Genomic, Proteomic, and Biochemical Analyses of Oleaginous Mucor circinelloides: Evaluating Its Capability in Utilizing Cellulolytic Substrates for Lipid Production.
Wei, H., Wang, W., Yarbrough, J. M., Baker, J. O., Laurens, L., Van Wychen, S., Chen, X., Taylor II, L. E., Xu, Q., Himmel, M. E. & Zhang, M. (2013). PloS One, 8(9), e71068.
Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.
Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production.
Chang, J. J., Ho, F. J., Ho, C. Y., Wu, Y. C., Hou, Y. H., Huang, C. C., Shih, M. C. & Li, W. H. (2013). Biotechnology Biofuels, 6(1), 19-31.
Background: Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed. Results: To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-based gene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1, 4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol. Conclusion: Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host.
Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes.
Zhang, D., VanFossen, A. L., Pagano, R. M., Johnson, J. S., Parker, M. H., Pan, S., Gray, N. B., Hancock, E., Hagen, D. J., Lucero, H. A., Shen, B., Lessard, P. A., Ely, C., Moriarty, M., Ekborg, N. A., Bougri, O., Samoylov, V., Lazar, G. & Raab, R. M. (2011). BioEnergy Research, 4(4), 276-286.
Significant amounts of cell wall degrading (CWD) enzymes are required to degrade lignocellulosic biomass into its component sugars. One strategy for reducing exogenous enzyme production requirements is to produce the CWD enzymes in planta. For this work, various CWD enzymes were expressed in maize (Zea mays). Following growth and dry down of the plants, harvested maize stover was tested to determine the impact of the expressed enzymes on the production of glucose and xylose using different exogenous enzyme loadings. In this study, a consolidated pretreatment and hydrolysis process consisting of a moderate chemical pretreatment at temperatures below 75°C followed by enzymatic hydrolysis using an in-house enzyme cocktail was used to evaluate engineered transgenic feedstocks. The carbohydrate compositional analysis showed no significant difference in the amounts of glucan and xylan between the transgenic maize plants expressing CWD enzyme(s) and the control plants. Hydrolysis results demonstrated that transgenic plants expressing CWD enzymes achieved up to 141% higher glucose yield and 172% higher xylose yield over the control plants from enzymatic hydrolysis under the experimental conditions. The hydrolytic performance of a specific xylanase (XynA) expressing transgenic event (XynA.2015.05) was heritable in the next generation, and the improved properties can be achieved even with a 25% reduction in exogenous enzyme loading. Simultaneous saccharification and fermentation of biomass hydrolysates from two different transgenic maize lines with yeast (Saccharomyces cerevisiae D5A) converted 65% of the biomass glucan into ethanol, versus only a 42% ethanol yield with hydrolysates from control plants, corresponding to a 55% improvement in ethanol production.
Competitive sorption kinetics of inhibited endo-and exoglucanases on a model cellulose substrate.
Maurer, S. A., Bedbrook, C. N. & Radke, C. J. (2012). Langmuir, 28(41), 14598-14608.
For the first time, the competitive adsorption of inhibited cellobiohydrolase I (Cel7A, an exoglucanase) and endoglucanase I (Cel7B) from T. longibrachiatum is studied on cellulose. Using quartz crystal microgravimetry (QCM), sorption histories are measured for individual types of cellulases and their mixtures adsorbing to and desorbing from a model cellulose surface. We find that Cel7A has a higher adsorptive affinity for cellulose than does Cel7B. The adsorption of both cellulases becomes irreversible on time scales of 30–60 min, which are much shorter than those typically used for industrial cellulose hydrolysis. A multicomponent Langmuir kinetic model including first-order irreversible binding is proposed. Although adsorption and desorption rate constants differ between the two enzymes, the rate at which each surface enzyme irreversibly binds is identical. Because of the higher affinity of Cel7A for the cellulose surface, when Cel7A and Cel7B compete for surface sites, a significantly higher bulk concentration of Cel7B is required to achieve comparable surface enzyme concentrations. Because cellulose deconstruction benefits significantly from the cooperative activity of endoglucanases and cellobiohydrolases on the cellulose surface, accounting for competitive adsorption is crucial to developing effective cellulase mixtures.
Droplet-based microfluidic platform for heterogeneous enzymatic assays.
Chang, C., Sustarich, J., Bharadwaj, R., Chandrasekaran, A., Adams, P. D. & Singh, A. K. (2013). Lab Chip, 13(9), 1817-1822.
Heterogeneous enzymatic reactions are used in many industrial processes including pulp and paper, food, and biofuel production. Industrially-relevant optimization of the enzymes used in these processes requires assaying them with insoluble substrates. However, platforms for high throughput heterogeneous assays do not exist thereby severely increasing the cost and time of enzyme optimization, or leading to the use of assays with soluble substrates for convenient, but non-ideal, optimization. We present an innovative approach to perform heterogeneous reactions in a high throughput fashion using droplet microfluidics. Droplets provide a facile platform for heterogeneous reactions as internal recirculation allows rapid mixing of insoluble substrates with soluble enzymes. Moreover, it is easy to generate hundreds or thousands of picoliter droplets in a small footprint chip allowing many parallel reactions. We validate our approach by screening combinations of cellulases with real-world insoluble substrates, and demonstrate that the chip-based screening is in excellent agreement with the conventional screening methods, while offering advantages of throughput, speed and lower reagent consumption. We believe that our approach, while demonstrated for a biofuel application, provides a generic platform for high throughput monitoring of heterogeneous reactions.
High-throughput enzymatic hydrolysis of lignocellulosic biomass via in-situ regeneration.
Bharadwaj, R., Wong, A., Knierim, B., Singh, S., Holmes, B. M., Auer, M., Simmons, B. A., Adams, P. D. & Singh, A. K. (2011). Bioresource Technology, 102(2), 1329-1337.
The high cost of lignocellulolytic enzymes is one of the main barriers towards the development of economically competitive biorefineries. Enzyme engineering can be used to significantly increase the production rate as well as specific activity of enzymes. However, the success of enzyme optimization efforts is currently limited by a lack of robust high-throughput (HTP) cellulase screening platforms for insoluble pretreated lignocellulosic substrates. We have developed a cost-effective microplate based HTP enzyme-screening platform for ionic liquid (IL) pretreated lignocellulose. By performing in-situ biomass regeneration in micro-volumes, we can volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for engineering novel IL-tolerant cellulases. Our platform only requires straightforward liquid-handling steps and allows the integration of biomass regeneration, washing, saccharification, and imaging steps in a single microtiter plate. The proposed method can be used to screen individual cellulases as well as to develop novel cellulase cocktails.
Surface kinetics for cooperative fungal cellulase digestion of cellulose from quartz crystal microgravimetry.
Maurer, S. A., Brady, N. W., Fajardo, N. P. & Radke, C. J. (2013). Journal of Colloid and Interface Science, 394, 498-508.
The kinetic behavior of aqueous cellulase on insoluble cellulose is best quantified through surface-based assays on a well-defined cellulose substrate of known area. We use a quartz crystal microbalance (QCM) to measure the activity of binary mixtures of Trichoderma longibrachiatum cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) on spin-coated cellulose films. By extending a previous surface kinetic model for cellulase activity, we obtain rate constants for competitive adsorption of Cel7A and Cel7B, their irreversible binding, their complexation with the cellulose surface, and their cooperative cellulolytic activity. The activity of the two cellulases is linked through the formation of cellulose chain ends by Cel7B that provide complexation sites from which Cel7A effects cellulose chain scission. Although the rate-limiting step in Cel7A activity is complexation, Cel7B activity is limited by adsorption to the cellulose surface. A 2:1 bulk mass ratio of aqueous Cel7A:Cel7B, corresponding to a 4:1 surface mass ratio, effects the greatest rate of cellulose degradation across a range of cellulase concentrations at 25°C. We find that surface chain-end concentration is a major predictor of Cel7A activity. Disruption of the hydrogen-bonding structure of cellulose by Cel7B enhances the activity of Cel7A on the cellulose surface.
Domain engineering of Saccharomyces cerevisiae exoglucanases.
Moses, S. B. G., Otero, R. R. C. & Pretorius, I. S. (2005). Biotechnology Letters, 27(5), 355-362.
To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg-1 respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.
Dissecting and Reconstructing Synergism in situ visualization of cooperativity among cellulases.
Ganner, T., Bubner, P., Eibinger, M., Mayrhofer, C., Plank, H. & Nidetzky, B. (2012). Journal of Biological Chemistry, 287(52), 43215-43222.
Cellulose is the most abundant biopolymer and a major reservoir of fixed carbon on earth. Comprehension of the elusive mechanism of its enzymatic degradation represents a fundamental problem at the interface of biology, biotechnology, and materials science. The interdependence of cellulose disintegration and hydrolysis and the synergistic interplay among cellulases is yet poorly understood. Here we report evidence from in situ atomic force microscopy (AFM) that delineates degradation of a polymorphic cellulose substrate as a dynamic cycle of alternating exposure and removal of crystalline fibers. Direct observation shows that chain-end-cleaving cellobiohydrolases (CBH I, CBH II) and an internally chain-cleaving endoglucanase (EG), the major components of cellulase systems, take on distinct roles: EG and CBH II make the cellulose surface accessible for CBH I by removing amorphous-unordered substrate areas, thus exposing otherwise embedded crystalline-ordered nanofibrils of the cellulose. Subsequently, these fibrils are degraded efficiently by CBH I, thereby uncovering new amorphous areas. Without prior action of EG and CBH II, CBH I was poorly active on the cellulosic substrate. This leads to the conclusion that synergism among cellulases is morphology-dependent and governed by the cooperativity between enzymes degrading amorphous regions and those targeting primarily crystalline regions. The surface-disrupting activity of cellulases therefore strongly depends on mesoscopic structural features of the substrate: size and packing of crystalline fibers are key determinants of the overall efficiency of cellulose degradation.
New glycosidase substrates for droplet-based microfluidic screening.
Najah, M., Mayot, E., Mahendra-Wijaya, I. P., Griffiths, A. D., Ladame, S. & Drevelle, A. (2013). Analytical Chemistry, 85(20), 9807-9814.
Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30°C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems.
Modified cellobiohydrolase-cellulose interactions following treatment with lytic polysaccharide monooxygenase CelS2 (ScLPMO10C) observed by QCM-D.
Selig, M. J., Vuong, T. V., Gudmundsson, M., Forsberg, Z., Westereng, B., Felby, C. & Master, E. R. (2015). Cellulose, 22(4), 2263-2270.
The beneficial mechanisms of the lytic polysaccharide monooxygenase CelS2 (ScLPMO10C) from Streptomyces coelicolor on interactions between cellulose and the processive cellulase cellobiohydrolase I (Cel7A) were investigated by quartz crystal microbalance with dissipation. Initial binding of CelS2 to cellulose coated SiO2 sensors at 40°C was rapid, but minimal, and was followed by modest positive changes in oscillation frequencies of the quartz crystal sensors that were attributed to mass loss from the cellulose surface. The presence of oxidized cellulose on the CelS2 treated sensors was verified by X-ray photoelectron spectroscopy. Subsequent binding of purified Cel7A from Trichoderma longibrachiatum at 23°C was significantly less in overall extent to CelS2 treated sensors than to untreated controls despite identical initial binding rates and dissipation-change: frequency-change ratios (an indication of the rigidity of the newly forming layer). Moreover, initial Cel7A binding to the treated sensors was followed by positive overall frequency changes indicating potential increased hydrolytic removal of cellulose mass by cellobiohydrolase action at 23°C. Furthermore, secondary binding of Cel7A to the control sensors was coupled with considerable changes in dissipation (indicating greater surface viscoelasticity) not observed during the initial binding phase; the extent of this secondary binding was observed to be negligible during Cel7A interaction with the CelS2 treated sensors. This drastic difference in more viscoelastic secondary binding suggests a reduction in loose non-productive cellobiohydrolase binding following CelS2 treatment.
Comparative insights into the saccharification potentials of a relatively unexplored but robust Penicillium funiculosum glycoside hydrolase 7 cellobiohydrolase.
Ogunmolu, F. E., Jagadeesha, N. B. K., Kumar, R., Kumar, P., Gupta, D. & Yazdani, S. S. (2017). Biotechnology for Biofuels, 10(71).
Background: GH7 cellobiohydrolases (CBH1) are vital for the breakdown of cellulose. We had previously observed the enzyme as the most dominant protein in the active cellulose-hydrolyzing secretome of the hypercellulolytic ascomycete—Penicillium funiculosum (NCIM1228). To understand its contributions to cellulosic biomass saccharification in comparison with GH7 cellobiohydrolase from the industrial workhorse—Trichoderma reesei, we natively purified and functionally characterized the only GH7 cellobiohydrolase identified and present in the genome of the fungus. Results: There were marginal differences observed in the stability of both enzymes, with P. funiculosum (PfCBH1) showing an optimal thermal midpoint (Tm) of 68°C at pH 4.4 as against an optimal Tm of 65°C at pH 4.7 for T. reesei (TrCBH1). Nevertheless, PfCBH1 had an approximate threefold lower binding affinity (Km), an 18-fold higher turnover rate (kcat), a sixfold higher catalytic efficiency as well as a 26-fold higher enzyme-inhibitor complex equilibrium dissociation constant (Ki) than TrCBH1 on p-nitrophenyl-β-D-lactopyranoside (pNPL). Although both enzymes hydrolyzed cellooligomers (G2–G6) and microcrystalline cellulose, releasing cellobiose and glucose as the major products, the propensity was more with PfCBH1. We equally observed this trend during the hydrolysis of pretreated wheat straws in tandem with other core cellulases under the same conditions. Molecular dynamic simulations conducted on a homology model built using the TrCBH1 structure (PDB ID: 8CEL) as a template enabled us to directly examine the effects of substrate and products on the protein dynamics. While the catalytic triads—EXDXXE motifs—were conserved between the two enzymes, subtle variations in regions enclosing the catalytic path were observed, and relations to functionality highlighted. Conclusion: To the best of our knowledge, this is the first report about a comprehensive and comparative description of CBH1 from hypercellulolytic ascomycete—P. funiculosum NCIM1228, against the backdrop of the same enzyme from the industrial workhorse—T. reesei. Our study reveals PfCBH1 as a viable alternative for CBH1 from T. reesei in industrial cellulase cocktails.
Pure enzyme cocktails tailored for the saccharification of sugarcane bagasse pretreated by using different methods.
Kim, I. J., Lee, H. J. & Kim, K. H. (2017). Process Biochemistry, In press.
The compositions and physical properties of pretreated lignocellulose vary depending on pretreatment methods; therefore, enzyme cocktails specific to pretreatments are desired for efficient saccharification of lignocellulose. Here, enzyme cocktails consisting of three pure lignocellulolytic enzymes endoglucanase (EG), cellobiohydrolase (CBH) and endoxylanase (XN) with a fixed amount of β-glucosidase were tailored for acid- and alkali-pretreated sugarcane bagasse (ACID and ALKALI, respectively). Based on a mixture design, the optimal mass ratios of EG, CBH, and XN were determined to be 61.25:38.73:0.02 and 53.99:34.60:11.41 for ACID and ALKALI, respectively. The optimized enzyme cocktail yielded a higher or comparable amount of reducing sugars from the hydrolysis of ACID and ALKALI when compared to that obtained using commercial cellulase mixtures. Using the commercial and easily available pure enzymes, this simple method for the in-house preparation of an enzyme cocktail specific to pretreated lignocellulose consisting of only four enzymes with a high level of hydrolysis will be helpful for achieving enzymatic saccharification in the lignocellulose-based biorefinery.