A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans
© The Author(s) 2018
Received: 27 December 2017
Accepted: 9 March 2018
Published: 22 March 2018
Acetylated 4-O-(methyl)glucuronoxylan (GX) is the main hemicellulose in deciduous hardwood, and comprises a β-(1→4)-linked xylopyranosyl (Xylp) backbone substituted by both acetyl groups and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Whereas enzymes that target singly acetylated Xylp or doubly 2,3-O-acetyl-Xylp have been well characterized, those targeting (2-O-MeGlcpA)3-O-acetyl-Xylp structures in glucuronoxylan have remained elusive.
An unclassified carbohydrate esterase (FjoAcXE) was identified as a protein of unknown function from a polysaccharide utilization locus (PUL) otherwise comprising carbohydrate-active enzyme families known to target xylan. FjoAcXE was shown to efficiently release acetyl groups from internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, an activity that has been sought after but lacking in known carbohydrate esterases. FjoAcXE action boosted the activity of α-glucuronidases from families GH67 and GH115 by five and nine times, respectively. Moreover, FjoAcXE activity was not only restricted to GX, but also deacetylated (3-O-Araf)2-O-acetyl-Xylp of feruloylated xylooligomers, confirming the broad substrate range of this new carbohydrate esterase.
This study reports the discovery and characterization of the novel carbohydrate esterase, FjoAcXE. In addition to cleaving singly acetylated Xylp, and doubly 2,3-O-acetyl-Xylp, FjoAcXE efficiently cleaves internal 3-O-acetyl-Xylp linkages in (2-O-MeGlcpA)3-O-acetyl-Xylp residues along with densely substituted and branched xylooligomers; activities that until now were missing from the arsenal of enzymes required for xylan conversion.
Enzymatic conversion of xylans to monosaccharides, oligosaccharides, or defined polymeric structures requires the concerted action of carbohydrate-active enzymes (CAZymes; http://www.cazy.org) from multiple CAZyme families . CAZymes that target side groups of GX and GAX include the following: α-l-arabinofuranosidases from glycoside hydrolase (GH) families GH3, GH43, GH51, GH54, and GH62, α-glucuronidases from the families GH67 and GH115, and acetyl xylan esterases (AcXEs) that have been primary reported in carbohydrate esterase (CE) families CE1, CE4–CE6, and CE16 [14–18]. Whereas α-glucuronidases from family GH115 target the α-(1→2)-linkage between MeGlcpA and mono-substituted Xylp at internal and end positions of GX, GH67 activity is restricted to such linkages at the non-reducing end of xylan and/or corresponding oligosaccharides . Similarly, CEs acting on GX have been grouped according to the positions they target (e.g., acting on O-2 and O-3 monoacetylated Xylp, versus targeting 2,3-di-O-acetylated Xylp) . Certain family CE16 enzymes also show activity towards non-reducing (2-O-MeGlcpA)3-O-acetyl-Xylp positions [19–22]; however, in these cases, acetyl group migration from O-3 to O-4 on the non-reducing Xylp could not be ruled out [5, 19, 23]. Critically, to date, there are no known esterases that efficiently target internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, nor α-glucuronidases that release MeGlcpA from acetylated Xylp, which hinders selective release of MeGlcpA substituents for material applications and full conversion of GX to fermentable monosaccharides [7, 19, 24]. Similarly, CEs capable of targeting acetyl groups adjacent to arabinosyl substituents and oligomeric structures in GAX remain to be uncovered.
Polysaccharide utilization loci (PULs) comprise a physically linked set of bacterial genes that encode CAZymes and other proteins that work in concert to modify and degrade specific polysaccharides and/or oligosaccharides . Predicted PULs were introduced to the CAZyme database in 2015 (http://www.cazy.org/PULDB/) , and have emerged as especially rich regions within genome and metagenome sequences for enzyme discovery. For example, within the last year alone, novel activities towards pectin , xylan , galactomannan , chitin , and β-glucans  were discovered.
Herein, we mined PUL sequences with the specific aim to finally uncover carbohydrate esterases that efficiently target (2-O-MeGlcpA)3-O-acetyl-Xylp positions internal to GX. Briefly, we identified PULs listed in the PULDB that encode CAZyme families known to target GX (i.e., GH10, GH115), and recombinantly expressed those sequences marked as having unknown function along with a predicted signal sequence for secretion. Ultimately, FjoAcXE encoded by Flavobacterium johnsoniae was selected and shown to cleave acetyl groups of internal and terminal (2-O-MeGlcpA)3-O-acetyl-Xylp structures of GX, leading to synergistic impacts on measured activities for both family GH67 and GH115 α-glucuronidases. The discovery of FjoAcXE thus completes the arsenal of CAZymes required for GX conversion to defined structures or full hydrolysis to monosaccharides. Moreover, the deacetylation activity of FjoAcXE on xylooligomers from corn fiber confirmed its ability to target acetylated Xylp structures also substituted by an oligomeric side chain, demonstrating the relevance of this unclassified CE activity for the conversion of diverse, complex xylans.
para-Nitrophenol (pNP) alkyl esters were purchased from the following sources: p-nitrophenyl acetate (C2) (Sigma, St. Louis, MO, USA; N8130), p-nitrophenyl butyrate (C4) (Sigma, N9876), p-nitrophenyl hexanoate (C6) (TCI-EP, Tokyo, Japan; H0484), p-nitrophenyl octanoate (C8) (Sigma, 21742), p-nitrophenyl decanoate (C10) (Sigma, N0252), p-nitrophenyl dodecanoate (C12) (Sigma, 61716), p-nitrophenyl myristate (C14) (Sigma, 70124), and p-nitrophenyl palmitate (C16) (Sigma, N2752). In addition, 4-methylumbelliferyl acetate (4-MUA) was purchased from Sigma (M0883) and 4-methylumbelliferone (4-MU) was purchased from Aldrich (M1381).
Acetylated monosaccharide substrates were purchased from the following sources: β-d-glucose pentaacetate (Aldrich, 285943), β-d-xylopyranose tetraacetate (Synthose Inc., Toronto, ON, Canada; TX534) and 1,2,3,4-tetra-O-acetyl-l-rhamnopyranose (Synthose Inc., TH212). Acetylated glucurono-xylooligosaccharides (Ac-XOS) were isolated by steam extraction of milled chips from Eucalyptus wood , or mixed hardwood kindly provided by Prof. Bradley Saville (University of Toronto, Canada) . Isolation and characterization of xylooligosaccharides from corn fibers were previously described .
The family GH115 α-glucuronidase from Amphibacillus xylanus (AxyAgu115) was purified as previously described . The family CE6 acetylxylan esterase from Orpinomyces sp. (E-AXEAO) was purchased from Megazyme (Bray, Ireland); the family GH67 α-glucuronidase from Cellvibrio japonicus (PRO-E0069; CjGlcA67A) was purchased from PROZOMIX Ltd (Haltwhistle, UK).
The PUL database (PULDB, http://www.cazy.org/PULDB)  was searched for PULs that contained at least two of the predicted xylan-active CAZyme families of interest, such as GH10, GH43, GH67, and GH115. From the obtained list of PULs, all proteins of unknown function were extracted and analyzed using Signal P4.1 ; those containing a predicted signal sequence for secretion were further analyzed based on the presence of Pfam domains, sequence length, and the type of CAZymes present on the corresponding PUL. Based on these criteria, FjoAcXE (PULDB ID: Fjoh_3879; GenBank ID: ABQ06890.1) was selected for recombinant protein expression. A structural model of FjoAcXE was built using PHYRE2.0  for the CBM-like domain, using PDB 2O14 as template, and Modeller 9.19  for the CE/catalytic domain using PDB 2O14 and PDB 1K7C . The model was displayed with PyMOLv22.214.171.124 Edu (PyMOL Molecular Graphics System, Schrödinger, LLC).
Gene synthesis and molecular cloning
The gene encoding FjoAcXE lacking the predicted signal sequence (residues 1–21) was codon optimized for expression in Escherichia coli K12 using IDT Codon Optimization Tool (http://www.idtdna.com/CodonOpt). Fifteen base pair extensions homologous to the p15TV-L vector (GenBank ID: EF456736.1) (T7, 5′-TTGTATTTCCAGGGC and T7term, 5′-CAAGCTTCGTCATCA) were added to each end of the sequence and the gene was synthesized as gBlock® gene fragments (Integrated DNA Technologies, Inc., Coralville, IA, USA). gBlock fragments were cloned into p15TV-L using the In-Fusion® HD EcoDry™ Cloning Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA). The resulting plasmid was transformed into E. coli HST08 Stellar™ Competent Cells (Clontech Laboratories, Inc.), and the sequence was verified using DNA sequencing service at the Center of Applied Genomics at the SickKids Hospital (Toronto, ON, Canada).
Protein expression and purification
E. coli BL21(λDE3) codon plus strain harboring p15TV-L-FjoAcXE was propagated at 37 °C in 8 L of Luria–Bertani (LB) Broth–Miller (BioShop) supplemented with 33 µg/mL chloramphenicol and 100 µg/mL ampicillin until the OD600 reached 0.6–0.8. Cultures were cooled on ice for 5 min; additional 33 µg/mL chloramphenicol and 100 µg/mL ampicillin were added, and recombinant expression was induced overnight at 16 °C with 0.5 mM isopropyl β-d-1-thiogalactopyranoside. Cells were harvested by centrifugation at 8967×g (Beckman Coulter, JLA-8.1000) for 15 min at 4 °C and the pellet (approx. 25 g fresh weight) was frozen at − 80 °C. The pellet was then suspended in binding buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol and 5 mM imidazole) and the cells were disrupted by sonication (100 amplitude, 5 s ON and 5 s OFF for 20 min). Cell debris was removed by centrifugation at 27,167×g (Beckman Coulter, JA-25.5 rotor) for 15 min at 4 °C and supernatant was filtered through Acrodisc® Syringe Filters with 0.45-µm Supor® membrane (Pall Corporation).
The sample was loaded onto 5-mL HisTrap HP (GE Healthcare) pre-equilibrated with binding buffer. A combination of step-wise and gradient elutions using elution buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol and 300 mM imidazole) was performed on Biologic DuoFlow™ chromatography system (BioRad) where the column was washed with 4 column volumes (CV) of 100% binding buffer, and then increasingly substituted with elution buffer as follows: 5% elution buffer for 4 CV, 10% elution buffer for 6 CV and then gradient elution from 10 to 100% elution buffer over 12 CV. All steps were done at 1 mL/min, protein elution was monitored at A280 nm and 2 mL fractions were collected throughout the entire run. The resulting fractions were analyzed by 12% SDS-PAGE; selected fractions were pooled and then exchanged to 25 mM HEPES (pH 8.0) using 10 kDA Jumbosep™ centrifugal devices (Pall Corporation). The sample was then further purified using a 1.3-mL UNO™ Q ion exchange column (BioRad) pre-equilibrated with 25 mM HEPES pH 8.0 (Buffer A). Following a wash using 11.5 CV of Buffer A, a step-wise gradient elution was performed at 1 mL/min on a Biologic DuoFlow™ using 25 mM HEPES pH 8.0 with 1 M NaCl as elution buffer (Buffer B), where 0–50% of Buffer B was passed over 15.4 CV, and then up to 100% Buffer B over 6 CV. The samples were collected as 1-mL fractions throughout the entire run and purity was checked with 12% SDS-PAGE. The purified sample was exchanged to 10 mM HEPES pH 7.5 containing 300 mM NaCl before being flash frozen in liquid nitrogen and stored at − 80 °C.
Protein concentration was measured using the Bradford assay with bovine serum albumin as a standard . The identity of purified FjoAcXE was then confirmed by peptide mass fingerprinting using an easy-nLC-1000 (ThermoFisher Scientific, Bremen, Germany) equipped with a 10.5-cm PicoTip Emitter Silica Tip packed in-house with C18 media coupled online to a Q-Exactive mass spectrometer (ThermoFisher Scientific) .
Enzyme activity measurements using pNP aliphatic fatty acid esters and 4-MUA
200 mM stock solutions for pNP alkyl esters from C2 to C12 were prepared in 100% DMSO, whereas pNP substrates from C14 to C16 were prepared in 1:1 (v/v) of isopropanol:acetonitrile. Reactions were performed in 50 mM HEPES (pH 7.0) with 2 mM substrate and were initiated by adding 0.5 µg of FjoAcXE; the final reaction volume was 200 µL. Reactions were conducted at pH 7.0 to minimize non-enzymatic de-esterification of pNP substrates over the prolonged incubation (i.e., 2 h). Reactions continued for 2 h at 30 °C, and absorbance was measured continuously at 410 nm. Reaction mixtures without FjoAcXE were used as a blank.
Kinetic parameters of FjoAcXE were determined using pNP-acetate and 4-MUA. For pNP-acetate, reactions contained approximately 0.06 µg (1.32 × 10−6 µmol) of FjoAcXE in 50 mM HEPES (pH 8.0) and were initiated by adding 0.05 mM to 10 mM pNP-acetate. The final reaction volume was 200 µL, and absorbance at 410 nm was read continuously for 40 min at 30 °C.
For 4-MUA, reactions contained approximately 0.02 µg (4.41×10−7 µmol) of FjoAcXE in 50 mM HEPES (pH 8.0) and final of 10% DMSO. The enzyme dose was chosen to ensure a linear relationship between reaction time and product release ; reactions were then initiated by adding 0.01–1.5 mM of 4-MUA. The final reaction volume was 200 µL, and absorbance at 354 nm was read continuously for 40 min at 30 °C. Kinetic parameters were calculated using Michaelis–Menten equation and Graphpad Prism 5 software (La Jolla, CA, USA).
Optimum reaction conditions
The pH optimum of FjoAcXE was tested using 100 mM Tris, 50 mM MES, 50 mM acetic acid, and 50 mM sodium acetate trihydrate buffer with pH range of 3.5–9.5. Reactions containing 0.5 mM 4-MUA were initiated by adding 5 µg of FjoAcXE, and continued for 20 min at 30 °C; the final reaction volume was 200 µL. 4-MUA was selected to determine the pH optimum of FjoAcXE given the relative stability of this substrate under alkaline conditions . For all reactions using 4-MUA, absorbance was measured at 354 nm and the reaction mixture without FjoAcXE was used as a blank.
To measure pH stability, solutions containing 4 µg of FjoAcXE were incubated for 16 h at 4 °C in 100 mM Tris, 50 mM MES, 50 mM acetic acid, and 50 mM sodium acetate trihydrate buffer adjusted to pH 3.5–9.5; the final solution volume was 40 µL. After incubation, residual enzyme activity was tested in reactions containing 0.5 mM 4-MUAc in 50 mM HEPES (pH 8.0). Reactions were initiated by adding 1 µg of FjoAcXE from each treatment and continued for 20 min at 30 °C; the final reaction volume was 200 µL.
The temperature stability of FjoAcXE was tested by suspending 4 µg of FjoAcXE in 50 mM HEPES (pH 8.0) and incubating each suspension (40 µL final volume) for up to 16 h at 20, 30, or 40 °C, or up to 60 min at 50, 60, or 70 °C. After the incubation, residual activity was tested in reactions containing 0.5 mM 4-MUAc in 50 mM HEPES (pH 8.0). Similar to pH stability, reactions were initiated by adding 1 µg of FjoAcXE from each treatment, and continued for 20 min at 30 °C; the final reaction volume was 200 µL.
Effect of divalent ions, detergents, and organic solvents
The metal-free apoenzyme of FjoAcXE was prepared as described in Wang et al. . Briefly, 0.05 g of CHELEX 100 (Sigma) was added to 1 mg of FjoAcXE and incubated at room temperature for 20 min. The effect of metal ions was tested in reactions containing 0.5 mM 4-MUA in 50 mM HEPES (pH 8.0). Reactions were initiated by adding 5 µg of FjoAcXE, and continued for 20 min at 30 °C in the presence of 1 mM of the following metal ions: Ag2+, Ca2+, Cd2+, Co2+, Cu2+, Fe3+, Mg2+, Mn2+, Ni2+, and Zn2+ (all as chloride salts, except for Ag2+, which was a nitrate). The final reaction volume was 200 µL, and control samples included CHELEX treated and untreated FjoAcXE with and without 20 mM EDTA. Absorbance was measured at 354 nm and the reaction mixture without FjoAcXE was used as a blank.
The chemical stability of FjoAcXE was tested in reactions containing 0.5 mM 4-MUAc in 50 mM HEPES (pH 8.0). Reactions were initiated by adding 0.5 µg of FjoAcXE, and continued for 20 min at 30 °C in the presence of 30% (v/v) ethanol, 30% (v/v) isopropanol, 1% (w/v) SDS, 1% (w/v) dithiothreitol (DTT), 1% (v/v) Tween-20, 1% (v/v) Tween-80, and 1% (v/v) TritonX-100; the final reaction volume was 200 µL.
Activity on acetylated monosaccharides
Reactions comprised 50 mM HEPES (pH 8.0) and 0.1% (w/v) of β-d-glucose pentaacetate (final concentration 2.5 mM), β-d-xylopyranose tetraacetate (final concentration 3.14 mM), or 1,2,3,4-tetra-O-acetyl-l-rhamnopyranose (final concentration 3 mM). Reactions were initiated by adding 0.5 µg of FjoAcXE; the final reaction volume was 30 µL. Reactions continued for 20 min at 30 °C, and were stopped by boiling for 10 min. The samples were quickly centrifuged and release of acetic acid was then measured in the supernatant using the Acetic Acid kit (K-ACETRM, Megazyme). Reaction mixtures without FjoAcXE were used as a blank for each substrate.
1H-NMR analysis of FjoAcXE activity on acetylated (glucurono)-xylooligosaccharides
1H-NMR was performed to monitor the activity of FjoAcXE and AxyAgu115A on Ac-XOS substrates. Reactions comprised 1% (w/v) acetylated (glucurono)-xylooligosaccharides in 50 mM HEPES buffer (pH 7.0) and 10 µg of each protein; the final reaction volume was 400 µL. The reaction continued for 20 h at 30 °C and gentle shaking. Reaction mixtures without enzyme were used as negative controls. Following the incubation, samples were filtered through Acrodisc® syringe filters with 0.2-µm Supor® membrane (Pall Corporation), and lyophilized. The samples were then dissolved in 300 µL D2O and transferred into 3-mm NMR tubes (Norell) for analysis using an Agilent DD2 700 MHz spectrometer equipped with a triple resonance HCN cold probe with a scan number of 64, relaxation delay of 1 s and acquisition time of 4.5 s. The data were obtained using VnmrJ 4.0 (Agilent) and analyzed with MestReNova 10.0 (Mestrelab Research). The HDO peak at 4.790 was used as internal standard. The change in signal intensity in the regions between 5.4 and 4.4 ppm corresponding to acetylated Xylp residues in the anomeric region of the spectrum, and 2.30–2.05 ppm region corresponding to the acetyl group methyl protons were used to assign proton chemical shifts, as reported in Uhliariková et al. .
Impact of FjoAcXE on α-glucuronidase activity towards (glucurono)-xylooligosaccharides
FjoAcXE, CjGlcA67A, AxyAgu115A, and E-AXEAO were tested alone and as pairs of carbohydrate esterase and α-glucuronidase activities. Reactions comprised 1% (w/v) Ac-XOS in 50 mM HEPES (pH 7.0), which is within one pH unit from the optimum pH of each enzyme. Reactions were initiated by adding 0.5 µg of each enzyme; the final reaction volume was 30 µL. Reactions continued for 20 min at 30 °C and were stopped by boiling for 10 min. The samples were quickly centrifuged and release of acetic acid and d-glucuronic acid were measured using Acetic Acid (K-ACETRM, Megazyme) and d-glucuronic acid/d-galacturonic acid (K-URONIC, Megazyme) assay kits, respectively. Reaction mixtures without enzymes were used as blanks.
Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF) analysis of FjoAcXE activity on feruloylated xylooligosaccharides
Reactions comprised 50 mM HEPES (pH 8.0), 1% (w/v) of feruloylated xylooligomers from corn fiber, and were initiated by adding 1 µg of FjoAcXE; the final reaction volume was 50 µL. Reactions continued for 20 min at 30 °C with gentle shaking, and were stopped by boiling the samples at 100 °C for 10 min. Reaction mixtures without enzyme were used as negative controls. One microliter of each reaction sample (after desalting with AG 1-X8 anion exchange resin; Bio-Rad, Hercules, CA, USA) was then mixed with 2 µL matrix solution [2,5-dihydroxybenzoic acid, 10 mg/mL H2O/ACN (3:7)] on a stainless-steel metal plate and allowed to dry under a constant stream of air. MALDI-TOF was performed using an Ultra-flex3 instrument (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser of 337 nm and operated in the positive mode. The system was controlled by FlexAnalysis software. Calibration was performed with a mixture of maltodextrins 480–3000 Da (Elicityl, Crolles, France). After a delayed extraction time of 130 ns, positive ions were accelerated with 22 kV voltage and detected using reflector mode.
Results and discussion
Candidate selection and recombinant protein production
General properties of FjoAcXE
Recombinant FjoAcXE was functionally expressed in E. coli with an N-terminal His6-tag and purified to homogeneity with approximate yield of 6 mg/g of fresh cell pellet. The predicted molecular mass of recombinant FjoAcXE is 45.2 kDa, which was consistent with that estimated by SDS-PAGE (Additional file 3: Fig. S3). Whereas no or negligible activity was measured on tested polysaccharides and pNP-glycosides (Additional file 4: Fig. S4, Additional file 5: Fig. S5, respectively), clear activity was observed on pNP-acetate followed by pNP-butyrate (Additional file 6: Fig. S6); notably, lack of activity on longer chain alkyl esters is consistent with esterase rather than lipase activity .
Kinetic parameters of FjoAcXE on 4-MUA and pNP-acetate
Specific activity (µmol/min/mg)
kcat/Km (s−1 mM−1)
126.3 ± 5.9
95.1 ± 4.4
0.8 ± 0.1
160.7 ± 4.0
120.8 ± 3.0
0.9 ± 0.1
Activity of FjoAcXE on acetylated mono- and oligosaccharides
MALDI-TOF analysis of FjoAcXE towards xylooligomers from corn fiber
An unclassified protein from a polysaccharide utilization locus predicted to promote xylan deconstruction was biochemically characterized and shown to harbor novel carbohydrate esterase activity, which was missing from the arsenal of enzymes required for GX conversion. In addition to promoting α-glucuronidase activity through release of acetyl groups from internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, FjoAcXE also targeted (3-O-Araf)2-O-acetyl-Xylp of feruloylated xylooligomers. The novel ability of FjoAcXE to release acetyl groups of Xylp backbone residues that are further substituted by either MeGlcpA or a neutral oligomeric side chain could be explained by the loop regions predicted to surround the catalytic site of the enzyme and to play a role in substrate preference. On-going efforts to solve the FjoAcXE structure, and to characterize predicted homologs of FjoAcXE, will help confirm these predictions and likely uncover additional protein features that determine the broad substrate range of this new carbohydrate esterase activity.
FMR selected, recombinantly expressed, purified, and characterized FjoAcXE, and wrote the manuscript. EJ analyzed FjoAcXE activity on feruloylated xylo-oligosaccharides by MALDI-TOF/TOF and contributed to data interpretation. PS developed and interpreted the FjoAcXE model structure. MT and MAK provided characterized acetylated glucurono-xylooligosaccharides and feruloylated xylo-oligosaccharides, respectively, and assisted with NMR and mass spectrometry analyses. RY and WW contributed to the basic characterization of FjoAcXE characterization, provision of AxyAgu115A, and 1H NMR analyses. ERM conceived and coordinated the study. All authors contributed to the revision of manuscript. All authors read and approved the final manuscript.
We thank Professor B. Saville (University of Toronto) for preparing hot water extracted glucuronoxylan from mixed hardwood, which was used to advance the characterization of FjoAcXE.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article [and its additional files].
Consent for publication
Ethics approval and consent to participate
This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) for the Strategic Network Grant “Industrial Biocatalysis Network,” the European Research Council (ERC) Consolidator Grant to ERM (BHIVE—648925), and an NSERC Canada Graduate Scholarship Masters Award and the Ontario Graduate Scholarship to FMR.
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