Engineered Lignin in Poplar Biomass Facilitates Cu-Catalyzed

Jan 22, 2018 - ... of Biochemistry, University of Wisconsin−Madison, 433 Babcock Drive, ... Zip-lignin and wild-type poplar were subjected to copper...
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Engineered Lignin in Poplar Biomass Facilitates Cu-Catalyzed Alkaline-Oxidative Pretreatment Aditya Bhalla, Namita Bansal, Sivakumar Pattathil, Muyang Li, Wei Shen, Chrislyn Particka, Steven D Karlen, Thanaphong Phongpreecha, Rachel R. Semaan, Eliana GonzalesVigil, John Ralph, Shawn Mansfield, Shi-You Ding, David B. Hodge, and Eric L. Hegg ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02067 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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Engineered Lignin in Poplar Biomass Facilitates Cu-Catalyzed Alkaline-Oxidative Pretreatment Aditya Bhalla1,2†, Namita Bansal1,2†, Sivakumar Pattathil3,4, Muyang Li1,5, Wei Shen1,5, Chrislyn A. Particka1, Steven D. Karlen6,7, Thanaphong Phongpreecha1,9, Rachel R. Semaan2, Eliana Gonzales-Vigil7, John Ralph6,7, Shawn D. Mansfield8, Shi-You Ding1,5, David B. Hodge1,9,10,11*, Eric L. Hegg1,2** 1

DOE Great Lakes Bioenergy Research Center, Michigan State University, 1129 Farm

Lane, East Lansing, MI 48824, USA 2

Department of Biochemistry & Molecular Biology, Michigan State University, 603

Wilson Road, East Lansing, MI 48824, USA 3

Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road,

Athens, GA 30602, USA 4

BioEnergy Science Center, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge,

TN 37831, USA 5

Department of Plant Biology, Michigan State University, 612 Wilson Road, East

Lansing, MI 48824, USA 6

DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, 1552

University Avenue, Madison, WI 53726, USA 7

Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive,

Madison, WI 53706, USA 8

Department of Wood Science, University of British Columbia, 4030-2424 Main Mall,

Vancouver, BC V6T 1Z4, Canada

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Department of Chemical Engineering & Materials Science, Michigan State University,

428 S. Shaw Lane, East Lansing, MI 48824 USA 10

Department of Biosystems & Agricultural Engineering, Michigan State University, 524

S. Shaw Lane, East Lansing, MI 48824 USA 11

Division of Sustainable Process Engineering, Luleå University of Technology, SE-98187,

Luleå, Sweden *E-mail: [email protected] (D. Hodge). Current address: Chemical and Biological Engineering Department, Montana State University, PO Box 173920, Bozeman, MT 59717 **E-mail: [email protected] (E. Hegg)

Keywords Alkaline hydrogen peroxide, Biofuels, Copper, Ester bonds, Glycome profiling, Microscopy, Oxidation, Zip-lignin

Abstract Both untransformed poplar and genetically modified “zip-lignin” poplar, in which additional ester bonds were introduced into the lignin backbone, were subjected to mild alkaline and copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment. Our hypothesis was that the lignin in zip-lignin poplar would be removed more easily than lignin in untransformed poplar during this alkaline pretreatment, resulting in higher sugar yields following enzymatic hydrolysis. We observed improved glucose and xylose 2 ACS Paragon Plus Environment

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hydrolysis yields for zip-lignin poplar compared to untransformed poplar following both alkaline-only pretreatment (56% for untransformed poplar compared to 67% for ziplignin poplar) and Cu-AHP pretreatment (77% for untransformed poplar compared to 85% for zip-lignin poplar). Compositional analysis, glycome profiling, and microscopy all supported the notion that the ester linkages increase delignification and improve sugar yields. Essentially no differences were noted in the molecular weight distributions of solubilized lignins between the zip-lignin poplar and the control line. Significantly, when zip-lignin poplar was utilized as the feedstock, hydrogen peroxide, catalyst, and enzyme loadings could all be substantially reduced while maintaining high sugar yields.

Introduction Lignocellulosic biomass is a potential feedstock for the production of next-generation biofuels.1 Lignin, a complex polymer found in plant cell walls, provides strength, hydrophobicity, and rigidity to the secondary cell walls of vascular plants. As one of the primary contributors to the recalcitrance of plant cell walls during biochemical processing, lignin restricts cellulolytic enzymes from accessing and hydrolyzing cell wall polysaccharides.2 To circumvent this recalcitrance, various pretreatment approaches, including acidic, alkaline, organosolv, and ionic liquid pretreatments, have been explored.3-6 Unfortunately, many of these pretreatments present technical and economic challenges to implementation due to their requirements for high energy and chemical inputs, and they are therefore currently not economically viable.7,8 If the recalcitrance due to lignin in the cell wall is reduced, however, it may be possible to decrease the severity of the pretreatments and thus improve the economics of lignocellulosic biofuel production.

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One approach to reducing lignin recalcitrance is to modify the lignin content, composition, and/or structure by altering the expression of genes in the lignin biosynthetic pathway.9,10 Although plants with reduced lignin content have increased cell wall digestibility, they often exhibit poor vascular function, decreased strength, and/or reduced growth.2,11 An alternative approach is to create so-called “designer lignins”10,12-13 in which the chemical structure of lignin is altered to decrease recalcitrance without altering either the function or the content of lignin in engineered plants. Various strategies have been proposed, including lignins with shorter chains,14 modified lignin monomer ratios (resulting in altered lignin syringyl:guaiacyl composition),15 and reduced crosslinking to carbohydrates.14,16-17 Poplar (Populus alba x grandidentata) lines expressing the ferulate monolignol transferase (FMT) gene from Angelica sinensis were recently developed.18 These zip-lignin poplars incorporate ester linkages into the lignin backbone, resulting in lignin that is more amenable to deconstruction methods, especially alkaline pretreatments that are particularly effective at hydrolyzing ester bonds.19-20 We recently demonstrated that the copper-catalyzed alkaline hydrogen peroxide (CuAHP) pretreatment method is effective at delignifying recalcitrant poplar biomass.7,21-22 In this manuscript, we report that Cu-AHP pretreatment of zip-lignin Line 7 transgenic poplar (hereafter referred to as Line 7)18 results in higher sugar yields following enzymatic hydrolysis compared to the wild-type poplar line P39 (hereafter referred to as WT). Glycome profiling and microscopy were employed to correlate the changes in the cell wall ultrastructure to their respective sugar yields following pretreatment of both Line 7 and WT. Lignin characterization was performed to identify both differences between Line 7 and WT and the impacts of pretreatment on the lignins. Finally, we

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demonstrated that it is possible to reduce the amount of hydrogen peroxide, catalyst, and enzyme loadings when pretreating Line 7 and still achieve high sugar yields following enzymatic hydrolysis.

Experimental Section Biomass and compositional analysis The biomass used in this study was from WT Line P39 poplar (Populus alba x grandidentata) and Line 7 zip-lignin poplar expressing the AsFMT gene that were grown in a greenhouse at the University of British Columbia, Vancouver, British Columbia 18. Poplar stems from 6-month-old, greenhouse-grown trees were harvested, debarked, and milled (Wiley MiniMill, Thomas Scientific, Swedesboro, NJ) to pass through a 2 mm screen prior to performing compositional analysis. A modeling study estimated the weight percentage of monolignol ferulate in Line 7 to be ~17.2%, compared to 1.7% in WT; thus, this model indicated that Line 7 has approximately 10-fold more zips.23 The composition of the structural carbohydrates and the acid-insoluble lignin (Klason lignin) of untreated, alkaline-only pretreated, and Cu-AHP pretreated samples were determined according to the National Renewable Energy Laboratory’s analytical procedures,24 with the difference that an Aminex HPX-87H HPLC column coupled to an HPLC fit with a refractive index detector was used to determine the percentages of structural carbohydrates. The xylose percentage yields from the samples are reported as a cumulative percentage of xylose, mannose, and galactose due to an inability of the HPX87H column to resolve these sugars. The chemical composition of the lignin before and after pretreatment was assayed using a combination of heteronuclear single-quantum

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coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy,25 derivatization followed by reductive cleavage (DFRC),26 and gel-permeation chromatography (GPC). Pretreatment Three different pretreatment methods were employed in this study; all were carried out for 24 h at 30 °C to be consistent with previous studies.7, 27 Alkaline-only pretreatment was performed by incubating 0.25 g biomass with 2.5 mL aqueous solution of NaOH (100 mg NaOH/g biomass) at 10% solids loading. Cu-AHP pretreatment (reference Cu-AHP) was performed in 2.5 mL of liquid reaction volume at 10% (wt/liquid vol) solids loading. The following concentrations of reactants were utilized during reference Cu-AHP pretreatment. The catalyst loading was set at 1 mM for copper and 2 mM for 2,2′-bipyridine (bpy), hydrogen peroxide loadings were set at 100 mg/g of poplar and NaOH loadings were set at 100 mg/g of poplar biomass. Distilled water was added to each reaction mixture to maintain a final solids loading of 10% (wt/liquid vol). Cu-AHP pretreatment with fed-batch addition of H2O2 (fed-batch Cu-AHP) was performed as described above for the reference Cu-AHP case, except that (a) the H2O2 loadings varied from 50-100 mg/g of biomass depending on the specific experiment and (b) the H2O2 was gradually added to the reaction mixture over a 10-h period instead of all at once.7 Following the final addition of H2O2, the mixture was incubated as described for an additional 14 h (24 h total reaction time). In certain studies, bpy and H2O2 concentrations were reduced stepwise to determine the effect on final glucose and xylose yields. Enzymatic hydrolysis

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After pretreatment, samples were slowly titrated with 72% (w/w) H2SO4 to adjust the pH to 5.0 prior to the addition of 1 M citric acid buffer (pH 5.0) at a final concentration of 50 mM. An enzyme cocktail consisting of stock solutions of Cellic® CTec3 (197.3mg/g) and HTec3 (170.5 mg/g) was kindly provided by Novozymes A/S (Bagsværd, DK) and used for the hydrolysis reactions at a final loading of 15 mg protein/g glucan each (i.e., 30 mg/g total enzyme loading) unless otherwise noted. The protein content was provided by the manufacturer. In certain circumstances, total enzyme levels were decreased (the CTec3:HTec3 ratio remained 1:1) to determine the effect of reduced enzyme loading on final glucose and xylose yields. The total aqueous volume of the reaction was then adjusted to 5 mL by adding deionized water to attain a solids loading of 5%. The samples were incubated at 50 °C for 72 h with orbital shaking at 210 rpm. Following enzymatic hydrolysis, the amounts of glucose and xylose released in the supernatant were quantified by high-performance liquid chromatography (HPLC) (Agilent 1260 Series equipped with an Infinity refractive index detector) using an Aminex HPX-87H column operating at 65 °C, a mobile phase of 5.0 mM H2SO4, and a flow rate of 0.6 mL/min. Different concentrations of pure glucose and xylose samples were prepared to obtain standard curves to calculate the sugar concentrations in the samples. The sugar yields (glucose and xylose) were calculated by dividing the amount of released sugar by the total sugar content (as monomer) of the biomass (dry weight basis) prior to pretreatment. Microscopy For imaging analysis, stem segments of WT and Line 7 poplar were cut with a microtome to yield ~50 µm cross-section slices, then subjected to alkaline-only pretreatment and fed-

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batch Cu-AHP pretreatment with 100 mg H2O2/g biomass as described above. Samples were imaged using two microscope systems. The Stimulated Raman Scattering (SRS) microscope used was a custom-built imaging system described previously.28-29 SRS offers relatively quantitative signals based on vibrational spectroscopy. The aromatic ring stretching band at 1600 cm-1 was used to map lignin content in the cell walls of both WT and Line 7 poplar. Fluorescence microscopy was applied to image the binding of a recombinantly-expressed cellulose-specific carbohydrate binding module (CBM) from Clostridium thermocellum tagged with the green fluorescent protein (GFP).30-31 The substrate accessibility to the enzyme could therefore be determined by the relative fluorescence signal of GFP-CBM3 in the cell walls. For CBM binding, the samples were incubated for 30 min at room temperature in the dark with 1 µg GFP-CBM3 in 200 µL deionized water, then washed with several volumes of additional deionized water to remove the unbound GFP-CBM3, and imaged in water using a 488 nm laser as an excitation source. Fluorescence images were recorded using the Olympus IX73 microscope system equipped with a DP80 digital camera. Glycome profiling Prior to glycome profiling, samples of WT and Line 7 poplar were subjected to reference Cu-AHP pretreatment. Preparation of cell wall materials from untreated and pretreated samples, and their glycome profiling analyses, was conducted as described previously.3233

Briefly, cell wall materials were sequentially extracted with increasingly harsh

reagents: ammonium oxalate (50 mM; pH 5.0), sodium carbonate (50 mM; pH 10.0) containing 0.5% sodium borohydride (w/v), 1 M KOH (potassium hydroxide) containing 1% sodium borohydride (w/v), 4 M KOH with 1% sodium borohydride (w/v), acidic

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sodium chlorite (100 mM), and finally 4 M KOH PC (4 M KOH post-chlorite extraction) containing 1% sodium borohydride (w/v). Sequential cell wall extracts obtained represented cell wall glycans extracted on the basis of the relative tightness to which they were bound to the cell walls and thus reflected the extractability status of various glycans. These extracts were subsequently ELISA-screened with a comprehensive suite of 155 cell wall-directed monoclonal antibodies (mAbs)32 that facilitated monitoring the abundance of most major non-cellulosic plant cell wall glycan epitopes. To perform ELISA, 15 µL of sample (0.3 µg glucose-equivalent of carbohydrates) was coated onto ELISA wells in a 384-well format (Costar 3700, Corning Inc., Corning, NY, USA) and assays were conducted with a robotic system (Thermo Fisher Scientific Inc. Waltham, MA, USA). The ELISA responses of mAbs were represented in the form of heat maps. Lignin isolation Precipitated lignins (PL) were obtained by filtering the pretreatment liquor through Whatman filter paper grade 1 and acidifying the filtrate to a pH of 1.9 – 2.0 by adding 72% (w/w) H2SO4 while vigorously stirring. The mixture was then centrifuged at 4,700 × g, and the liquid was poured out. The precipitated lignin was washed with pH 2 water by three cycles of vortexing, centrifuging, and decanting. The washed PL was then lyophilized to obtain the final product. Enzyme lignins (EL) were prepared from ball-milled cell wall materials as described previously.34 Briefly, oven dried untreated biomass or pretreated biomass solid residues were ball-milled at ambient temperature with a Fritsch pulverisette 7 (1 g, 20 mL ZrO2 jar, 10 × 10 mm ZrO2 ball bearings, 600 rpm for 10 min, 5 min rest, 47 cycles, reverse on). Ball-milled powder (~570–700 mg) was transferred to 50 mL centrifuge

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tubes, suspended in 40 mL of 50 mM sodium acetate buffer (pH 5.0), and treated with crude cellulases (40 mg, Cellulysin®, EMD Biosciences, CA). The samples were incubated at 35 °C on a shaker table shaking at 225 rpm for 3 days. After incubation, solids were pelleted by centrifugation (8,800 × g, 20 min, Sorvall biofuge primo centrifuge), and the supernatant decanted. The pelleted solids were then treated a second time with fresh cellulases (40 mg) for 3 days, pelleted, and washed with RO water (3 × 40 mL). The washed, pelleted solids (EL) were dried at 50 °C under vacuum and used without further purification for NMR, DFRC, and GPC assays. NMR analysis Heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker Biospin (Billerica, MA) Avance 700 MHz spectrometer equipped with a 5-mm quadruple-resonance 1H/31P/13C/15N QCI gradient cryoprobe with inverse geometry (proton coils closest to the sample). Samples were prepared in DMSO-d6/pyridine-d5 (using high-quality ‘100%’ pyridine- d5, 4:1, v/v, 600 µL), as ball-milled whole-cell-wall (WCW) gels (50 mg for each sample), or as EL or PL (20 mg for each sample) solutions.25,35 Peak assignments were made by comparison with previously assigned spectra.25,36 Volume integration of contours in HSQC plots were set to a G+S+S'=100% basis for aromatic and aliphatic signals, using just the G2 and S2/6 correlations (and logically halving the S integrals). Aliphatic signals were set on an A+A'+B+B'+C+C'=100% basis using just the α-C/H correlations for each (and again halving the integrals for C and C' because a single resinol unit has two equivalent C/H pairs). DFRC analysis

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Incorporation of monolignol p-hydroxybenzoates and monolignol ferulates into the lignin was determined using the ether-cleaving ester-retaining DFRC method established for other monolignol conjugates.18-19, 37-40 Using a previously described protocol,41 the DFRC analysis was performed on both EL and PL samples (15–25 mg). Lignin molecular weight profile analysis: The isolated lignins (10 mg of each sample) were acetylated with acetic anhydride and pyridine (1:1, v/v, 1 mL). The lignin was dissolved in the acetic anhydride / pyridine mixture with the aid of sonication for 1.5 h. They were then placed on a speedvac to remove the solvent (50 °C, 1 h, 0.1 torr, 35 torr/min). The resulting films were dissolved in tetrahydrofuran. Each sample was found to have varying degrees of insoluble material; the alkaline pretreated lignins contained large visibly insoluble particles. The samples were then filtered through a 0.2 µm PTFE membrane, dried, and then prepared as 5, 2.5, and 1 mg/mL solutions and submitted for molecular weight profiling analysis. Molecular weights profiles were determined by gel-permeation chromatography (GPC) using a Shimadzu LC20 connected to a PSS PolarSil analytical linear S column (8 × 300 mm, 5 µm particle size) held at 40 °C. The mobile phase was degassed tetrahydrofuran filtered through an in line 0.2 µm polytetrafluoroethylene (PTFE) membrane at 1.0 mL/min. The GPC signal was acquired using a Shimadzu SPD-M20A UV-Vis flow cell photodiode array detector at 280 nm and processed using Wyatt Astra v. 7.1.2. software. The detector calibration constants were measured using 30 kDa polystyrene standard (MW = 30 kDa, MW /MN = 1.06, dn/dcTHF = 0.185, ε272 = 0.3677 mLg-1cm-1) and a fourth order conventional calibration curve was determined using a ReadyCal Kit from Sigma-Aldrich (Aldrich #76552, polystyrene MP 0.25–70 kDa).

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Results and Discussion: Alkaline-only and Cu-AHP pretreatment of zip-lignin lines Alkaline pretreatments have been shown to efficiently cleave ester crosslinks between lignin and xylan in grasses.42-43 We therefore hypothesized that treating zip-lignin poplar, which was engineered to contain ester linkages in the lignin backbone (an estimated 10fold more in Line 7 compared to WT),23 with an alkaline pretreatment should result in higher sugar yields following enzymatic hydrolysis relative to sugar yields from wildtype poplar. To test this hypothesis, we treated both Line 7 and WT poplar with an alkaline-only pretreatment (Figure 1). The results clearly demonstrated improved glucose and xylose yields for Line 7 (67% and 76%, respectively) compared to the WT poplar (56% and 71%, respectively).

Figure 1. A) Glucose yields and B) xylose yields obtained following enzymatic hydrolysis of WT and Line 7 poplar pretreated under alkaline-only or fed-batch Cu-AHP pretreatment (75 mg H2O2/g biomass). The points are the averages of three biological replicates, and error bars indicate ± standard deviations of the means.

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as Cu-AHP pretreatment was previously shown to improve the sugar yields substantially from poplar biomass compared to alkaline-only or AHP-only pretreatment.7 As predicted, improved glucose and xylose yields were observed for Line 7 biomass (85% and 91%, respectively) compared to WT biomass (77% and 84%, respectively) after fed-batch (75 mg H2O2/g biomass) Cu-AHP pretreatment followed by enzymatic hydrolysis (Figure 1). While the percent improvement in glucose hydrolysis yields between WT and Line 7 was comparable to alkaline-only pretreatment, the absolute magnitude of the hydrolysis is greater for Cu-AHP pretreatment relative to the alkaline-only pretreatment conditions used. It is well established that during pretreatment, removing or modifying recalcitrant lignin from the plant cell wall increases the accessibility of polysaccharides to cellulolytic enzymes and consequently increases sugar yields.44-46 To ascertain how alkaline-only and fed-batch Cu-AHP pretreatments altered the cell wall in Line 7 and WT, compositional analysis of the biomass was performed for both lines before and after the pretreatments. The results showed increased removal of Klason lignin from the cell wall of Line 7 biomass (29% and 53% of lignin removed) compared to WT biomass (22% and 50% of lignin removed) with alkaline and Cu-AHP pretreatments, respectively (Figure 2).

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Figure 2. Mass loss and cell wall compositional change associated with WT and Line 7 poplar subjected to alkaline-only and fed-batch Cu-AHP pretreatment (75 mg H2O2/g biomass). The values reported are the averages of the three biological replicates (except ash, which represents a single measurement), and the error bars indicate ± standard deviations of the means.

In addition to the total lignin content, the properties of the lignins can also impact the cell wall recalcitrance to enzymatic hydrolysis. For example, it is known that conversion of lignins to more hydrophilic lignins by oxidation47 or sulfonation48 can improve enzymatic hydrolysis yields. Furthermore, another complicating factor is that both lignin content and its free radical-scavenging capacity have been shown to impact the efficacy of enzyme cocktails containing a lytic polysaccharide monooxygenase49,50 such as the Cellic CTec3 enzyme cocktail used in this work.

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Detailed characterization of the lignins was next performed to determine differences in lignins and the impact of pretreatment on the lignins. Specifically, the lignins recovered from the residual solids (enzyme lignins; EL) and acid precipitation of lignins from the pretreatment liquors (precipitated lignins; PL) were subjected to characterization by 2D-HSQC NMR, derivatization followed by reductive cleavage (DFRC) analysis, and gel permeation chromatography (GPC) with the results presented in Table 1. The chemical composition as determined by NMR showed no difference between the aromatic composition in the ELs of Line 7 and WT (S/G = 1.74 for both; Table 1). Neither the aromatic unit ratio nor the interunit linkages (β–O–4, β–β, or β–5), in the cell-wall-bound lignins were significantly altered by the alkaline or Cu-AHP pretreatments (Table S1). The acid-precipitated lignins were enriched in S lignin units (S/G=3.22). DFRC analysis of the lignins confirmed that there was no difference in S/G ratio between the plant lines or as a function of pretreatment. Due the heterogeneous nature of lignins and the β-ether-cleaving action of DFRC, only a portion of the polymer is quantified in terms of moles release per gram of lignin. The levels of quantified total monolignol p-hydroxybenzoates revealed that alkaline pretreatment, and even more so for Cu-AHP pretreatment, reduced the p-hydroxybenzoate esters (Table 1). The zip-lignin total monolignol ferulate ester levels were also reduced by both pretreatments. The NMR and DFRC results indicated that while Cu-AHP did not significantly alter the chemical structure of the lignin, it did shorten the lignin chain length. To test this, the molecular weight profile of the EL and PL lignins were determined by GPC, Table 1 and Figure 3. There was no difference in molecular weight profiles between WT and Line 7;

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untreated–EL from both had weight-average molecular weights (MW) of 12 kDa. The EL from alkaline-only pretreatments decreased the MW (9 kDa) by ~10%, primarily to the loss of p-hydroxybenzoate and acetate esters, and decreased the polydispersity from 8 to 5. Cell-wall bound Cu-AHP lignin had an intermediate MW (10 kDa) with the same polydispersity as alkaline-EL, further supporting the observation that the cell-wall bound lignins after pretreatment were not significantly changed. Analysis of the PL fractions from the two pretreatments showed the presence of only small polymeric and oligomeric fragments were present (alkaline–PL MW < 4 kDa and CuAHP–PL MW < 1.6 kDa), Table 1. This supports the hypothesis that Cu-AHP pretreatment cleaves interunit linkages to make smaller alkali-soluble lignin fragments. The cell-wall residual lignins appear to have been shielded from the pretreatment solvent and therefore not altered. Interestingly, the lignins in the Cu-AHP pretreated residues exhibit higher values of MN and MP than the untreated or alkali-only pretreated poplars. One potential explanation for this is that the smaller polymers were extracted and digested leaving larger untreated lignin fragments.

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Figure 3. GPC chromatograms of isolated lignins from WT (solid line) and Line 7 (hashed line) poplar subjected to no treatment (untreated), alkaline-only, and fed-batch Cu-AHP pretreatment. Signal intensities of the EL samples correspond to their fractions of the untreated lignin. Elution volumes corresponding to the peak maximum (MP) for select polystyrene standards used for column calibration are shown for reference (vertical light gray lines).

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Table 1. Lignin characterization by 2D HSQC NMR, DFRC, and GPC of WT and Line 7 poplar stem tissue. %S; syringyl, %G; guaiacyl, and pBA; p-hydroxybenzoate are 2D-HSQC NMR volume integral percentages on a G+S+S'=100% basis. DFRC quantified lignin subunits H, 4-hydroxycinnamyl alcohol; G, coniferyl alcohol; S, sinapyl alcohol; G-pBA, coniferyl p-hydroxybenzoate; S-pBA, sinapyl p-hydroxybenzoate; G-FA, coniferyl ferulate; and S-FA, sinapyl ferulate. GPC determined: MW, weight-average molecular weight; MN, number-average molecular weight; MP, peak molecular weight; MW/MN, polydispersity; based on polystyrene standards.

NMR %G* *

%S S/G %pBA*

Untreated–EL WT Line7

Alkaline–EL WT Line 7

Cu-AHP–EL WT Line 7

Alkaline–PL WT Line 7

Cu-AHP–PL WT Line 7

37%

37%

35%

34%

37%

36%

29%

29%

35%

24%

63% 1.74 12%

63% 1.72 10%

65% 1.89 6%

66% 1.92 6%

63% 1.74 12%

64% 1.78 5%

71% 2.44 11%

71% 2.47 9%

65% 1.89 6%

76% 3.22 12%

2.6±0.1 349±11 741±7 0.35±0.01 70.3±5.2 trace N/D

2.6±0.0 369±22 820±40 0.26±0.05 68.6±4.8 0.09±0.01 0.20±0.02

2.3±0.0 329±8 683±6 0.17±0.02 43.2±1.0 trace N/D

2.3±0.0 334±10 708±23 0.18±0.03 45.3±2.3 0.11±0.01 0.19±0.02

1.6±0.0 214±2 814±14 0.31±0.07 72.6±6.7 N/D N/D

1.5±0.0 177±4 526±2 0.28±0.04 47.6±1.7 N/D trace

1.7±0.1 164±2 658±5 0.30±0.07 69.3±3.5 N/D N/D

1.7±0.0 164±2 646±26 0.07±0.05 30.1±21.3 trace 0.21±0.01

8.9 1.7 5.2 5.3

8.8 1.7 5.1 5.3

10 1.9 5.3 5.2

10.6 2.0 5.5 5.4

4.1 0.9 1.8 4.6

4.3 0.9 1.9 4.8

1.5 0.4 0.3 3.9

1.5 0.4 0.4 3.8

DFRC (µmol/g Lignin ± SEM) H ** 2.4±0.1 2.3±0.0 G ** 287±18 287±1 S ** 543±27 583±1 0.60±0.05 0.43±0.10 G-pBA** 83.8±1.5 76.1±4.5 S-pBA** 0.27±0.02 0.39±0.01 G-FA** Trace 0.31±0.01 S-FA** GPC (acetylated lignins in THF) MW (kDa) 12.1 12.2 MN (kDa) 1.5 1.4 MP (kDa) 4.1 3.9 MW / MN 8.2 8.4

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To obtain additional insight into the differing responses of Line 7 and WT poplar to pretreatment and enzymatic hydrolysis, glycome profiling studies and direct imaging using two microscopic systems were performed. To test our hypothesis of improved sugar yields in Line 7 compared to WT due to increased lignin removal, the amount of residual lignin in the cell walls after both alkaline-only and fed-batch Cu-AHP pretreatment was compared between the two poplar lines using SRS imaging (Figure 4AF). The results for untreated WT and Line 7 poplar demonstrated similar lignin intensity and distribution patterns, as they mainly differ in lignin composition rather than lignin content (Figure 4A & 4B).18 (Interestingly, the variation in the apparent “sharpness” of the lignin images for WT versus Line 7 poplar suggest possible minor differences in lignin distribution.) For fed-batch Cu-AHP pretreated samples, improved lignin removal in Line 7 compared to WT was observed based on the reduced SRS lignin signals, consistent with the higher sugar yield of Line 7 following Cu-AHP pretreatment and enzymatic hydrolysis (Figures 4E & 4F). These results support the cell wall compositional analysis results (Figure 2), in which increased lignin solubilization was observed for Cu-AHP-treated Line 7 compared to Cu-AHP treated WT biomass.

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Figure 4. Lignin content (A-F) and cellulose accessibility (G-L) imaged by SRS microscopy based on aromatic ring stretching band at 1600 cm-1, and fluoresce microscopy of GFP-CBM3 binding, respectively. All images were taken in the same scale, scale bar (A) = 10 µm. WT is wild type poplar line P39 and Line 7 is zip-lignin Line 7 transgenic poplar. GFP-CBM3 is a recombinant protein previously produced based on a cellulose-specific carbohydrate binding module (a family 3 CBM from Clostridium thermocellum) tagged with green fluorescent protein (GFP). Alkaline refers to alkaline-only pretreated biomass and Cu-AHP refers to fed-batch Cu-AHP pretreated biomass (100 mg H2O2/g biomass). The CBM3-GFP labelling strategy has been widely used to detect the accessible cellulose present in plant cell walls.29, 31 This method allows for observation of the differences in accessibility of cellulose to cellulolytic enzymes by comparison with the accessibility of GFP-CBM3.51 Both alkaline-only and Cu-AHP pretreatment of WT and Line 7 (Figures 4 I-L) biomass significantly enhance CBM binding compared to

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untreated biomass (Figures 4G & 4H). Notably, there was more CBM binding observed for Cu-AHP treated Line 7 (Figure 4L) when compared to Cu-AHP treated WT (Figure 4K) biomass. The increase in the fluorescence intensity indicates that the presence of ester bonds in zip-lignin poplar facilitates Cu-AHP pretreatment and potentially enhances the accessibility of cellulolytic enzymes to poplar secondary cell walls. The results concur with previous studies reporting that lignin removal results in improved cellulose accessibility.52-53 Glycome profiling Glycome profiling was next employed to assess changes in non-cellulosic glycan composition and extractability for untreated and reference Cu-AHP-pretreated WT and Line 7. Specifically, the non-cellulosic glycans derived from six sequential extractions [oxalate, carbonate, 1 M KOH, 4 M KOH, chlorite delignification, and 4 M KOH postchlorite extraction (KOH PC)] from the four plant cell wall samples (untreated and CuAHP pretreated WT and Line 7 zip-lignin poplar) were screened against a panel of glycan-directed mAbs. The binding results and total glycan content of the extracts for a subset of epitopes for xyloglucan (XG), xylan, homogalacturonan (HG), and rhamnogalacturonan 1 (RG-I) is presented in Figure 5. The complete glycome profile is provided as Supplemental Table S2‡.

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Figure 5. Subset of glycome profiles (xyloglucan, xylan, and pectin backbones) for both wild-type poplar (WT) and zip-lignin poplar (Line 7) that is either untreated (blue) or subjected to reference Cu-AHP (100 mg H2O2/g biomass) pretreatment (pink). The scales for the heat maps in each subplot are normalized to the maximum value. The gravimetric amounts of carbohydrate materials extracted during each extraction step are represented as bar graphs on the top panels above heat maps. XG, xyloglucan; HG, homogalacturonan; RG, rhamnogalacturonan. The complete glycome profiles are provided in Table S2‡.

A number of notable results can be observed in the glycome profiles. First, the abundance of xylan epitopes in the oxalate extract is elevated in Line 7 for both untreated and pretreated samples. This is consistent with the findings from our previous work that revealed that improved hydrolysis yields in diverse hardwoods (including hybrid poplar) subjected to alkaline and alkaline-oxidative pretreatments can be correlated to improved non-cellulose glycan extractability by oxalate.27 Second, pretreatment significantly increases the amount of extractable glycans in the oxalate extract (Figure 4, top panel). Relative to the pretreated WT, the pretreated Line 7 exhibits a higher abundance of xylan 22 ACS Paragon Plus Environment

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epitopes in the 1 M and 4 M KOH extracts and a lower abundance in the 4 M KOH PC extract. This result may be due to improved lignin disruption (and removal) during pretreatment for the zip-lignin line that consequently improves the extractability of xylan. As more xylan is removed in the 1 M and 4 M KOH extractions for the Cu-AHP pretreated zip-lignin line, less is available for extraction following chlorite delignification. In other words, the xylan epitopes in the 4 M KOH PC extract have shifted to the more mild extracts for the pretreated zip-lignin line, further indicating that zip-lignin poplar is less recalcitrant than WT poplar. Evaluating the potential of zip-lignin biomass at low chemical inputs of Cu-AHP Due to the reduced recalcitrance of zip-lignin Line 7 poplar relative to WT, we hypothesized that we should be able to lower the chemical inputs and severity of our CuAHP process and still maintain high sugar yields. Of the chemical inputs used in the CuAHP process, bpy is the most expensive (approximately 40% of the total cost), followed by enzymes (approximately 20%) and H2O2 (approximately 10%). We first tested the effect of different bpy loadings (0 - 1 mM) on the final sugar yields following enzymatic hydrolysis at a total enzyme loading of 30 mg/g glucan. Our results exhibited noteworthy differences in sugar yields between Line 7 and WT at all bpy concentrations (Figure 6). Glucose yields for Line 7 at 0.25 mM bpy concentration (81%) were nearly identical to those obtained from WT at 1 mM bpy concentration, while xylose yields for Line 7 at 025 mM bpy concentration were higher (91%) than those for WT at 1mM bpy concentration (87%). These results indicate that when Line 7 biomass is used, bpy loadings can be lowered dramatically while still maintaining high glucose and xylose yields.

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Figure 6. Impact of bipyridine loadings on hydrolysis yields of A) glucose and B) xylose following fed-batch Cu-AHP pretreatment (100 mg H2O2/g biomass). The data points are the averages of three independent experiments, and the error bars, which are smaller than the data point symbols, indicate ± standard deviations of the means.

We also determined the effect of reducing H2O2 on final glucose and xylose hydrolysis yields.54 Previously, in a study using native poplar, we demonstrated that a 10% reduction in glucose yields was observed when the oxidant loading was reduced to 50 mg/g biomass from 75 mg/g biomass.7 In this study, however, our results demonstrated that glucose yields remained high (83% and 79%, respectively) when Line 7 biomass was treated with just 50 mg or 25 mg H2O2/g biomass (Figure 7). Xylose yields also remained above 90% with both 50 mg and 25 mg H2O2/g biomass. In fact, Line 7 consistently gave ~10% higher glucose yields and ~8% higher xylose yields relative to WT poplar regardless of the H2O2 concentration employed.

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Figure 7. Impact of H2O2 loadings on hydrolysis yields of A) glucose and B) xylose following fed-batch Cu-AHP pretreatment. The data points are the averages of three independent experiments, and the error bars indicate ± standard deviations of the means.

A third set of experiments was performed to examine the effect of different concentrations of enzymes on final sugar hydrolysis yields at an H2O2 loading of 50 mg/g biomass (Figure 8). The results revealed higher glucose and xylose yields for Line 7 (83% and 93%, respectively) compared to WT (75% and 84%, respectively) at 30 mg total enzyme/g glucan. Furthermore, nearly identical glucose yields were obtained from Line 7 treated at just 20 mg/g glucan compared to WT treated at 30 mg/g glucan. The effect of zips was most notable at 20 mg/g glucan, where there was a 17% difference in glucose yields between Line 7 (73%) and WT (56%). The difference in xylose yield between WT and Line 7 was smaller than the difference in glucose yield, although Line 7 still gave xylose yields between 7 and 9% higher than WT.

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Figure 8. Impact of total enzyme loadings (CTec3:HTec3 = 1:1) on hydrolysis yields of A) glucose and B) xylose following Cu-AHP pretreatment (50 mg H2O2/g biomass) utilizing fed-batch Cu-AHP pretreatment. The data points are the averages of three independent experiments, and the error bars, which are smaller than the data point symbols, indicate ± standard deviations of the means.

Other recent studies have evaluated the effect of different lignin modifications and pretreatment technologies on biomass saccharification, as reviewed.10, 55-57 Hybrid poplar trees with reduced lignin content due to a downregulation of the enzyme that catalyzes the first step of the monolignol-specific pathway led to increased saccharification with no pretreatment as well as after two alkaline and one acid pretreatments.58 Likewise, Cai et al.59 observed increased release of simple sugars from poplar hybrids with reduced lignin content and altered lignin structure following alkaline pretreatment as well as with no pretreatment. Switchgrass modified to have both reduced lignin content and a lower S:G ratio resulted in increased ethanol yields following dilute acid pretreatment and fermentation.60 Consistent with the aforementioned studies, our data clearly reveal that zip-lignin engineered into poplar facilitates Cu-AHP pretreatment and has the potential to

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significantly reduce the costs involved in the deconstruction of biomass to fermentable sugars without reducing lignin content. In conclusion, we have demonstrated that utilizing zip-lignin poplar results in an improvement in the efficacy of Cu-AHP pretreatment. The glucose and xylose hydrolysis yields following enzymatic hydrolysis of the zip-lignin poplar Line 7 were higher compared to the yields from the WT biomass. Glycome profiling, microscopy, and compositional analysis results all support the hypothesis that the lignin in these engineered poplar trees is easier to remove during pretreatment. Consistent with these results, CBM binding experiments confirmed that the crystalline cellulose is more accessible to hydrolytic enzymes in zip-lignin poplar than in WT poplar. The decreased recalcitrance of the zip-lignin poplar allowed us to decrease the chemical inputs, severity of the Cu-AHP pretreatment process, and/or decrease the enzyme requirements to achieve comparable sugars yields, thereby validating the potential of zip-lignin biomass as an improved feedstock for the biofuels and bioproducts industries.

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Associated Content Supporting Information The following Supporting Information is available free of charge on the ACS Publications Website: Table S1. Relative levels, determined from 2D-NMR volume integrals, of various units in the cell wall biomass of WT and Line 7. Table S2. Full glycome profile in Excel format for both Line 7 zip-lignin poplar and WT poplar. Figure S1. Lignin components present in WT and Line 7 poplar EL and PL samples. For dimeric units, the bond formed during the radical coupling step is bolded. Figure S2. Aromatic region of the HSQC NMR spectra of WT and Line 7 poplar EL and PL samples. Figure S3. Aliphatic region of the HSQC NMR spectra of WT and Line 7 poplar EL and PL samples.

Author Information Corresponding Authors *E-mail: [email protected] (E. Hegg) *E-mail: [email protected] (D. Hodge) Author Contributions All authors were involved in biomass and/or data collection and/or manuscript preparation, and have given approval to the final version of the manuscript. †These authors contributed equally to this work. Notes The authors declare the following competing financial interest: J. Ralph and S. D. Mansfield (Feruloyl-CoA:Monolignol Transferase – US2013/0203973); D. B. Hodge, E. L. Hegg, A. Bhalla, and N. Bansal (Multi-Ligand Metal Complexes and Methods of 28 ACS Paragon Plus Environment

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Using Same to Perform Oxidative Catalytic Pretreatment of Lignocellulosic Biomass – 2015/0352540 A1). As a holder of these patents, we may benefit financially from advances in the technology discussed in this manuscript.

Acknowledgements This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494).

List of Abbreviations bpy: 2,2′-bipyridine CBM: carbohydrate binding module Cu-AHP: copper-catalyzed alkaline hydrogen peroxide pretreatment DFRC: derivatization followed by reductive cleavage EL: enzyme lignins FMT: ferulate monolignol transferase gene GFP: green fluorescent protein GPC: gel-permeation chromatography HG: homogalacturonan HPLC: high-performance liquid chromatography HSQC: heteronuclear single-quantum coherence KOH PC: KOH post-chlorite extraction Line 7: zip-lignin Line 7 transgenic poplar mAbs: monoclonal antibodies MN: number-average molecular weight MP: peak molecular weight 29 ACS Paragon Plus Environment

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MW: weight-average molecular weight NMR: nuclear magnetic resonance PL: precipitated lignins PTFE: polytetrafluoroethylene RG-I: rhamnogalacturonan I SRS: Stimulated Raman Scattering WCW: whole-cell-wall WT: wild-type poplar line P39 XG: xyloglucan

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30. Dagel, D. J.; Liu, Y.-S.; Zhong, L.; Luo, Y.; Himmel, M. E.; Xu, Q.; Zeng, Y.; Ding, S.-Y.; Smith, S., In situ imaging of single carbohydrate-binding modules on cellulose microfibrils. J. Phys. Chem. B 2011, 115 (4), 635-641. 31. Ding, S. Y.; Xu, Q.; Ali, M. K.; Baker, J. O.; Bayer, E. A.; Barak, Y.; Lamed, R.; Sugiyama, J.; Rumbles, G.; Himmel, M. E., Versatile derivatives of carbohydratebinding modules for imaging of complex carbohydrates approaching the molecular level of resolution. BioTechniques 2006, 41 (4), 435-442. 32. Pattathil, S.; Avci, U.; Miller, J. S.; Hahn, M. G., Immunological approaches to plant cell wall and biomass characterization: Glycome Profiling. Methods Mol. Biol. 2012, 908, 61-72. 33. DeMartini, J. D.; Pattathil, S.; Avci, U.; Szekalski, K.; Mazumder, K.; Hahn, M. G.; Wyman, C. E., Application of monoclonal antibodies to investigate plant cell wall deconstruction for biofuels production. Energy Environ. Sci. 2011, 4 (10), 4332-4339. 34. Chang, H.-m.; Cowling Ellis, B.; Brown, W., Comparative studies on cellulolytic enzyme lignin and milled wood lignin of sweetgum and spruce. Holzforschung 1975, 29 (5), 153-159. 35. Kim, H.; Ralph, J.; Akiyama, T., Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6. BioEnergy Res. 2008, 1 (1), 56-66. 36. Ralph, S. A.; Landucci, L. L.; Ralph, J. NMR databases of lignin and cell wall model compounds. https://www.glbrc.org/databases_and_software/nmrdatabase. 37. Lu, F.; Ralph, J., The DFRC method for lignin analysis. 1. New method for β-aryl ether cleavage: lignin model studies. J. Agric. Food. Chem. 1997, 45 (12), 4655-4660. 38. Lu, F.; Ralph, J., Detection and determination of p-coumaroylated units in lignins. J. Agric. Food. Chem. 1999, 47 (5), 1988-1992. 39. Lu, F.; Karlen, S. D.; Regner, M.; Kim, H.; Ralph, S. A.; Sun, R.-C., Naturally phydroxybenzoylated lignins in palms. Bioenergy Res. 2015, 8, 934-952. 40. Smith, R. A.; Cass, C. L.; Mazaheri, M.; Sekhon, R. S.; Heckwolf, M.; Kaeppler, H.; de Leon, N.; Mansfield, S. D.; Kaeppler, S. M.; Sedbrook, J. C.; Karlen, S. D.; Ralph, J., Suppression of CINNAMOYL-CoA REDUCTASE increases the level of monolignol ferulates incorporated into maize lignins. Biotechnol. Biofuels 2017, 10 (1), 109. 41. Karlen, S. D.; Smith, R. A.; Kim, H.; Padmakshan, D.; Bartuce, A.; Mobley, J. K.; Free, H. C. A.; Smith, B. G.; Harris, P. J.; Ralph, J., Highly decorated lignins in leaf tissues of the Canary Island date palm Phoenix canariensis. Plant Physiol. 2017, 175 (3), 1058-1067. 42. Chundawat, S. P. S.; Vismeh, R.; Sharma, L. N.; Humpula, J. F.; da Costa Sousa, L.; Chambliss, C. K.; Jones, A. D.; Balan, V.; Dale, B. E., Multifaceted characterization of cell wall decomposition products formed during ammonia fiber expansion (AFEX) and dilute acid based pretreatments. Bioresour. Technol. 2010, 101 (21), 8429-8438. 43. Li, M.; Heckwolf, M.; Crowe, J. D.; Williams, D. L.; Magee, T. D.; Kaeppler, S. M.; de Leon, N.; Hodge, D. B., Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize (Zea mays L.) lines. J. Exp. Bot. 2015, 66 (14), 4305-4315. 44. Mooney, C. A.; Mansfield, S. D.; Touhy, M. G.; Saddler, J. N., The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods. Bioresour. Technol. 1998, 64 (2), 113-119.

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45. Yu, Z.; Jameel, H.; Chang, H.-m.; Park, S., The effect of delignification of forest biomass on enzymatic hydrolysis. Bioresour. Technol. 2011, 102 (19), 9083-9089. 46. Stoklosa, R. J.; Hodge, D. B., Fractionation and improved enzymatic deconstruction of hardwoods with alkaline delignification. BioEnergy Res, 2015, 8 (3), 1224-1234. 47. Nakagame, S.; Chandra, R. P.; Kadla, J. F.; Saddler, J. N., Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin. Biotechnol. Bioeng. 2011, 108 (3), 538-548. 48. Del Rio, L. F.; Chandra, R. P.; Saddler, J. N., The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings. Biotechnol. Bioeng. 2011, 108 (7), 1549-1558. 49. Dimarogona, M.; Topakas, E.; Olsson, L.; Christakopoulos, P., Lignin boosts the cellulase performance of a GH-61 enzyme from Sporotrichum thermophile. Bioresour. Technol. 2012, 110, 480-487. 50. Rodríguez-Zúñiga, U. F.; Cannella, D.; Giordano, R. d. C.; Giordano, R. d. L. C.; Jørgensen, H.; Felby, C., Lignocellulose pretreatment technologies affect the level of enzymatic cellulose oxidation by LPMO. Green Chem. 2015, 17 (5), 2896-2903. 51. Lacayo, C. I.; Hwang, M. S.; Ding, S. Y.; Thelen, M. P., Lignin depletion enhances the digestibility of cellulose in cultured xylem cells. PLoS One 2013, 8 (7), e68266. 52. Sun, Q. N.; Foston, M.; Meng, X. Z.; Sawada, D.; Pingali, S. V.; O'Neill, H. M.; Li, H. J.; Wyman, C. E.; Langan, P.; Ragauskas, A. J.; Kumar, R., Effect of lignin content on changes occurring in poplar cellulose ultrastructure during dilute acid pretreatment. Biotechnol. Biofuels 2014, 7, 150. 53. Loque, D.; Scheller, H. V.; Pauly, M., Engineering of plant cell walls for enhanced biofuel production. Curr. Opin. Plant Biol. 2015, 25, 151-161. 54. Banerjee, G.; Car, S.; Scott-Craig, J. S.; Hodge, D. B.; Walton, J. D., Alkaline peroxide pretreatment of corn stover: effects of biomass, peroxide, and enzyme loading and composition on yields of glucose and xylose. Biotechnol. Biofuels 2011, 4 (1), 16. 55. Vanholme, R.; Morreel, K.; Ralph, J.; Boerjan, W., Lignin engineering. Curr. Opin. Plant Biol. 2008, 11 (3), 278-285. 56. Gallego-Giraldo, L.; Shadle, G.; Shen, H.; Barros-Rios, J.; Fresquet Corrales, S.; Wang, H.; Dixon, R. A., Combining enhanced biomass density with reduced lignin level for improved forage quality. Plant Biotechnol. J. 2016, 14 (3), 895-904. 57. Bonawitz, N. D.; Chapple, C., The genetics of lignin biosynthesis: connecting genotype to phenotype. Annu. Rev. Genet. 2010, 44, 337-363. 58. Van Acker, R.; Leple, J. C.; Aerts, D.; Storme, V.; Goeminne, G.; Ivens, B.; Legee, F.; Lapierre, C.; Piens, K.; Van Montagu, M. C. E.; Santoro, N.; Foster, C. E.; Ralph, J.; Soetaert, W.; Pilate, G.; Boerjan, W., Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (2), 845-850. 59. Cai, Y. H.; Zhang, K. W.; Kim, H.; Hou, G. C.; Zhang, X. B.; Yang, H. J.; Feng, H.; Miller, L.; Ralph, J.; Liu, C. J., Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase. Nat. Commu. 2016, 7, 11989. 60. Fu, C.; Mielenz, J. R.; Xiao, X.; Ge, Y.; Hamilton, C. Y.; Rodriguez, M., Jr.; Chen, F.; Foston, M.; Ragauskas, A.; Bouton, J.; Dixon, R. A.; Wang, Z. Y., Genetic 34 ACS Paragon Plus Environment

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manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (9), 3803-3808.

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Table of Contents/Abstract Graphic For Table of Contents Use Only

Zip-lignin and wild-type poplar were subjected to copper-catalyzed alkaline hydrogen peroxide pretreatment; sugar yields following enzymatic hydrolysis were higher for ziplignin poplar.

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