/acssuschemeng.7b01202 ACS Sustainable Chem. Eng. 2017, 5, 6247−6252

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treated substrates and possibly due to the additional formation of pseudolignin during acid pretreatment that further contributes to protein adsorption. Therefore, it is likely that harsher pretreatment chemistry that result in residual lignins would still be relevant to our enzyme redesign approach to overcome lignin inhibition and will be a subject of future studies. From a sustainability perspective, a well-cited technoeconomic analysis shows that the cost savings by decreasing enzyme loading by 2-fold would be between $0.26−0.57 per gallon ethanol equivalent.30 Removing lignin inhibition is one way to decrease enzyme loadings provided that the cellulases also maintain equivalent activity. However, these current enzyme designs do not maintain sufficient activity on insoluble substrates to lower overall loading. Thus, immediate next steps focus on tailoring the negatively charged surface to fine-tune the balance between cellulose activity and lignin inhibition and to further investigate the relationship between activity on cellulose and pH, salt concentration, lignin source and pretreatment, and protein surface potential. In the current study we made designs containing 10−15 mutations to negatively charged amino acids, as our previous study9 indicated that designs in this portion of the surface potential phase space would have minimal lignin inhibition. However, a more modest number of mutations in further designs may be warranted. As robust computational protocols exist to supercharge proteins without impacting expression yields, function, or stability,7,31 designing additional active cellulases with variegated electrostatic surface potentials would be relatively trivial.

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REFERENCES

(1) Berlin, A.; Balakshin, M.; Gilkes, N.; Kadla, J.; Maximenko, V.; Kubo, S.; Saddler, J. Inhibition of cellulase, xylanase and betaglucosidase activities by softwood lignin preparations. J. Biotechnol. 2006, 125 (2), 198−209. (2) Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; Davis, M. F.; Davison, B. H.; Dixon, R. A.; Gilna, P.; Keller, M.; Langan, P.; Naskar, A. K.; Saddler, J. N.; Tschaplinski, T. J.; Tuskan, G. A.; Wyman, C. E. Lignin Valorization: Improving Lignin Processing in the Biorefinery. Science 2014, 344 (6185), 1246843. (3) Yang, B.; Wyman, C. E. BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol. Bioeng. 2006, 94 (4), 611−617. (4) Lou, H. M.; Zhu, J. Y.; Lan, T. Q.; Lai, H. R.; Qiu, X. Q. pHInduced Lignin Surface Modification to Reduce Nonspecific Cellulase Binding and Enhance Enzymatic Saccharification of Lignocelluloses. ChemSusChem 2013, 6 (5), 919−927. (5) Rahikainen, J. L.; Evans, J. D.; Mikander, S.; Kalliola, A.; Puranen, T.; Tamminen, T.; Marjamaa, K.; Kruus, K. Cellulase−lignin interactionsThe role of carbohydrate-binding module and pH in non-productive binding. Enzyme Microb. Technol. 2013, 53 (5), 315− 321. (6) Vermaas, J. V.; Petridis, L.; Qi, X.; Schulz, R.; Lindner, B.; Smith, J. C. Mechanism of lignin inhibition of enzymatic biomass deconstruction. Biotechnol. Biofuels 2015, 8, 217−223. (7) Strobel, K. L.; Pfeiffer, K. A.; Blanch, H. W.; Clark, D. S. Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition. Biotechnol. Bioeng. 2016, 113 (6), 1369− 1374. (8) Strobel, K. L.; Pfeiffer, K. A.; Blanch, H. W.; Clark, D. S. Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin. J. Biol. Chem. 2015, 290 (37), 22818−22826. (9) Haarmeyer, C. N.; Smith, M. D.; Chundawat, S. P. S.; Sammond, D.; Whitehead, T. A. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein. Biotechnol. Bioeng. 2017, 114, 740−750. (10) Yarbrough, J. M.; Mittal, A.; Mansfield, E.; Taylor, L. E., II; Hobdey, S. E.; Sammond, D. W.; Bomble, Y. J.; Crowley, M. F.; Decker, S. R.; Himmel, M. E.; Vinzant, T. B. New perspective on glycoside hydrolase binding to lignin from pretreated corn stover. Biotechnol. Biofuels 2015, 8, 214. (11) Nordwald, E. M.; Brunecky, R.; Himmel, M. E.; Beckham, G. T.; Kaar, J. L. Charge Engineering of Cellulases Improves Ionic Liquid Tolerance and Reduces Lignin Inhibition. Biotechnol. Bioeng. 2014, 111, 1541−1549. (12) Gitlin, I.; Carbeck, J. D.; Whitesides, G. M. Why are proteins charged? Networks of charge-charge interactions in proteins measured by charge ladders and capillary electrophoresis. Angew. Chem., Int. Ed. 2006, 45 (19), 3022−60. (13) Lawrence, M. S.; Phillips, K. J.; Liu, D. R. Supercharging proteins can impart unusual resilience. J. Am. Chem. Soc. 2007, 129 (33), 10110−10112. (14) Miklos, A. E.; Kluwe, C.; Der, B. S.; Pai, S. P.; Sircar, A.; Hughes, R. A.; Berrondo, M.; Xu, J. Q.; Codrea, V.; Buckley, P. E.; Calm, A. M.; Welsh, H. S.; Warner, C. R.; Zacharko, M. A.; Carney, J. P.; Gray, J. J.; Georgiou, G.; Kuhlman, B.; Ellington, A. D. Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies. Chem. Biol. 2012, 19 (4), 449−455. (15) Leaver-Fay, A.; Tyka, M.; Lewis, S. M.; Lange, O. F.; Thompson, J.; Jacak, R.; Kaufman, K.; Renfrew, P. D.; Smith, C. A.; Sheffler, W.; Davis, I. W.; Cooper, S.; Treuille, A.; Mandell, D. J.; Richter, F.; Ban, Y. E.; Fleishman, S. J.; Corn, J. E.; Kim, D. E.; Lyskov, S.; Berrondo, M.; Mentzer, S.; Popovic, Z.; Havranek, J. J.; Karanicolas, J.; Das, R.; Meiler, J.; Kortemme, T.; Gray, J. J.; Kuhlman, B.; Baker, D.; Bradley, P. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 2011, 487, 545−74. (16) Deng, K.; Takasuka, T. E.; Heins, R.; Cheng, X. L.; Bergeman, L. F.; Shi, J.; Aschenbrener, R.; Deutsch, S.; Singh, S.; Sale, K. L.;

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01202.

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Research Article

DNA and amino acid sequences of all proteins and effect of pH on CelE-CD hydrolysis yield for PASC (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Phone: (517) 432-2097. E-mail: [email protected]. *Phone: (848)-445-3678. E-mail: shishir.chundawat@rutgers. edu. ORCID

Timothy A. Whitehead: 0000-0003-3177-1361 Funding

This work was supported by US National Science Foundation Award #1236120 CBET (to T.A.W. and S.P.S.C.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank E. Maurer for help with protein expression and purification, C. Haarmeyer for initial computational designs, Dr. Leonardo Sousa (Michigan State University) for assistance with lignin extraction, Dr. D. Sammond (NREL) for helpful comments, and Prof. Brian Fox (University of Wisconsin Madison) for providing the CelE wild-type plasmid. 6251

DOI: 10.1021/acssuschemeng.7b01202 ACS Sustainable Chem. Eng. 2017, 5, 6247−6252

Research Article

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DOI: 10.1021/acssuschemeng.7b01202 ACS Sustainable Chem. Eng. 2017, 5, 6247−6252