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ScienceDirect Protein engineering of cellulases Andreas S Bommarius1,2,3, Minjeong Sohn1,2,4, Yuzhi Kang1,2, Jay H Lee4 and Matthew J Realff1 This review covers the topic of protein engineering of cellulases, mostly after 2009. Two major trends that are identified in this work are: first, the increased importance of results from computational protein engineering to drive ideas in the field, as experimental ideas and results often are still scarce, and, second, the further development of helper proteins for cellulose hydrolysis, such as lytic polysaccharide monooxygenase (LPO). The discussion in this work focuses both on improved attributes of cellulases and on the domains of cellulase that have been improved. Addresses 1 School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA 2 Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA 3 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA 4 School of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon, South Korea
The present review presents recent progress classified by: first, protein, second, protein domain, that is, catalytic unit, linker sequence, and binding domain, third, protein engineering technique, and fourth, attribute targeted for optimization, such as catalytic activity, processivity or thermostability. As results differ depending on the domain involved, improvements of performance attributes are ordered along the line of domains (Figure 1).
Recent trends This review uncovers the following trends on cellulases that will shape the field for time to come: Results of computational protein engineering are increasingly important to drive ideas in the field, especially in absence of or in case of scarce experimental data.
Corresponding authors: Bommarius, Andreas S ([email protected]) and Lee, Jay H ([email protected])
Helper proteins, such as lytic polysaccharide monooxygenase (LPO) define progress on the topic of commercial mixtures for enzymatic hydrolysis of cellulase and its components.
Current Opinion in Biotechnology 2014, 29:139–145
The following trends are outside the scope of this review:
This review comes from a themed issue on Cell and pathway engineering Edited by Tina Lu¨tke-Eversloh and Keith EJ Tyo
0958-1669/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.copbio.2014.04.007
Overview and introduction This review focuses on recent protein engineering efforts, both site-specific and random, mostly since 2009, on a range of proteins important for the hydrolysis of lignocellulosic materials, not only exocellulases and endocellulases but also helper proteins, such as lytic polysaccharide monooxygenase (LPO), swollenins, and expansins. The subject recently has been reviewed by Wilson [1], Clarke et al. [2], and Kellermann et al. [3]. Major advances during the reporting period can be found on the subjects of, first, the development of LPO [4], second, the thermostabilization of Cel6A and Cel7A via SCHEMAguided recombination [5,6], and, third, the computationally-based findings of the importance of the linker sequence for binding onto cellulose surfaces [7–9]. www.sciencedirect.com
(i) Kinetics and kinetic models of cellulose action; for these, the reader is encouraged to consult the review by Bansal et al. [10] as well as the models developed by Levine et al. [11] and Fox et al. [12], Cruys-Bagger et al. [13,14], Kurasin and Valjamae [15], and Bansal et al. [16]; (ii) The discussion of influences of crystallinity, accessibility, or mass transfer limitations. The reader is referred to the works by Hall et al. [17], Ponni et al. [18], Kumar et al. [19], Ioelovich [20], and Roberts et al. [21], respectively; (iii) Discussion of hemicellulases and esterases.
Protein engineering of the catalytic domain (CD) Processivity
The three-dimensional structure of the TrCel7A catalytic domain shows a long active site tunnel harboring four tryptophan residues. They are known to be essential for hydrophobic stacking interactions for the glucosyl units at the tunnel entrance (Trp-40), at the center of the tunnel (Trp-38), and around the catalytic sites (Trp-367 and Trp376) and these interactions are linked to effective and continuous hydrolysis of crystalline cellulose in a processive mode. Nakamura et al. found that the tryptophan Current Opinion in Biotechnology 2014, 29:139–145
140 Cell and pathway engineering
Figure 1
Carbohydrate -Binding Module
Catalytic Module
Cellulase Activity Thermostability Processivity Binding
Linker
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Improvement of performance attributes depends on the cellulose domain involved.
located at the tunnel entrance is important for processivity of degradation of crystalline cellulose [22]. They evaluated enzyme specific activity of WT and Trp-40 mutant on amorphous and crystalline cellulose revealing that Trp-40 is involved in recruiting cellulose chain reducing ends into the active site tunnel to initiate processive hydrolysis. Synergism with endocellulases and processivity
Vuong and Wilson [23] examined the exocellulase Cel6B properties of processivity and synergism with endocellulases from the Thermobifida fusca soil bacterium. Six residues not involved in the active site but in the active site tunnel were targeted for mutation, as summarized in Table 1a. Although processivity and oligosaccharide production were increased by a factor of 2–3 in some variants, the synergism with a native Cel5A was reduced such that the exocellulase exhibited lower processivity than the wild type. It was postulated that the decrease in side chain volume allowed freer movement of the cellulose chain along the catalytic tunnel. The lack of synergism was attributed to a potential reduction in the activity on the easily hydrolysable soluble chains produced by the endocellulase. An experiment with reducing-end attacking exocellulases Cel48A did show increased synergism, suggesting that the simple synergism model of endocellulases producing more reducing ends for Current Opinion in Biotechnology 2014, 29:139–145
exocellulases to attack is insufficient to explain the observed phenomena. Engineering of glycosylation for CD
Adney et al. examined the effects of N-linked glycans of the catalytic domain on cellulase activity by genetically removing or adding the N-glycosyl modification [24]. The removal of N-linked glycans located at both N384A of TrCel7A and N388A of Penicillium funiculosum Cel7A (PfCel7A) exhibited improved hydrolysis activity for crystalline cellulosic substrate. Similarly, the addition of a new N-glycosylation motif to PfCel7A (A196S) near the proximal end of the active site tunnel enhanced enzyme activity for cellulose degradation. These results suggest that glycosylation could be a target for improvement of cellulase performance. Reduced inhibition by glucose
Cellulases are often inhibited by their end product, cellobiose, and to a lesser degree by the glucose produced by further hydrolysis. Lavigne et al. [25] engineered Cel6A enzyme to reduce glucose inhibition, thus allowing cellulose hydrolysis to proceed at a rate closer to the maximum. They selected amino acids at position 103, 136, 186, 365, or 410 of TrCel6A enzyme for mutation and showed 1.8-fold to 6.73-fold reductions in glucose www.sciencedirect.com
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Table 1 Protein engineering on cellobiohydrolases Cellulase
Residue
Proposed function
Evidence f
Source
(a) Catalytic domain W135, W376, W269, W272
TrCel6A a
Free energy of substrate binding
Relative binding affinity changed by orders of magnitude.
[9]
W40
TrCel7A
Recruitment of substrate chains into the active site tunnel.
Experimental variation in cellobiose production from different substrates
[22]
W376
TrCel7A
Hydrophobic stacking interactions for glucosyl units (catalytic center), free energy of product binding Free energy of product binding
Change in free energy of product binding in computational mutation experiment.
[33]
R251 D259 D262 Y381 Y103, L136, S186, G365, R410
TrCel6A
N282
b
TfCel6B
Tunnel entrance sugar binding (+2 position)
R180
Tunnel exit sugar binding (4 position)
L230
Processivity
W464
Substrate binding
D512
Loop flexibility
M514
Change in free energy of product binding in computational mutation experiment.
Reduce inhibition by glucose
[25]
Mutants have less bulky side chains N282A, N282D Mutant 2 processive, activity N282A/D 3 on PASC, 2/1.5 on CMC Mutants have less bulky side chains R180K, R180A twice processivity, no significant increase in activity. L230A minor processive gain 2.5 active on PASC W464A/Y 7/2 activity on CMC 0.25/ 0.34 activity on BMCC D512A 1.74 on PASC 2.4 on PC, 5.68 on CMC 0.34 on BMCC M514A 1.5 on CMC 1.3 on BMCC, M514Q 1.74 on CMC 1.25 on BMCC
[23]
N384 for Tr N388 for Pf A196 for Pf
TrCel7A c PfCel7A
Glycan in peptide loop of the active site tunnel
1.7 hydrolysis activity on BMCC
[24]
C313S
d
HiCel6A
Thermostability
Increase thermostability by 8.5 8C, activity by 10
[29]
S329G
e
CtCel8A
Thermostability
Increase thermostability by 7.0 8C, half-life at 85 8C by 8.5
[28]
TrCel6A, TrCel7A
Glycosylation in linker
R!E/S!T, reduce lignin binding and/or increase hydrolysis rate
[36]
Processivity
Energy minima corresponding to a cellobiose unit along hydrophobic face of cellulose
[34]
(b) Linker All R and S in the linker
(c) Carbohydrate-binding modules Y5, Q7, N29, Y32 TrCel7A
a
TrCel7A: Trichoderma reesei Cel7A. TfCel6B: Thermobifida fusca Cel6B. c PfCel7A: Penicillium funiculosum Cel7A. d HiCel6A: Humicola insolens Cel6A. e CtCel8A: Clostridium thermocellum Cel8A. f CMC, carboxymethyl cellulose; PASC, phosphoric acid-swollen cellulose; BMCC, bacterial microcrystalline cellulose; PC, phosphoric acid-treated cotton. b
inhibition. Such improved cellulases could be useful for integrated hydrolysis processes which benefit from constant cellulase activity in presence of glucose, pending direct evidence of improvement of overall hydrolysis rate. www.sciencedirect.com
Thermal stabilization via protein engineering
Thermostable cellulases are useful in biofuel processes, as they feature increased specific activity, higher levels of stability, inhibition of microbial growth, or increase in Current Opinion in Biotechnology 2014, 29:139–145
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mass transfer rate due to lower fluid viscosity. A recent review [26] describes state-of-the-art protein engineering protocols, which are based on combinatorial and datadriven techniques, to achieve thermostable cellulases. Such techniques and protocols are envisioned to be important for other improved cellulase properties, such as solvent, salt, and extreme pH tolerance. The Arnold group at Caltech built on their highly successful structureguided recombination (SCHEMA) of three fungal cellobiohydrolases 2 (CBH2) [6]. In chimeras of CBH2, stability was found to be additive with respect to certain sequence blocks via a linear mathematical model which also resulted in a successful single mutation, C313S [27]. A similar approach worked on Cel8A and on CBH1: in the former case, the half-life at 85 8C of Cel8A of Clostridium thermocellum was enhanced 8.5 in the single variant S329G [28]; in the latter case, 38% (6 of 16) predicted thermostabilized chimeras with up to 37 altered amino acid positions compared to the wildtype from Talaromyces emersonii hydrolyzed solid cellulose at 70 8C, 5 8C higher than the wildtype CBH1 [29]. Through utilization of FoldX and the consensus sequence approach, further mutations were found in five homologous CBH1 enzymes and 41 aligned CBH1 sequences to improve the temperature of optimum activity Topt to 10 8C above the wild-type T. emersonii [5]. In the fungal family 6 cellobiohydrolases (Cel6A), free cysteines, in contrast to disulfide bonds, were found to contribute to irreversible thermal inactivation in both wildtype Cel6A from Hypocrea jecorina and Humicola insolens as well as engineered thermostable Cel6A from these parents [30]. The consensus approach was also successful in two other recent studies. Alignment of five thermophilic CBH2 proteins resulted in 15 advantageous positions out of 45 non-consensus sequence positions in the Phanerochaete chrysosporium protein and multifold stabilization at 50 8C [31]. The consensus approach on family 8 endoglucanases (Cel8A) yielded a single mutation, G283P, which in conjunction with three previously identified variations led to a 14-fold increased half-life at 85 8C [32].
change of the free energy of binding [9]. The positions inside the tunnel did not exhibit large changes in free energy but were important in stabilizing the fluctuations associated with movement of cellobiose substrate moieties. The conclusion was that given the different factors involved in the aromatic–carbohydrate interactions, it may be difficult to generalize protein engineering across glycolytic hydrolase families to improve ligand binding and processivity based on tunnel position alone.
Protein engineering of the linker The linkers from cellulase enzymes are thought to connect glycoside hydrolases to carbohydrate-binding domain flexibly, and their sequences are observed to have diversity in amino acid content as well as length so that their functional role has remained unclear. To gain insight into the function of the linker and to maximize the effect on enzyme performance, linker engineering can be targeted at linker length, amino acid content, and glycosyl modification since each one contributes to linker flexibility, structure, and interaction with the substrate. Studies have demonstrated that linker length is optimized on type of structural domain and that the linker contained glycine, proline, serine, and threonine residues [7,8]. Scott et al. [36] constructed linker variants to obtain lignin resistant cellulase enzyme by altering linker amino acids (arginine or serine to glutamate or threonine, respectively) so that linker peptide-engineered cellulase showed increases in hydrolysis activity in the presence of lignin along with decreasing lignin binding. Payne et al. [37] constructed two simulation models for TrCel7A and TrCel6A and showed that O-glycosylated linker dynamically and nonspecifically binds to the hydrophobic face of cellulose. They suggested both through simulation and experimentation that O-glycan attached on serine and threonine residues served not only to extend peptide conformation, but also to affect linker binding directly to cellulose, causing enhanced binding affinity.
Protein engineering of the carbohydratebinding module (CBM)
Computational investigation of CDs
Proximity and targeting
Cellulase functionality is complex because of both its multiple domain structure and the different physicochemical phenomena that are involved in cellulose digestion. This has prompted computational molecular dynamics studies of different aspects of cellulase behavior, and its different subunits, to improve understanding and develop targets for protein engineering [7– 9,33,34,35]. In a recent paper, steered molecular dynamics simulations of TrCel7A were used to hypothesize that certain positions, listed in Table 1a, play key roles in cellobiose and glucose binding to the catalytic tunnel [33]. These were mutated in silico and the change in the free energy of binding during product expulsion computed. Mutations of the aromatic residues at different tunnel positions showed significant differences in the
CBMs display no hydrolytic function and the widely accepted role of CBMs is their binding affinity to both soluble and insoluble carbohydrate substrates. Through addition or deletion of the CBM, ligand-binding capacity can be altered. It has been reported that single-module polysaccharide hydrolases, such as cellobiohydrolase and mannanase, exhibit higher catalytic activity as well as thermostability when attached to heterologous CBMs [38–41]. Upon fusion to other non-cellulolytic enzymes, the targeting effect of CBM can bring the chimera to within close proximity of the carbohydrate-containing substrate, which in turn improves the appended enzymes activity on their substrates in close association with carbohydrates. Ravalason et al. [42] demonstrated that biobleaching efficiency can be drastically enhanced by
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forming a chimeric enzyme comprising laccase and family 1 CBM, through which the effective chimera concentration on insoluble lignin/cellulose surface can be enriched. However, higher ligand-binding capacity/affinity does not guarantee an improved cellulase activity as the ultra-tight binding could become a restriction for the dynamic motion of the enzymes [43]. Through the comparison study of a single-CBM and double-CBM processive endoglucanase Cel9A, the same mutations introduced to the latter were found to be significantly less advantageous. This is possibly because of the existence of a strong wild-type binding domain in addition to the one being engineered [44]. Besides the critical residues responsible for carbohydrate adsorption, the binding affinity of CBMs is modulated in part by the glycosylation pattern. Taylor et al. [35] through computational investigation found a 140-fold increase in binding attributable to the addition of an artificial glycan on serine locates on the planar binding face of TrCel7A. The improvement on binding and the ultimate effect on hydrolysis efficiency await experimental validation. Nonetheless, the molecular simulation results present an alternative direction for protein engineering of cellulases.
Table 1c. Depending on the different modular structures of cellulases, the mode of processivity can be enzymespecific. Nevertheless, the recognition of the protein– carbohydrate interaction face and critical residues for binding in the CBM could provide the most direct evidence for its role in processivity. Besides the proximity, targeting and processivity effect, novel disruptive functions of CBMs have been discovered, such as hydrogen bond splitting and cellulose decrystallization effects [52,53]. However, to our knowledge, disruptive functionality has not been targeted for protein engineering.
Protein engineering of the cellulase booster
In addition to the positive ligand-recognition function, recently, the role of CBMs at high substrate consistency was investigated for both cellulose and lignocelluloses. Even more than pure cellulose, the lignin-containing plant cell wall is highly recalcitrant and resistant to biological degradation. Cellulase deprived of CBMs exhibits similar or even higher hydrolysis yield at high substrate loadings when compared to wild-type cellulase, especially for lignocelluloses. It was thus postulated that CBMs partly contribute to non-productive binding to lignin [45,46]. However, more direct evidence on CBMs’ preferential adsorption onto lignin is needed to support the hypothesis and demonstrate the generic applicability for CBM-containing cellulases.
Novel non-hydrolytic proteins, which are capable of significantly enhancing the activity of cellulases through reducing the crystallinity of cellulose, have been studied recently. These proteins potentially lead to reduction in the quantity of relatively expensive cellulase added to enzymatic hydrolysis. Expansin-like proteins produced by fungi and bacteria, called swollenins, are known to be capable of loosening or disrupting the cellulose [54–56]. LPOs were functionally identified as participating in the degradation of cellulose by catalyzing oxidative cleavage of polysaccharide [4]. These proteins bind to the surfaces of crystalline cellulose, where they introduce chain breaks in the polysaccharide chains promoting deagglomeration and dispersion. Then the cellulosic matrix becomes susceptible to the action of a cellulase, which significantly accelerates hydrolysis of the sugars. Three types of cellulases have been displayed on yeast cell surfaces as well as expansin-like proteins, resulting in simultaneous saccharification and fermentation. This resulted in the production of 3.4 g/L ethanol from amorphous cellulose [57]. Combining cellulase and helper protein for pretreatment and fermentation has a great potential in reduction of enzyme cost for biofuels production and opening new targets for cellulase engineering.
Processivity
Conclusions
The impact of CBMs on the mechanism of enzyme processivity is not completely understood [47]. Among the recently published work, processivity is believed mainly to be controlled by the residues of the CDs [44,48,49]. However, controversial results are also reported regarding the role of CBMs in processivity [50]. Recently, the CBM of a family 5 endoglucanases was identified to play a vital role in enzyme processivity through comparison with the CBM-less variant [51] and computational simulation results also provide evidence that family 1 CBMs are responsible for processivity [34]. The TrCel7A CBD exhibits thermodynamically stable regions corresponding to the length of a cellobiose unit, which thus is the critical length scale of the processive TrCel7A [34]. The residues recognized to be responsible for the processivity length scale of TrCel7A are listed in
The complex nature of cellulase functionality has led to recent protein engineering studies designed to inform us on our understanding of the contribution of different protein subunits to the overall protein function. Computational studies can be particularly effective in this regard, because information that is inaccessible to experimental studies can be obtained. Feedback between experimental probing of sequences designed by computational studies to test hypotheses of sequence-to-function mapping shows significant promise to tease apart processivity, substrate binding, accessibility, and kinetic limitations of cellulases. Equipped with greater understanding of which functionalities limit cellulase rates on different substrates, a greater variety of protein engineering tools can be brought to bear on these tightly coupled complicated enzymes with higher probabilities of success in improving their function.
Non-productive binding to lignin
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144 Cell and pathway engineering
Acknowledgements Minjeong Sohn and the other authors gratefully acknowledge support from the Advanced Biomass Center (ABC) program at KAIST, Daejeon, South Korea. Yuzhi Kang acknowledges support from the Institute of Paper Science and Technology (IPST) at the Georgia Institute of Technology.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Wilson DB: Cellulases and biofuels. Curr Opin Biotechnol 2009, 20:295-299.
2.
Clarke ND: Protein engineering for bioenergy and biomassbased chemicals. Curr Opin Struct Biol 2010, 20:527-532.
3.
Kellermann SJ, Rentmeister A: Current developments in cellulase engineering. ChemBioEng Rev 2014, 1:6-13.
4.
Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG: Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 2012, 5:1-13.
5.
Komor RS, Romero PA, Xie CB, Arnold FH: Highly thermostable fungal cellobiohydrolase I (Cel7A) engineered using predictive methods. Protein Eng Des Sel 2012, 25:827-833.
6.
Heinzelman P, Snow CD, Wu I, Nguyen C, Villalobos A, Govindarajan S, Minshull J, Arnold FH: A family of thermostable fungal cellulases created by structure-guided recombination. Proc Natl Acad Sci USA 2009, 106:5610-5615. This frequently cited paper introduced recombination of whole cellulose domains as a method for thermal stabilization. Not only could the catalytic domain be significantly stabilized but a regression of the data demonstrated additivity of mutations for stabilization.
7.
8.
9.
Beckham GT, Bomble YJ, Matthews JF, Taylor CB, Resch MG, Yarbrough JM, Decker SR, Bu L, Zhao X, McCabe C: The Oglycosylated linker from the Trichoderma reesei family 7 cellulase is a flexible, disordered protein. Biophys J 2010, 99:3773-3781. Sammond DW, Payne CM, Brunecky R, Himmel ME, Crowley MF, Beckham GT: Cellulase linkers are optimized based on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation. PLoS ONE 2012, 7:e48615. Payne CM, Bomble Y, Taylor CB, McCabe C, Himmel ME, Crowley MF, Beckham GT: Multiple functions of aromaticcarbohydrate interactions in a processive cellulase examined with molecular simulation. J Biol Chem 2011, 286:41028-41035.
10. Bansal P, Hall M, Realff MJ, Lee JH, Bommarius AS: Modeling cellulase kinetics on lignocellulosic substrates. Biotechnol Adv 2009, 27:833-848. 11. Levine SE, Fox JM, Blanch HW, Clark DS: A mechanistic model of the enzymatic hydrolysis of cellulose. Biotechnol Bioeng 2010, 107:37-51. 12. Fox JM, Levine SE, Clark DS, Blanch HW: Initial- and processivecut products reveal cellobiohydrolase rate limitations and the role of companion enzymes. Biochemistry 2012, 51:442-452. 13. Cruys-Bagger N, Elmerdahl J, Praestgaard E, Borch K, Westh P: A steady-state theory for processive cellulases. FEBS J 2013, 280:3952-3961. 14. Cruys-Bagger N, Tatsumi H, Ren GR, Borch K, Westh P: Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain. Biochemistry 2013, 52:8938-8948. 15. Kurasin M, Valjamae P: Processivity of cellobiohydrolases is limited by the substrate. J Biol Chem 2011, 286:169-177. 16. Bansal P, Vowell BJ, Hall M, Realff MJ, Lee JH, Bommarius AS: Elucidation of cellulose accessibility, hydrolysability and reactivity as the major limitations in the enzymatic hydrolysis of cellulose. Bioresour Technol 2012, 107:243-250. Current Opinion in Biotechnology 2014, 29:139–145
17. Hall M, Bansal P, Lee JH, Realff MJ, Bommarius AS: Cellulose crystallinity—a key predictor of the enzymatic hydrolysis rate. FEBS J 2010, 277:1571-1582. 18. Ponni R, Kontturi E, Vuorinen T: Accessibility of cellulose: Structural changes and their reversibility in aqueous media. Carbohydr Polym 2013, 93:424-429. 19. Kumar L, Arantes V, Chandra R, Saddler J: The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility. Bioresour Technol 2012, 103:201-208. 20. Ioelovich M: Accessibility and crystallinity of cellulose. Bioresources 2009, 4:1168-1177. 21. Roberts KM, Lavenson DM, Tozzi EJ, McCarthy MJ, Jeoh T: The effects of water interactions in cellulose suspensions on mass transfer and saccharification efficiency at high solids loadings. Cellulose 2011, 18:759-773. 22. Nakamura A, Tsukada T, Auer S, Furuta T, Wada M, Koivula A, Igarashi K, Samejima M: The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7a is important for initiation of degradation of crystalline cellulose. J Biol Chem 2013, 288:13503-13510. Mutates Trp-40 involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis, measures differences in kinetic rate constants and combines this with molecular simulation to propose mechanistic explanation. 23. Vuong TV, Wilson DB: Processivity, synergism, and substrate specificity of Thermobifida fusca Cel6B. Appl Environ Microbiol 2009, 75:6655-6661. Performs specific mutations of the catalytic domain and demonstrates substrate specific engineering of cellulase. Shows that synergism between cellulases can be disrupted by these mutations. 24. Adney WS, Jeoh T, Beckham GT, Chou Y-C, Baker JO, Michener W, Brunecky R, Himmel ME: Probing the role of Nlinked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis. Cellulose 2009, 16:699-709. 25. Lavigne J, Hill C, Tremblay A, St-Pierre P, Tomashek J. Cellulase variants with reduced inhibition by glucose. WO Patent 2,009,089,630; 2009. 26. Anbar M, Bayer EA: Approaches for improving thermostability characteristics in cellulases. Cellulases 2012, 510:261-271. 27. Heinzelman P, Snow CD, Smith MA, Yu XL, Kannan A, Boulware K, Villalobos A, Govindarajan S, Minshull J, Arnold FH: SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability. J Biol Chem 2009, 284:26229-26233. 28. Anbar M, Lamed R, Bayer EA: Thermostability enhancement of Clostridium thermocellum cellulosomal endoglucanase Cel8A by a single glycine substitution. Chemcatchem 2010, 2:9971003. 29. Heinzelman P, Komor R, Kanaan A, Romero P, Yu XL, Mohler S, Snow C, Arnold F: Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination. Protein Eng Des Sel 2010, 23:871-880. 30. Wu I, Heel T, Arnold FH: Role of cysteine residues in thermal inactivation of fungal Cel6A cellobiohydrolases. Biochim Biophys Acta-Proteins Proteomic 2013, 1834:1539-1544. 31. Ito Y, Ikeuchi A, Imamura C: Advanced evolutionary molecular engineering to produce thermostable cellulase by using a small but efficient library. Protein Eng Des Sel 2013, 26:73-79. 32. Anbar M, Gul O, Lamed R, Sezerman UO, Bayer EA: Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Appl Environ Microbiol 2012, 78:3458-3464. 33. Bu L, Beckham GT, Shirts MR, Nimlos MR, Adney WS, Himmel ME, Crowley MF: Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy methods. J Biol Chem 2011, 286:1816118169. www.sciencedirect.com
Protein engineering of cellulases Bommarius et al. 145
34. Beckham GT, Matthews JF, Bomble YJ, Bu LT, Adney WS, Himmel ME, Nimlos MR, Crowley MF: Identification of amino acids responsible for processivity in a family 1 carbohydratebinding module from a fungal cellulase. J Phys Chem B 2010, 114:1447-1453. This work identified the potential role of CBMs in the processivity of T. reesei Cel7A by computational simulation. 35. Taylor CB, Talib MF, McCabe C, Bu LT, Adney WS, Himmel ME, Crowley MF, Beckham GT: Computational investigation of glycosylation effects on a family 1 carbohydrate-binding module. J Biol Chem 2012, 287:3147-3155. This work utilizes molecular simulation to study the effect of glycosylation on the TrCel7A CBM. Both native and artificial glycosylation showed substantial enhancement on binding compared to nonglycosylated CBM. 36. Scott BR, St-Pierre P, Lavigne JA, Masri N, White TC, Tomashek JJ. Novel lignin-resistant cellulase enzymes. WO Patent 2,010,096,931; 2010. The mutant of arginine and sernine substituted to glutamic acid and threonine, respectively, exhibited increased hydrolysis activity in presence of lignin and decreased lignin binding. The result of this study indicated that protein engineering of linker domain of cellobiohydrolases can yield lignin resistance. 37. Payne CM, Resch MG, Chen L, Crowley MF, Himmel ME, Taylor LE, Sandgren M, Sta˚hlberg J, Stals I, Tan Z: Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci 2013, 110:14646-14651. This study suggests that O-glycosylated linker domains of cellobiohydrolases (TrCel7A and TrCel6A) bind to the hydrophobic face of cellulose microfibrils through molecular dynamics simulations. In order to validate their model they measured binding affinity of TrCel7A CBM and the glycosylated TrCel7A CBM-linker on cellulose, in consistent to prediction the linker showed a higher binding affinity. The result of this study indicated that glycosylated linker play a role in direct interaction with crystalline cellulose. 38. Voutilainen SP, Nurmi-Rantala S, Penttila M, Koivula A: Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 2013. 39. Hall M, Rubin J, Behrens SH, Bommarius AS: The cellulosebinding domain of cellobiohydrolase Cel7A from Trichoderma reesei is also a thermostabilizing domain. J Biotechnol 2011, 155:370-376. 40. Tang CD, Li JF, Wei XH, Min R, Gao SJ, Wang JQ, Yin X, Wu MC: Fusing a carbohydrate-binding module into the Aspergillus usamii beta-mannanase to improve its thermostability and cellulose-binding capacity by in silico design. PLoS ONE 2013:8. 41. Thongekkaew J, Ikeda H, Masaki K, Iefuji H: Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates. Enzyme Microb Technol 2013, 52:241-246. 42. Ravalason H, Herpoel-Gimbert I, Record E, Bertaud F, Grisel S, de Weert S, van den Hondel CA, Asther M, Petit-Conil M, Sigoillot JC: Fusion of a family 1 carbohydrate binding module of Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulp biobleaching. J Biotechnol 2009, 142:220-226. 43. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ: Carbohydratebinding modules: fine-tuning polysaccharide recognition. Biochem J 2004, 382:769-781. 44. Li Y, Irwin DC, Wilson DB: Increased crystalline cellulose activity via combinations of amino acid changes in the family 9
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catalytic domain and family 3c cellulose binding module of Thermobifida fusca Cel9A. Appl Environ Microbiol 2010, 76:2582-2588. This work elucidates the significance of the binding balance between CD and CBM as well as within CD binding cleft. 45. Le Costaouec T, Pakarinen A, Varnai A, Puranen T, Viikari L: The role of carbohydrate binding module (CBM) at high substrate consistency: comparison of Trichoderma reesei and Thermoascus aurantiacus Cel7A (CBHI) and Cel5A (EGII). Bioresour Technol 2013, 143:196-203. 46. Varnai A, Siika-Aho M, Viikari L: Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs. Biotechnol Biofuels 2013, 6:30. 47. Li Y, Irwin DC, Wilson DB: Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A. Appl Environ Microbiol 2007, 73:3165-3172. This work identifies the critical residues for processivity within the CD of endoglucanase Cel9A. 48. Igarashi K, Koivula A, Wada M, Kimura S, Penttila M, Samejima M: High speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose. J Biol Chem 2009, 284: 36186-36190. An important piece of work visualizing the processive motion of TrCel7A and its variants on insoluble cellulose substrates. 49. Chiriac AI, Cadena EM, Vidal T, Torres AL, Diaz P, Pastor FI: Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture. Appl Microbiol Biotechnol 2010, 86:1125-1134. 50. Irwin D, Shin DH, Zhang S, Barr BK, Sakon J, Karplus PA, Wilson DB: Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis. J Bacteriol 1998, 180:1709-1714. 51. Zheng F, Ding S: Processivity and enzymatic mode of a glycoside hydrolase family 5 endoglucanase from Volvariella volvacea. Appl Environ Microbiol 2013, 79:989-996. 52. Ciolacu D, Kovac J, Kokol V: The effect of the cellulosebinding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers. Carbohydr Res 2010, 345:621-630. 53. Hall M, Bansal P, Lee JH, Realff MJ, Bommarius AS: Biological pretreatment of cellulose: enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases. Bioresour Technol 2011, 102:2910-2915. 54. Kim IJ, Ko HJ, Kim TW, Choi IG, Kim KH: Characteristics of the binding of a bacterial expansin (BsEXLX1) to microcrystalline cellulose. Biotechnol Bioeng 2013, 110:401-407. 55. Lee HJ, Lee S, Ko H-j, Kim KH, Choi I-G: An expansin-like protein from Hahella chejuensis binds cellulose and enhances cellulase activity. Mol Cells 2010, 29:379-385. 56. Ja¨ger G, Girfoglio M, Dollo F, Rinaldi R, Bongard H, Commandeur U, Fischer R, Spiess AC, Bu¨chs J: How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis. Biotechnol Biofuels 2011, 4:33. 57. Nakatani Y, Yamada R, Ogino C, Kondo A: Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose. Microb Cell Fact 2013, 12:66.
Current Opinion in Biotechnology 2014, 29:139–145
ScienceDirect Protein engineering of cellulases Andreas S Bommarius1,2,3, Minjeong Sohn1,2,4, Yuzhi Kang1,2, Jay H Lee4 and Matthew J Realff1 This review covers the topic of protein engineering of cellulases, mostly after 2009. Two major trends that are identified in this work are: first, the increased importance of results from computational protein engineering to drive ideas in the field, as experimental ideas and results often are still scarce, and, second, the further development of helper proteins for cellulose hydrolysis, such as lytic polysaccharide monooxygenase (LPO). The discussion in this work focuses both on improved attributes of cellulases and on the domains of cellulase that have been improved. Addresses 1 School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA 2 Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA 3 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA 4 School of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon, South Korea
The present review presents recent progress classified by: first, protein, second, protein domain, that is, catalytic unit, linker sequence, and binding domain, third, protein engineering technique, and fourth, attribute targeted for optimization, such as catalytic activity, processivity or thermostability. As results differ depending on the domain involved, improvements of performance attributes are ordered along the line of domains (Figure 1).
Recent trends This review uncovers the following trends on cellulases that will shape the field for time to come: Results of computational protein engineering are increasingly important to drive ideas in the field, especially in absence of or in case of scarce experimental data.
Corresponding authors: Bommarius, Andreas S ([email protected]) and Lee, Jay H ([email protected])
Helper proteins, such as lytic polysaccharide monooxygenase (LPO) define progress on the topic of commercial mixtures for enzymatic hydrolysis of cellulase and its components.
Current Opinion in Biotechnology 2014, 29:139–145
The following trends are outside the scope of this review:
This review comes from a themed issue on Cell and pathway engineering Edited by Tina Lu¨tke-Eversloh and Keith EJ Tyo
0958-1669/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.copbio.2014.04.007
Overview and introduction This review focuses on recent protein engineering efforts, both site-specific and random, mostly since 2009, on a range of proteins important for the hydrolysis of lignocellulosic materials, not only exocellulases and endocellulases but also helper proteins, such as lytic polysaccharide monooxygenase (LPO), swollenins, and expansins. The subject recently has been reviewed by Wilson [1], Clarke et al. [2], and Kellermann et al. [3]. Major advances during the reporting period can be found on the subjects of, first, the development of LPO [4], second, the thermostabilization of Cel6A and Cel7A via SCHEMAguided recombination [5,6], and, third, the computationally-based findings of the importance of the linker sequence for binding onto cellulose surfaces [7–9]. www.sciencedirect.com
(i) Kinetics and kinetic models of cellulose action; for these, the reader is encouraged to consult the review by Bansal et al. [10] as well as the models developed by Levine et al. [11] and Fox et al. [12], Cruys-Bagger et al. [13,14], Kurasin and Valjamae [15], and Bansal et al. [16]; (ii) The discussion of influences of crystallinity, accessibility, or mass transfer limitations. The reader is referred to the works by Hall et al. [17], Ponni et al. [18], Kumar et al. [19], Ioelovich [20], and Roberts et al. [21], respectively; (iii) Discussion of hemicellulases and esterases.
Protein engineering of the catalytic domain (CD) Processivity
The three-dimensional structure of the TrCel7A catalytic domain shows a long active site tunnel harboring four tryptophan residues. They are known to be essential for hydrophobic stacking interactions for the glucosyl units at the tunnel entrance (Trp-40), at the center of the tunnel (Trp-38), and around the catalytic sites (Trp-367 and Trp376) and these interactions are linked to effective and continuous hydrolysis of crystalline cellulose in a processive mode. Nakamura et al. found that the tryptophan Current Opinion in Biotechnology 2014, 29:139–145
140 Cell and pathway engineering
Figure 1
Carbohydrate -Binding Module
Catalytic Module
Cellulase Activity Thermostability Processivity Binding
Linker
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Improvement of performance attributes depends on the cellulose domain involved.
located at the tunnel entrance is important for processivity of degradation of crystalline cellulose [22]. They evaluated enzyme specific activity of WT and Trp-40 mutant on amorphous and crystalline cellulose revealing that Trp-40 is involved in recruiting cellulose chain reducing ends into the active site tunnel to initiate processive hydrolysis. Synergism with endocellulases and processivity
Vuong and Wilson [23] examined the exocellulase Cel6B properties of processivity and synergism with endocellulases from the Thermobifida fusca soil bacterium. Six residues not involved in the active site but in the active site tunnel were targeted for mutation, as summarized in Table 1a. Although processivity and oligosaccharide production were increased by a factor of 2–3 in some variants, the synergism with a native Cel5A was reduced such that the exocellulase exhibited lower processivity than the wild type. It was postulated that the decrease in side chain volume allowed freer movement of the cellulose chain along the catalytic tunnel. The lack of synergism was attributed to a potential reduction in the activity on the easily hydrolysable soluble chains produced by the endocellulase. An experiment with reducing-end attacking exocellulases Cel48A did show increased synergism, suggesting that the simple synergism model of endocellulases producing more reducing ends for Current Opinion in Biotechnology 2014, 29:139–145
exocellulases to attack is insufficient to explain the observed phenomena. Engineering of glycosylation for CD
Adney et al. examined the effects of N-linked glycans of the catalytic domain on cellulase activity by genetically removing or adding the N-glycosyl modification [24]. The removal of N-linked glycans located at both N384A of TrCel7A and N388A of Penicillium funiculosum Cel7A (PfCel7A) exhibited improved hydrolysis activity for crystalline cellulosic substrate. Similarly, the addition of a new N-glycosylation motif to PfCel7A (A196S) near the proximal end of the active site tunnel enhanced enzyme activity for cellulose degradation. These results suggest that glycosylation could be a target for improvement of cellulase performance. Reduced inhibition by glucose
Cellulases are often inhibited by their end product, cellobiose, and to a lesser degree by the glucose produced by further hydrolysis. Lavigne et al. [25] engineered Cel6A enzyme to reduce glucose inhibition, thus allowing cellulose hydrolysis to proceed at a rate closer to the maximum. They selected amino acids at position 103, 136, 186, 365, or 410 of TrCel6A enzyme for mutation and showed 1.8-fold to 6.73-fold reductions in glucose www.sciencedirect.com
Protein engineering of cellulases Bommarius et al. 141
Table 1 Protein engineering on cellobiohydrolases Cellulase
Residue
Proposed function
Evidence f
Source
(a) Catalytic domain W135, W376, W269, W272
TrCel6A a
Free energy of substrate binding
Relative binding affinity changed by orders of magnitude.
[9]
W40
TrCel7A
Recruitment of substrate chains into the active site tunnel.
Experimental variation in cellobiose production from different substrates
[22]
W376
TrCel7A
Hydrophobic stacking interactions for glucosyl units (catalytic center), free energy of product binding Free energy of product binding
Change in free energy of product binding in computational mutation experiment.
[33]
R251 D259 D262 Y381 Y103, L136, S186, G365, R410
TrCel6A
N282
b
TfCel6B
Tunnel entrance sugar binding (+2 position)
R180
Tunnel exit sugar binding (4 position)
L230
Processivity
W464
Substrate binding
D512
Loop flexibility
M514
Change in free energy of product binding in computational mutation experiment.
Reduce inhibition by glucose
[25]
Mutants have less bulky side chains N282A, N282D Mutant 2 processive, activity N282A/D 3 on PASC, 2/1.5 on CMC Mutants have less bulky side chains R180K, R180A twice processivity, no significant increase in activity. L230A minor processive gain 2.5 active on PASC W464A/Y 7/2 activity on CMC 0.25/ 0.34 activity on BMCC D512A 1.74 on PASC 2.4 on PC, 5.68 on CMC 0.34 on BMCC M514A 1.5 on CMC 1.3 on BMCC, M514Q 1.74 on CMC 1.25 on BMCC
[23]
N384 for Tr N388 for Pf A196 for Pf
TrCel7A c PfCel7A
Glycan in peptide loop of the active site tunnel
1.7 hydrolysis activity on BMCC
[24]
C313S
d
HiCel6A
Thermostability
Increase thermostability by 8.5 8C, activity by 10
[29]
S329G
e
CtCel8A
Thermostability
Increase thermostability by 7.0 8C, half-life at 85 8C by 8.5
[28]
TrCel6A, TrCel7A
Glycosylation in linker
R!E/S!T, reduce lignin binding and/or increase hydrolysis rate
[36]
Processivity
Energy minima corresponding to a cellobiose unit along hydrophobic face of cellulose
[34]
(b) Linker All R and S in the linker
(c) Carbohydrate-binding modules Y5, Q7, N29, Y32 TrCel7A
a
TrCel7A: Trichoderma reesei Cel7A. TfCel6B: Thermobifida fusca Cel6B. c PfCel7A: Penicillium funiculosum Cel7A. d HiCel6A: Humicola insolens Cel6A. e CtCel8A: Clostridium thermocellum Cel8A. f CMC, carboxymethyl cellulose; PASC, phosphoric acid-swollen cellulose; BMCC, bacterial microcrystalline cellulose; PC, phosphoric acid-treated cotton. b
inhibition. Such improved cellulases could be useful for integrated hydrolysis processes which benefit from constant cellulase activity in presence of glucose, pending direct evidence of improvement of overall hydrolysis rate. www.sciencedirect.com
Thermal stabilization via protein engineering
Thermostable cellulases are useful in biofuel processes, as they feature increased specific activity, higher levels of stability, inhibition of microbial growth, or increase in Current Opinion in Biotechnology 2014, 29:139–145
142 Cell and pathway engineering
mass transfer rate due to lower fluid viscosity. A recent review [26] describes state-of-the-art protein engineering protocols, which are based on combinatorial and datadriven techniques, to achieve thermostable cellulases. Such techniques and protocols are envisioned to be important for other improved cellulase properties, such as solvent, salt, and extreme pH tolerance. The Arnold group at Caltech built on their highly successful structureguided recombination (SCHEMA) of three fungal cellobiohydrolases 2 (CBH2) [6]. In chimeras of CBH2, stability was found to be additive with respect to certain sequence blocks via a linear mathematical model which also resulted in a successful single mutation, C313S [27]. A similar approach worked on Cel8A and on CBH1: in the former case, the half-life at 85 8C of Cel8A of Clostridium thermocellum was enhanced 8.5 in the single variant S329G [28]; in the latter case, 38% (6 of 16) predicted thermostabilized chimeras with up to 37 altered amino acid positions compared to the wildtype from Talaromyces emersonii hydrolyzed solid cellulose at 70 8C, 5 8C higher than the wildtype CBH1 [29]. Through utilization of FoldX and the consensus sequence approach, further mutations were found in five homologous CBH1 enzymes and 41 aligned CBH1 sequences to improve the temperature of optimum activity Topt to 10 8C above the wild-type T. emersonii [5]. In the fungal family 6 cellobiohydrolases (Cel6A), free cysteines, in contrast to disulfide bonds, were found to contribute to irreversible thermal inactivation in both wildtype Cel6A from Hypocrea jecorina and Humicola insolens as well as engineered thermostable Cel6A from these parents [30]. The consensus approach was also successful in two other recent studies. Alignment of five thermophilic CBH2 proteins resulted in 15 advantageous positions out of 45 non-consensus sequence positions in the Phanerochaete chrysosporium protein and multifold stabilization at 50 8C [31]. The consensus approach on family 8 endoglucanases (Cel8A) yielded a single mutation, G283P, which in conjunction with three previously identified variations led to a 14-fold increased half-life at 85 8C [32].
change of the free energy of binding [9]. The positions inside the tunnel did not exhibit large changes in free energy but were important in stabilizing the fluctuations associated with movement of cellobiose substrate moieties. The conclusion was that given the different factors involved in the aromatic–carbohydrate interactions, it may be difficult to generalize protein engineering across glycolytic hydrolase families to improve ligand binding and processivity based on tunnel position alone.
Protein engineering of the linker The linkers from cellulase enzymes are thought to connect glycoside hydrolases to carbohydrate-binding domain flexibly, and their sequences are observed to have diversity in amino acid content as well as length so that their functional role has remained unclear. To gain insight into the function of the linker and to maximize the effect on enzyme performance, linker engineering can be targeted at linker length, amino acid content, and glycosyl modification since each one contributes to linker flexibility, structure, and interaction with the substrate. Studies have demonstrated that linker length is optimized on type of structural domain and that the linker contained glycine, proline, serine, and threonine residues [7,8]. Scott et al. [36] constructed linker variants to obtain lignin resistant cellulase enzyme by altering linker amino acids (arginine or serine to glutamate or threonine, respectively) so that linker peptide-engineered cellulase showed increases in hydrolysis activity in the presence of lignin along with decreasing lignin binding. Payne et al. [37] constructed two simulation models for TrCel7A and TrCel6A and showed that O-glycosylated linker dynamically and nonspecifically binds to the hydrophobic face of cellulose. They suggested both through simulation and experimentation that O-glycan attached on serine and threonine residues served not only to extend peptide conformation, but also to affect linker binding directly to cellulose, causing enhanced binding affinity.
Protein engineering of the carbohydratebinding module (CBM)
Computational investigation of CDs
Proximity and targeting
Cellulase functionality is complex because of both its multiple domain structure and the different physicochemical phenomena that are involved in cellulose digestion. This has prompted computational molecular dynamics studies of different aspects of cellulase behavior, and its different subunits, to improve understanding and develop targets for protein engineering [7– 9,33,34,35]. In a recent paper, steered molecular dynamics simulations of TrCel7A were used to hypothesize that certain positions, listed in Table 1a, play key roles in cellobiose and glucose binding to the catalytic tunnel [33]. These were mutated in silico and the change in the free energy of binding during product expulsion computed. Mutations of the aromatic residues at different tunnel positions showed significant differences in the
CBMs display no hydrolytic function and the widely accepted role of CBMs is their binding affinity to both soluble and insoluble carbohydrate substrates. Through addition or deletion of the CBM, ligand-binding capacity can be altered. It has been reported that single-module polysaccharide hydrolases, such as cellobiohydrolase and mannanase, exhibit higher catalytic activity as well as thermostability when attached to heterologous CBMs [38–41]. Upon fusion to other non-cellulolytic enzymes, the targeting effect of CBM can bring the chimera to within close proximity of the carbohydrate-containing substrate, which in turn improves the appended enzymes activity on their substrates in close association with carbohydrates. Ravalason et al. [42] demonstrated that biobleaching efficiency can be drastically enhanced by
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Protein engineering of cellulases Bommarius et al. 143
forming a chimeric enzyme comprising laccase and family 1 CBM, through which the effective chimera concentration on insoluble lignin/cellulose surface can be enriched. However, higher ligand-binding capacity/affinity does not guarantee an improved cellulase activity as the ultra-tight binding could become a restriction for the dynamic motion of the enzymes [43]. Through the comparison study of a single-CBM and double-CBM processive endoglucanase Cel9A, the same mutations introduced to the latter were found to be significantly less advantageous. This is possibly because of the existence of a strong wild-type binding domain in addition to the one being engineered [44]. Besides the critical residues responsible for carbohydrate adsorption, the binding affinity of CBMs is modulated in part by the glycosylation pattern. Taylor et al. [35] through computational investigation found a 140-fold increase in binding attributable to the addition of an artificial glycan on serine locates on the planar binding face of TrCel7A. The improvement on binding and the ultimate effect on hydrolysis efficiency await experimental validation. Nonetheless, the molecular simulation results present an alternative direction for protein engineering of cellulases.
Table 1c. Depending on the different modular structures of cellulases, the mode of processivity can be enzymespecific. Nevertheless, the recognition of the protein– carbohydrate interaction face and critical residues for binding in the CBM could provide the most direct evidence for its role in processivity. Besides the proximity, targeting and processivity effect, novel disruptive functions of CBMs have been discovered, such as hydrogen bond splitting and cellulose decrystallization effects [52,53]. However, to our knowledge, disruptive functionality has not been targeted for protein engineering.
Protein engineering of the cellulase booster
In addition to the positive ligand-recognition function, recently, the role of CBMs at high substrate consistency was investigated for both cellulose and lignocelluloses. Even more than pure cellulose, the lignin-containing plant cell wall is highly recalcitrant and resistant to biological degradation. Cellulase deprived of CBMs exhibits similar or even higher hydrolysis yield at high substrate loadings when compared to wild-type cellulase, especially for lignocelluloses. It was thus postulated that CBMs partly contribute to non-productive binding to lignin [45,46]. However, more direct evidence on CBMs’ preferential adsorption onto lignin is needed to support the hypothesis and demonstrate the generic applicability for CBM-containing cellulases.
Novel non-hydrolytic proteins, which are capable of significantly enhancing the activity of cellulases through reducing the crystallinity of cellulose, have been studied recently. These proteins potentially lead to reduction in the quantity of relatively expensive cellulase added to enzymatic hydrolysis. Expansin-like proteins produced by fungi and bacteria, called swollenins, are known to be capable of loosening or disrupting the cellulose [54–56]. LPOs were functionally identified as participating in the degradation of cellulose by catalyzing oxidative cleavage of polysaccharide [4]. These proteins bind to the surfaces of crystalline cellulose, where they introduce chain breaks in the polysaccharide chains promoting deagglomeration and dispersion. Then the cellulosic matrix becomes susceptible to the action of a cellulase, which significantly accelerates hydrolysis of the sugars. Three types of cellulases have been displayed on yeast cell surfaces as well as expansin-like proteins, resulting in simultaneous saccharification and fermentation. This resulted in the production of 3.4 g/L ethanol from amorphous cellulose [57]. Combining cellulase and helper protein for pretreatment and fermentation has a great potential in reduction of enzyme cost for biofuels production and opening new targets for cellulase engineering.
Processivity
Conclusions
The impact of CBMs on the mechanism of enzyme processivity is not completely understood [47]. Among the recently published work, processivity is believed mainly to be controlled by the residues of the CDs [44,48,49]. However, controversial results are also reported regarding the role of CBMs in processivity [50]. Recently, the CBM of a family 5 endoglucanases was identified to play a vital role in enzyme processivity through comparison with the CBM-less variant [51] and computational simulation results also provide evidence that family 1 CBMs are responsible for processivity [34]. The TrCel7A CBD exhibits thermodynamically stable regions corresponding to the length of a cellobiose unit, which thus is the critical length scale of the processive TrCel7A [34]. The residues recognized to be responsible for the processivity length scale of TrCel7A are listed in
The complex nature of cellulase functionality has led to recent protein engineering studies designed to inform us on our understanding of the contribution of different protein subunits to the overall protein function. Computational studies can be particularly effective in this regard, because information that is inaccessible to experimental studies can be obtained. Feedback between experimental probing of sequences designed by computational studies to test hypotheses of sequence-to-function mapping shows significant promise to tease apart processivity, substrate binding, accessibility, and kinetic limitations of cellulases. Equipped with greater understanding of which functionalities limit cellulase rates on different substrates, a greater variety of protein engineering tools can be brought to bear on these tightly coupled complicated enzymes with higher probabilities of success in improving their function.
Non-productive binding to lignin
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Current Opinion in Biotechnology 2014, 29:139–145
144 Cell and pathway engineering
Acknowledgements Minjeong Sohn and the other authors gratefully acknowledge support from the Advanced Biomass Center (ABC) program at KAIST, Daejeon, South Korea. Yuzhi Kang acknowledges support from the Institute of Paper Science and Technology (IPST) at the Georgia Institute of Technology.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Wilson DB: Cellulases and biofuels. Curr Opin Biotechnol 2009, 20:295-299.
2.
Clarke ND: Protein engineering for bioenergy and biomassbased chemicals. Curr Opin Struct Biol 2010, 20:527-532.
3.
Kellermann SJ, Rentmeister A: Current developments in cellulase engineering. ChemBioEng Rev 2014, 1:6-13.
4.
Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG: Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 2012, 5:1-13.
5.
Komor RS, Romero PA, Xie CB, Arnold FH: Highly thermostable fungal cellobiohydrolase I (Cel7A) engineered using predictive methods. Protein Eng Des Sel 2012, 25:827-833.
6.
Heinzelman P, Snow CD, Wu I, Nguyen C, Villalobos A, Govindarajan S, Minshull J, Arnold FH: A family of thermostable fungal cellulases created by structure-guided recombination. Proc Natl Acad Sci USA 2009, 106:5610-5615. This frequently cited paper introduced recombination of whole cellulose domains as a method for thermal stabilization. Not only could the catalytic domain be significantly stabilized but a regression of the data demonstrated additivity of mutations for stabilization.
7.
8.
9.
Beckham GT, Bomble YJ, Matthews JF, Taylor CB, Resch MG, Yarbrough JM, Decker SR, Bu L, Zhao X, McCabe C: The Oglycosylated linker from the Trichoderma reesei family 7 cellulase is a flexible, disordered protein. Biophys J 2010, 99:3773-3781. Sammond DW, Payne CM, Brunecky R, Himmel ME, Crowley MF, Beckham GT: Cellulase linkers are optimized based on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation. PLoS ONE 2012, 7:e48615. Payne CM, Bomble Y, Taylor CB, McCabe C, Himmel ME, Crowley MF, Beckham GT: Multiple functions of aromaticcarbohydrate interactions in a processive cellulase examined with molecular simulation. J Biol Chem 2011, 286:41028-41035.
10. Bansal P, Hall M, Realff MJ, Lee JH, Bommarius AS: Modeling cellulase kinetics on lignocellulosic substrates. Biotechnol Adv 2009, 27:833-848. 11. Levine SE, Fox JM, Blanch HW, Clark DS: A mechanistic model of the enzymatic hydrolysis of cellulose. Biotechnol Bioeng 2010, 107:37-51. 12. Fox JM, Levine SE, Clark DS, Blanch HW: Initial- and processivecut products reveal cellobiohydrolase rate limitations and the role of companion enzymes. Biochemistry 2012, 51:442-452. 13. Cruys-Bagger N, Elmerdahl J, Praestgaard E, Borch K, Westh P: A steady-state theory for processive cellulases. FEBS J 2013, 280:3952-3961. 14. Cruys-Bagger N, Tatsumi H, Ren GR, Borch K, Westh P: Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain. Biochemistry 2013, 52:8938-8948. 15. Kurasin M, Valjamae P: Processivity of cellobiohydrolases is limited by the substrate. J Biol Chem 2011, 286:169-177. 16. Bansal P, Vowell BJ, Hall M, Realff MJ, Lee JH, Bommarius AS: Elucidation of cellulose accessibility, hydrolysability and reactivity as the major limitations in the enzymatic hydrolysis of cellulose. Bioresour Technol 2012, 107:243-250. Current Opinion in Biotechnology 2014, 29:139–145
17. Hall M, Bansal P, Lee JH, Realff MJ, Bommarius AS: Cellulose crystallinity—a key predictor of the enzymatic hydrolysis rate. FEBS J 2010, 277:1571-1582. 18. Ponni R, Kontturi E, Vuorinen T: Accessibility of cellulose: Structural changes and their reversibility in aqueous media. Carbohydr Polym 2013, 93:424-429. 19. Kumar L, Arantes V, Chandra R, Saddler J: The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility. Bioresour Technol 2012, 103:201-208. 20. Ioelovich M: Accessibility and crystallinity of cellulose. Bioresources 2009, 4:1168-1177. 21. Roberts KM, Lavenson DM, Tozzi EJ, McCarthy MJ, Jeoh T: The effects of water interactions in cellulose suspensions on mass transfer and saccharification efficiency at high solids loadings. Cellulose 2011, 18:759-773. 22. Nakamura A, Tsukada T, Auer S, Furuta T, Wada M, Koivula A, Igarashi K, Samejima M: The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7a is important for initiation of degradation of crystalline cellulose. J Biol Chem 2013, 288:13503-13510. Mutates Trp-40 involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis, measures differences in kinetic rate constants and combines this with molecular simulation to propose mechanistic explanation. 23. Vuong TV, Wilson DB: Processivity, synergism, and substrate specificity of Thermobifida fusca Cel6B. Appl Environ Microbiol 2009, 75:6655-6661. Performs specific mutations of the catalytic domain and demonstrates substrate specific engineering of cellulase. Shows that synergism between cellulases can be disrupted by these mutations. 24. Adney WS, Jeoh T, Beckham GT, Chou Y-C, Baker JO, Michener W, Brunecky R, Himmel ME: Probing the role of Nlinked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis. Cellulose 2009, 16:699-709. 25. Lavigne J, Hill C, Tremblay A, St-Pierre P, Tomashek J. Cellulase variants with reduced inhibition by glucose. WO Patent 2,009,089,630; 2009. 26. Anbar M, Bayer EA: Approaches for improving thermostability characteristics in cellulases. Cellulases 2012, 510:261-271. 27. Heinzelman P, Snow CD, Smith MA, Yu XL, Kannan A, Boulware K, Villalobos A, Govindarajan S, Minshull J, Arnold FH: SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability. J Biol Chem 2009, 284:26229-26233. 28. Anbar M, Lamed R, Bayer EA: Thermostability enhancement of Clostridium thermocellum cellulosomal endoglucanase Cel8A by a single glycine substitution. Chemcatchem 2010, 2:9971003. 29. Heinzelman P, Komor R, Kanaan A, Romero P, Yu XL, Mohler S, Snow C, Arnold F: Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination. Protein Eng Des Sel 2010, 23:871-880. 30. Wu I, Heel T, Arnold FH: Role of cysteine residues in thermal inactivation of fungal Cel6A cellobiohydrolases. Biochim Biophys Acta-Proteins Proteomic 2013, 1834:1539-1544. 31. Ito Y, Ikeuchi A, Imamura C: Advanced evolutionary molecular engineering to produce thermostable cellulase by using a small but efficient library. Protein Eng Des Sel 2013, 26:73-79. 32. Anbar M, Gul O, Lamed R, Sezerman UO, Bayer EA: Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Appl Environ Microbiol 2012, 78:3458-3464. 33. Bu L, Beckham GT, Shirts MR, Nimlos MR, Adney WS, Himmel ME, Crowley MF: Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy methods. J Biol Chem 2011, 286:1816118169. www.sciencedirect.com
Protein engineering of cellulases Bommarius et al. 145
34. Beckham GT, Matthews JF, Bomble YJ, Bu LT, Adney WS, Himmel ME, Nimlos MR, Crowley MF: Identification of amino acids responsible for processivity in a family 1 carbohydratebinding module from a fungal cellulase. J Phys Chem B 2010, 114:1447-1453. This work identified the potential role of CBMs in the processivity of T. reesei Cel7A by computational simulation. 35. Taylor CB, Talib MF, McCabe C, Bu LT, Adney WS, Himmel ME, Crowley MF, Beckham GT: Computational investigation of glycosylation effects on a family 1 carbohydrate-binding module. J Biol Chem 2012, 287:3147-3155. This work utilizes molecular simulation to study the effect of glycosylation on the TrCel7A CBM. Both native and artificial glycosylation showed substantial enhancement on binding compared to nonglycosylated CBM. 36. Scott BR, St-Pierre P, Lavigne JA, Masri N, White TC, Tomashek JJ. Novel lignin-resistant cellulase enzymes. WO Patent 2,010,096,931; 2010. The mutant of arginine and sernine substituted to glutamic acid and threonine, respectively, exhibited increased hydrolysis activity in presence of lignin and decreased lignin binding. The result of this study indicated that protein engineering of linker domain of cellobiohydrolases can yield lignin resistance. 37. Payne CM, Resch MG, Chen L, Crowley MF, Himmel ME, Taylor LE, Sandgren M, Sta˚hlberg J, Stals I, Tan Z: Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci 2013, 110:14646-14651. This study suggests that O-glycosylated linker domains of cellobiohydrolases (TrCel7A and TrCel6A) bind to the hydrophobic face of cellulose microfibrils through molecular dynamics simulations. In order to validate their model they measured binding affinity of TrCel7A CBM and the glycosylated TrCel7A CBM-linker on cellulose, in consistent to prediction the linker showed a higher binding affinity. The result of this study indicated that glycosylated linker play a role in direct interaction with crystalline cellulose. 38. Voutilainen SP, Nurmi-Rantala S, Penttila M, Koivula A: Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 2013. 39. Hall M, Rubin J, Behrens SH, Bommarius AS: The cellulosebinding domain of cellobiohydrolase Cel7A from Trichoderma reesei is also a thermostabilizing domain. J Biotechnol 2011, 155:370-376. 40. Tang CD, Li JF, Wei XH, Min R, Gao SJ, Wang JQ, Yin X, Wu MC: Fusing a carbohydrate-binding module into the Aspergillus usamii beta-mannanase to improve its thermostability and cellulose-binding capacity by in silico design. PLoS ONE 2013:8. 41. Thongekkaew J, Ikeda H, Masaki K, Iefuji H: Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates. Enzyme Microb Technol 2013, 52:241-246. 42. Ravalason H, Herpoel-Gimbert I, Record E, Bertaud F, Grisel S, de Weert S, van den Hondel CA, Asther M, Petit-Conil M, Sigoillot JC: Fusion of a family 1 carbohydrate binding module of Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulp biobleaching. J Biotechnol 2009, 142:220-226. 43. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ: Carbohydratebinding modules: fine-tuning polysaccharide recognition. Biochem J 2004, 382:769-781. 44. Li Y, Irwin DC, Wilson DB: Increased crystalline cellulose activity via combinations of amino acid changes in the family 9
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catalytic domain and family 3c cellulose binding module of Thermobifida fusca Cel9A. Appl Environ Microbiol 2010, 76:2582-2588. This work elucidates the significance of the binding balance between CD and CBM as well as within CD binding cleft. 45. Le Costaouec T, Pakarinen A, Varnai A, Puranen T, Viikari L: The role of carbohydrate binding module (CBM) at high substrate consistency: comparison of Trichoderma reesei and Thermoascus aurantiacus Cel7A (CBHI) and Cel5A (EGII). Bioresour Technol 2013, 143:196-203. 46. Varnai A, Siika-Aho M, Viikari L: Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs. Biotechnol Biofuels 2013, 6:30. 47. Li Y, Irwin DC, Wilson DB: Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A. Appl Environ Microbiol 2007, 73:3165-3172. This work identifies the critical residues for processivity within the CD of endoglucanase Cel9A. 48. Igarashi K, Koivula A, Wada M, Kimura S, Penttila M, Samejima M: High speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose. J Biol Chem 2009, 284: 36186-36190. An important piece of work visualizing the processive motion of TrCel7A and its variants on insoluble cellulose substrates. 49. Chiriac AI, Cadena EM, Vidal T, Torres AL, Diaz P, Pastor FI: Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture. Appl Microbiol Biotechnol 2010, 86:1125-1134. 50. Irwin D, Shin DH, Zhang S, Barr BK, Sakon J, Karplus PA, Wilson DB: Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis. J Bacteriol 1998, 180:1709-1714. 51. Zheng F, Ding S: Processivity and enzymatic mode of a glycoside hydrolase family 5 endoglucanase from Volvariella volvacea. Appl Environ Microbiol 2013, 79:989-996. 52. Ciolacu D, Kovac J, Kokol V: The effect of the cellulosebinding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers. Carbohydr Res 2010, 345:621-630. 53. Hall M, Bansal P, Lee JH, Realff MJ, Bommarius AS: Biological pretreatment of cellulose: enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases. Bioresour Technol 2011, 102:2910-2915. 54. Kim IJ, Ko HJ, Kim TW, Choi IG, Kim KH: Characteristics of the binding of a bacterial expansin (BsEXLX1) to microcrystalline cellulose. Biotechnol Bioeng 2013, 110:401-407. 55. Lee HJ, Lee S, Ko H-j, Kim KH, Choi I-G: An expansin-like protein from Hahella chejuensis binds cellulose and enhances cellulase activity. Mol Cells 2010, 29:379-385. 56. Ja¨ger G, Girfoglio M, Dollo F, Rinaldi R, Bongard H, Commandeur U, Fischer R, Spiess AC, Bu¨chs J: How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis. Biotechnol Biofuels 2011, 4:33. 57. Nakatani Y, Yamada R, Ogino C, Kondo A: Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose. Microb Cell Fact 2013, 12:66.
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